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Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code

Open AccessPublished:June 09, 2020DOI:https://doi.org/10.1074/jbc.REV120.011666
      Cells must be able to cope with the challenge of folding newly synthesized proteins and refolding those that have become misfolded in the context of a crowded cytosol. One such coping mechanism that has appeared during evolution is the expression of well-conserved molecular chaperones, such as those that are part of the heat shock protein 70 (Hsp70) family of proteins that bind and fold a large proportion of the proteome. Although Hsp70 family chaperones have been extensively examined for the last 50 years, most studies have focused on regulation of Hsp70 activities by altered transcription, co-chaperone “helper” proteins, and ATP binding and hydrolysis. The rise of modern proteomics has uncovered a vast array of post-translational modifications (PTMs) on Hsp70 family proteins that include phosphorylation, acetylation, ubiquitination, AMPylation, and ADP-ribosylation. Similarly to the pattern of histone modifications, the histone code, this complex pattern of chaperone PTMs is now known as the “chaperone code.” In this review, we discuss the history of the Hsp70 chaperone code, its currently understood regulation and functions, and thoughts on what the future of research into the chaperone code may entail.
      The heat shock protein 70 (Hsp70) family consists of well-conserved yet functionally diverse molecular chaperones that are critical for nascent protein folding (
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      ). As an integral part of the cellular proteostasis machinery, Hsp70s are critical for maintaining cell viability in response to a large variety of cellular stresses, including high temperature, nutrient starvation, osmotic shock, oxidative stress, and DNA damage (
      • Rosenzweig R.
      • Nillegoda N.B.
      • Mayer M.P.
      • Bukau B.
      The Hsp70 chaperone network.
      ). Historically, studies on Hsp70s have primarily focused on intrinsic folding activity driven by the binding and hydrolysis of ATP, interaction with helper co-chaperones, and inducibility of expression under stress. More recently, evidence has accumulated that Hsp70s are highly modified at the post-translational level (
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      ,
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      The complex phosphorylation patterns that regulate the activity of Hsp70 and its cochaperones.
      ,
      • Griffith A.A.
      • Holmes W.
      Fine tuning: effects of post-translational modification on Hsp70 chaperones.
      ). These modifications fine-tune chaperone function, altering chaperone activity, localization, and selectivity. In the same way that modifications on histones are collectively called the “histone code,” we now refer to the complex array of post-translational modifications (PTMs) on chaperones as the “chaperone code.” In this review, we summarize in detail the current knowledge of how PTMs contribute to the regulation of Hsp70 family chaperones. We further offer a perspective on future directions and challenges the field may encounter in establishing and integrating the physiological impact of the Hsp70 chaperone code.
      The Hsp70 family of proteins is evolutionarily conserved and found in archaebacteria, prokaryotes, and eukaryotes (including plants and animals), establishing it as a crucial protein family in the phylogenetic tree of life (
      • Borchiellini C.
      • Boury-Esnault N.
      • Vacelet J.
      • Le Parco Y.
      Phylogenetic analysis of the Hsp70 sequences reveals the monophyly of Metazoa and specific phylogenetic relationships between animals and fungi.
      ,
      • Gupta R.S.
      • Singh B.
      Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus.
      ,
      • Wu B.
      • Hunt C.
      • Morimoto R.
      Structure and expression of the human gene encoding major heat shock protein HSP70.
      ). Hsp70s have been widely studied both in vitro and in vivo, using a combination of purified recombinant proteins, tissue culture setups, and model organism such as Saccharomyces cerevisiae (budding yeast). A testament to the high level of functional conservation is the ability of Hsp70s from diverse organisms (human, sea anemone, and plant) to maintain the cell viability of budding yeast when expressed as the sole cytosolic Hsp70 (
      • Werner-Washburne M.
      • Stone D.E.
      • Craig E.A.
      Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae.
      ). S. cerevisiae contains seven cytosolic Hsp70 isoforms: the four canonical chaperones Ssa1, Ssa2, Ssa3, and Ssa4 and the three ribosome-associated chaperones Ssb1, Ssb2, and Ssz1. In addition, there are three mitochondrial isoforms (Ssc1, Ssq1, and Ecm10) and one specific to endoplasmic reticulum (Kar2) (
      • Lotz S.K.
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      • Jones G.W.
      • Truman A.W.
      Not quite the SSAme: unique roles for the yeast cytosolic Hsp70s.
      ). Ssa1 and Ssa2 are constitutively expressed, whereas Ssa3 and Ssa4 are not present during normal growth but up-regulated in response to stress and in the stationary phase (
      • Boorstein W.R.
      • Craig E.A.
      Structure and regulation of the SSA4 HSP70 gene of Saccharomyces cerevisiae.
      ,
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      Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae.
      ,
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      Yeast Hsp70 RNA levels vary in response to the physiological status of the cell.
      ,
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      The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.
      ). Yeast mutants lacking all four canonical Hsp70 chaperones are not viable (
      • Kabani M.
      • Martineau C.N.
      Multiple hsp70 isoforms in the eukaryotic cytosol: mere redundancy or functional specificity?.
      ). Ssa1–4 are partially functionally redundant, with overexpression of any single Ssa isoform enough to provide cell viability in an ssa1–4Δ strain (
      • Werner-Washburne M.
      • Stone D.E.
      • Craig E.A.
      Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae.
      ).
      Human Hsp70s are encoded by a multigene family, which constitutes 17 genes and 30 pseudogenes (
      • Brocchieri L.
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      • Macario A.J.
      hsp70 genes in the human genome: conservation and differentiation patterns predict a wide array of overlapping and specialized functions.
      ). This multigene family gives rise to 13 gene products, which vary in their location, amino acid composition, and expression levels in the cell. Hsp70-1 (HSPA1A, Hsp70) and Hsp70-2 (HSPA1B, Hsp70) are the two major stress-inducible isoforms, which only differ from each other by two amino acids. The noninducible Ssz-like chaperone Hsp70-1t (HspA1L) is constitutively expressed and exhibits 90% identity to Hsp70-1. Hsp70-5 (HSPA5/BiP/GRP78) is localized to the endoplasmic reticulum and facilitates transport and folding of nascent polypeptides into the ER lumen. Hsp70-6 (HspA6) is an additional stress-inducible Hsp70 family member that has 85% homology to Hsp70-1 and is expressed in moderate levels in dendritic cells, monocytes, and natural killer cells but has no detectable basal expression level in other cells. HSPA7 has been considered a pseudogene transcribed in response to stress. Hsp70-8 (HSPA8, Hsc70, Hsp73, Hsc72) is the cognate Hsp70 family member that exhibits essential housekeeping functions such as folding of nascent polypeptides and misfolded proteins. Hsp70-9 (HSPA9, mortalin, GRP75, mtHsp70) is a mitochondrial Hsp70 isoform that bears a 46-amino acid target signal responsible for localization to the mitochondrial lumen (
      • Vos M.J.
      • Hageman J.
      • Carra S.
      • Kampinga H.H.
      Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families.
      ,
      • Zhu X.
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      • Gragerov A.
      • Ogata C.M.
      • Gottesman M.E.
      • Hendrickson W.A.
      Structural analysis of substrate binding by the molecular chaperone DnaK.
      ). Global knockouts of constitutively expressed Hsp70 isoform HSPA5, HSPA8, or HSPA9 are lethal, highlighting the central role of these chaperones in cellular physiology (
      • Daugaard M.
      • Rohde M.
      • Jäättelä M.
      The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions.
      ,
      • Daugaard M.
      • Jäättelä M.
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      Hsp70-2 is required for tumor cell growth and survival.
      ).

      Structural features of Hsp70 proteins

      Structurally, Hsp70s consists of a 45-kDa N-terminal nucleotide-binding domain (NBD) and a 28-kDa substrate-binding domain (SBD) that are connected by a flexible linker (Figure 1, Figure 2) (
      • Alderson T.R.
      • Kim J.H.
      • Markley J.L.
      Dynamical structures of Hsp70 and Hsp70-Hsp40 complexes.
      ,
      • Liu T.
      • Singh R.
      • Rios Z.
      • Bhushan A.
      • Li M.
      • Sheridan P.P.
      • Bearden S.E.
      • Lai J.C.K.
      • Agbenowu S.
      • Cao S.
      • Daniels C.K.
      Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.
      ). The NBD consists of four subdomains (IA, I, IIA, and IIB), which are required for the binding and hydrolysis of ATP to ADP. The SBD is further subdivided into a 15-kDa substrate-binding domain (SBDβ) and a 10-kDa helical lid domain (SBDα) acting as a flexible lid. The hydrolysis of ATP promotes NBD-conformational changes, which are transduced through the linker domain and trigger the clamping down of the lid domain onto unfolded protein clients, preventing substrate dissociation and providing an opportunity for the protein to obtain its native fold (
      • Mayer M.P.
      • Bukau B.
      Hsp70 chaperones: cellular functions and molecular mechanism.
      ). The release of ADP and subsequent binding of fresh ATP promotes the release of the folded substrate, setting Hsp70 up for the next round of folding.
      Figure thumbnail gr1
      Figure 1The post-translational modifications of mammalian Hsp70, Hsc70, and BiP and yeast Ssa1–4. Shown is a domain representation of the Hsp70 family members, with detected PTMs marked with appropriate residue numbers. PTMs are labeled as follows: phosphorylation in red, ubiquitination in green, acetylation in yellow, methylation in cyan, ADP-ribosylation in purple, and AMPylation in dark blue.
      Figure thumbnail gr2
      Figure 2Locations of PTMs on the Hsp70 structure. PTMs were mapped onto predicted structural models created by SWISS-MODEL based on Protein Data Bank entry 2KHO for each Hsp70 isoform (
      • Bertelsen E.B.
      • Chang L.
      • Gestwicki J.E.
      • Zuiderweg E.R.
      Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.
      ). PTMs are colored as in , except sites of multiple modification are labeled brown.

      Regulation of Hsp70 function

      The activity of Hsp70 family chaperones is tightly regulated. A first layer of regulation is provided by the rapid expression of stress-inducible Hsp70 isoforms in the presence of protein-unfolding stress. The up-regulation of Hsp70-1 and Hsp70-2 in the cytoplasm and BiP in the ER are key events during the induction of cellular stress responses, such as the heat shock response or the unfolded protein response in the ER (UPRER). These compartment-specific Hsp70 enrichments provide cells with enhanced protein (re)folding capacities to prevent or resolve stress-induced damage. Historically, Hsp70 was thought to exist primarily in a monomeric state, but recent evidence suggests that Hsp70 can form monomers, dimers, trimers, and higher-order oligomers (
      • Takakuwa J.E.
      • Nitika
      • Knighton L.E.
      • Truman A.W.
      Oligomerization of Hsp70: current perspectives on regulation and function.
      ). Although the functions of these high-order forms have not been fully delineated, they are clearly required for at least a subset of chaperone functions (
      • Takakuwa J.E.
      • Nitika
      • Knighton L.E.
      • Truman A.W.
      Oligomerization of Hsp70: current perspectives on regulation and function.
      ,
      • Trcka F.
      • Durech M.
      • Vankova P.
      • Chmelik J.
      • Martinkova V.
      • Hausner J.
      • Kadek A.
      • Marcoux J.
      • Klumpler T.
      • Vojtesek B.
      • Muller P.
      • Man P.
      Human stress-inducible Hsp70 has a high propensity to form ATP-dependent antiparallel dimers that are differentially regulated by cochaperone binding.
      ,
      • Sarbeng E.B.
      • Liu Q.
      • Tian X.
      • Yang J.
      • Li H.
      • Wong J.L.
      • Zhou L.
      • Liu Q.
      A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.
      ). The Hsp70 folding cycle is accelerated and regulated by a suite of co-chaperone proteins, including Hsp40s and nucleotide exchange factors (NEFs). Hsp40s are a heterogenous family of co-chaperones bearing a conserved J-domain required for binding to Hsp70 (
      • Kelley W.L.
      Molecular chaperones: how J domains turn on Hsp70s.
      ). These co-chaperones can bind to unfolded proteins via their C terminus to prevent aggregation and transport them to Hsp70 for folding/refolding. Hsp40s also play a more direct role in regulating Hsp70 by directly stimulating the ATP hydrolysis activity of Hsp70, locking the client into the closed SBD (
      • Kelley W.L.
      Molecular chaperones: how J domains turn on Hsp70s.
      ,
      • Voisine C.
      • Craig E.A.
      • Zufall N.
      • von Ahsen O.
      • Pfanner N.
      • Voos W.
      The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70.
      ). Once a client protein is folded, NEFs facilitate ADP release and mediate the exchange of ADP with ATP, which is required for the opening of the lid and the release of the folded client from the SBD (
      • Alderson T.R.
      • Kim J.H.
      • Markley J.L.
      Dynamical structures of Hsp70 and Hsp70-Hsp40 complexes.
      ,
      • Takayama S.
      • Reed J.C.
      Molecular chaperone targeting and regulation by BAG family proteins.
      ,
      • Höhfeld J.
      • Minami Y.
      • Hartl F.U.
      Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle.
      ,
      • Bascos N.A.D.
      • Mayer M.P.
      • Bukau B.
      • Landry S.J.
      The Hsp40 J-domain modulates Hsp70 conformation and ATPase activity with a semi-elliptical spring.
      ,
      • Malinverni D.
      • Jost Lopez A.
      • De Los Rios P.
      • Hummer G.
      • Barducci A.
      Modeling Hsp70/Hsp40 interaction by multi-scale molecular simulations and coevolutionary sequence analysis.
      ,
      • Kityk R.
      • Kopp J.
      • Mayer M.P.
      Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones.
      ). Another class of co-chaperones that regulate Hsp70 activity includes the tetratricopeptide repeat proteins Hip, Hop, and CHIP. Hip prevents the dissociation of ADP from Hsp70 binding to its NBD (
      • Höhfeld J.
      • Jentsch S.
      GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1.
      ,
      • Nelson G.M.
      • Prapapanich V.
      • Carrigan P.E.
      • Roberts P.J.
      • Riggs D.L.
      • Smith D.F.
      The heat shock protein 70 cochaperone hip enhances functional maturation of glucocorticoid receptor.
      ). Hop coordinates with both Hsp70 and Hsp90 and targets Hsp90 to the Hsp70-client protein complex (
      • Chen S.
      • Smith D.F.
      Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery.
      ,
      • Alvira S.
      • Cuéllar J.
      • Röhl A.
      • Yamamoto S.
      • Itoh H.
      • Alfonso C.
      • Rivas G.
      • Buchner J.
      • Valpuesta J.M.
      Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop.
      ,
      • Carrigan P.E.
      • Sikkink L.A.
      • Smith D.F.
      • Ramirez-Alvarado M.
      Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure.
      ). CHIP acts as an E3 ubiquitin ligase, ubiquitinating Hsp70 substrates, which results in their degradation by the proteasome (
      • Meacham G.C.
      • Lu Z.
      • King S.
      • Sorscher E.
      • Tousson A.
      • Cyr D.M.
      The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis.
      ). The number of highly related co-chaperones (e.g. 13 NEFs and 41 J-domain–containing proteins in humans) substantially exceeds the number of Hsp70 isoforms, consistent with the idea that they are critically involved in regulating the functional diversity of Hsp70s (
      • Kampinga H.H.
      • Craig E.A.
      The HSP70 chaperone machinery: J proteins as drivers of functional specificity.
      ).

      History of the Hsp70 chaperone code

      Early studies that identified modification of Hsp70 were generally descriptive in nature. PTMs, such as phosphorylation, were detected on chaperones, yet the sites of modification, their regulation, and their function remained elusive (
      • Hernando R.
      • Manso R.
      Muscle fibre stress in response to exercise: synthesis, accumulation and isoform transitions of 70-kDa heat-shock proteins.
      ,
      • Melling C.W.
      • Thorp D.B.
      • Milne K.J.
      • Noble E.G.
      Myocardial Hsp70 phosphorylation and PKC-mediated cardioprotection following exercise.
      ,
      • González B.
      • Manso R.
      Induction, modification and accumulation of HSP70s in the rat liver after acute exercise: early and late responses.
      ). A decrease in cost and increase in the resolution of proteomic experiments led to an abundance of PTMs being discovered on Hsp70s. In 2012, bioinformatic attempts to characterize regions of functionally important PTMs on proteins (“hotspots”) identified two such hotspots on Hsp70 (
      • Beltrao P.
      • Albanèse V.
      • Kenner L.R.
      • Swaney D.L.
      • Burlingame A.
      • Villén J.
      • Lim W.A.
      • Fraser J.S.
      • Frydman J.
      • Krogan N.J.
      Systematic functional prioritization of protein posttranslational modifications.
      ). The first region was located in the NBD (Thr-36 and Thr-38), whereas the second is present on the SBD (Thr-492, Ser-495, and Thr-499). Mutation of these sites led to viable cells that exhibited compromised chaperone function, including an inability to refold denatured proteins and increased global protein aggregation (
      • Beltrao P.
      • Albanèse V.
      • Kenner L.R.
      • Swaney D.L.
      • Burlingame A.
      • Villén J.
      • Lim W.A.
      • Fraser J.S.
      • Frydman J.
      • Krogan N.J.
      Systematic functional prioritization of protein posttranslational modifications.
      ). The first mechanistic study of an Hsp70 PTM was published later in 2012, when it was demonstrated that a conserved site on Hsp70 was phosphorylated by cell cycle kinases, leading to altered cyclin stability (
      • Cvoro A.
      • Dundjerski J.
      • Trajković D.
      • Matić G.
      The level and phosphorylation of Hsp70 in the rat liver cytosol after adrenalectomy and hyperthermia.
      ). Follow-up studies demonstrating that both C-terminal phosphorylation and methylation of Hsp70 can impact chaperone function led to the proposal of the “chaperone code” (
      • Cloutier P.
      • Coulombe B.
      Regulation of molecular chaperones through post-translational modifications: decrypting the chaperone code.
      ,
      • Truman A.W.
      • Kristjansdottir K.
      • Wolfgeher D.
      • Hasin N.
      • Polier S.
      • Zhang H.
      • Perrett S.
      • Prodromou C.
      • Jones G.W.
      • Kron S.J.
      CDK-dependent Hsp70 phosphorylation controls G1 cyclin abundance and cell-cycle progression.
      ,
      • Muller P.
      • Ruckova E.
      • Halada P.
      • Coates P.J.
      • Hrstka R.
      • Lane D.P.
      • Vojtesek B.
      C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.
      ). In this model, multiple cellular signals converge on Hsp70 (and indeed other chaperones such as Hsp90), leading to the fine-tuning of chaperone interactions and altered flow of information through cellular pathways.
      More recently, bioinformatics-based approaches, molecular weight shift analysis, isoelectric properties of protein, isotope labeling, and high-resolution MS have been used to uncover many more PTMs on the Hsp70 family of proteins, including acetylation, methylation, ubiquitination, SUMOylation, AMPylation, and ADP-ribosylation. Thankfully, several online resources facilitate tracking and interpreting the potential impact of Hsp70 PTMs. PhosphositePlus® (RRID:SCR_001837) is a comprehensive and curated online resource that collects information regarding the target proteins/residues and functional implications of abundant PTMs, such as phosphorylation, acetylation, methylation, ubiquitination, and O-glycosylation (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). The Global Proteome Machine Database (RRID:SCR_006617) is another excellent resource and is updated on a daily basis (
      • Craig R.
      • Cortens J.P.
      • Beavis R.C.
      Open source system for analyzing, validating, and storing protein identification data.
      ). Analysis of the available data from sites such as these reveals an astonishing number of PTMs on Hsp70s (Fig. 1). Below, we summarize the current state of knowledge of Hsp70 PTMs, including phosphorylation, acetylation, ubiquitination, AMPylation, ADP-ribosylation, and methylation, and their conservation and potential interplay.

      Hsp70 phosphorylation

      Over 100,000 phosphorylation events occur in mammalian cells, and improper protein phosphorylation is the cause of many human pathologies (
      • Pawson T.
      • Scott J.D.
      Protein phosphorylation in signaling—50 years and counting.
      ). Hsp70 family proteins are highly phosphorylated; a total of 88, 87, and 70 phosphorylated sites have been identified so far on Hsc70, BiP, and yeast Ssa1, respectively (Figure 1, Figure 2). Nevertheless, a mechanistic understanding of how individual phosphorylation events impact distinct Hsp70 functions remains elusive, with exceptions discussed below.

      Hsp70 phosphorylation in cell cycle progression and cell proliferation

      Cells must tightly coordinate growth and division in response to a variety of internal and external cues that include nutrient availability and genome integrity. These signals are sensed and propagated throughout cells via complex interlinked signal transduction pathways. Early studies on the cell cycle had demonstrated a role for the yeast co-chaperone Ydj1 in regulating the entry of the Cln3 G1 cyclin into the nucleus (
      • Verghese J.
      • Abrams J.
      • Wang Y.
      • Morano K.A.
      Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system.
      ). Cln3 possesses a J-domain–like region and competes with Ydj1 for binding to Ssa1. In 2012, Truman et al. (
      • Cvoro A.
      • Dundjerski J.
      • Trajković D.
      • Matić G.
      The level and phosphorylation of Hsp70 in the rat liver cytosol after adrenalectomy and hyperthermia.
      ) established that the molecular trigger for displacement of Ydj1 and recruitment of Cln3 was phosphorylation of Ssa1 on Thr-36 mediated by two related CDKs (Pho85 and Cdk1). These two kinases activate Thr-36 under different cellular conditions. During periods of nutrient scarcity, Pho85 phosphorylates Ssa1, promoting Cln3 recruitment to Ssa1 and its eventual destruction, leading to G1 cell cycle arrest. In cycling cells, transient Thr-36 phosphorylation of Ssa1 by Cdk1 promotes Cln3 destruction at the end of G2/M, resetting G1 cyclin levels in preparation for the beginning of the next cell cycle. Suggesting an evolutionarily conserved mechanism, phosphorylation of the equivalent Thr-38 residue on mammalian Hsc70 promotes Hsc70-cyclin D1 interaction and cyclin D1 destruction.
      Although clearly important for G1/S progression, Hsp70 phosphorylation can also impact later stages of the cell cycle. Polo-like kinase 1 (Plk1) is another serine/threonine kinase that regulates mitotic entry to cytokinesis and cell cycle progression (
      • Eckerdt F.
      • Strebhardt K.
      Polo-like kinase 1: target and regulator of anaphase-promoting complex/cyclosome-dependent proteolysis.
      ). In arsenic trioxide–treated mitotically arrested HeLa S3 cells, Plk1 phosphorylates Hsp70 at five sites: Thr-13, Thr-226, Ser-326, Ser-631, and Ser-633. The phosphomimetic mutants of Ser-631 and Ser-633 increase the proportion of cells arrested at mitosis. Thus, Plk1-mediated phosphorylation of Hsp70 plays a protective role against cell death by apoptosis. However, the significance of the other three phosphorylation sites by Plk1 remains obscure, and mechanisms by which Hsp70 provides protection remain to be explored. Hsp70 phosphorylated at Ser-631 and Ser-633 co-localizes with Plk1 at the centrosome. This association leads to an increase in microtubule stability, elongation of mitotic spindles, and mitotic arrest. Furthermore, when the interaction of Hsp70 and Plk1 is inhibited by 2-phenylethynesulfonamide or by inhibiting Plk1 activity by using BI2536, the apoptotic inhibition is released and leads to cell death. Hence, the phosphorylation of Hsp70 at Ser-631 and Ser-633 promotes Hsp70's role as a centrosomal chaperone (
      • Chen Y.J.
      • Lai K.C.
      • Kuo H.H.
      • Chow L.P.
      • Yih L.H.
      • Lee T.C.
      HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.
      ). Although the mechanism of this apoptotic release still remains unclear, the inhibition of Hsp70 phosphorylation or Plk1 inhibition might serve as a key to increase efficiency of arsenic trioxide as a chemotherapeutic drug. In addition to Plk1, the mitotic Nek6 kinase phosphorylates Hsp72 in the NBD at Thr-66, promoting recruitment of Hsp72 to the mitotic spindles (
      • O'Regan L.
      • Sampson J.
      • Richards M.W.
      • Knebel A.
      • Roth D.
      • Hood F.E.
      • Straube A.
      • Royle S.J.
      • Bayliss R.
      • Fry A.M.
      Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression.
      ). This phosphorylation event activates the alignment of chromosomes to the spindle equator by stabilizing kinetochore fibers. This is achieved via recruitment of two proteins, ch-TOG and TACC3, to kinetochore fibers. Phosphoinhibitory mutants of Hsp72 fail to localize at spindle poles, resulting in the destabilization of kinetochore fibers, leading to abnormal mitotic progression (
      • O'Regan L.
      • Sampson J.
      • Richards M.W.
      • Knebel A.
      • Roth D.
      • Hood F.E.
      • Straube A.
      • Royle S.J.
      • Bayliss R.
      • Fry A.M.
      Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression.
      ).
      Whereas it has been known for several years that chaperones can bind and regulate MAP kinase function (
      • Gong X.
      • Luo T.
      • Deng P.
      • Liu Z.
      • Xiu J.
      • Shi H.
      • Jiang Y.
      Stress-induced interaction between p38 MAPK and HSP70.
      ), a recent study suggests that mitogen-activated protein kinase pathway activity can in turn regulate chaperone function (
      • Lim S.
      • Kim D.G.
      • Kim S.
      ERK-dependent phosphorylation of the linker and substrate-binding domain of HSP70 increases folding activity and cell proliferation.
      ). Treatment of cells with epidermal growth factor triggers Hsp70 phosphorylation on Ser-385 and Ser-400. These sites are particularly interesting as they reside on and adjacent to the flexible linker that connects the NBD and SBD of Hsp70. Phosphorylation of Hsp70 at these two sites leads to an extended conformation of the protein, which in turn increases the binding affinity of the clients (
      • Lim S.
      • Kim D.G.
      • Kim S.
      ERK-dependent phosphorylation of the linker and substrate-binding domain of HSP70 increases folding activity and cell proliferation.
      ). Mutation of these two sites results in cells that have a reduced viability and growth rate. Although clearly dependent on the activity of extracellular signal–regulated kinase (ERK), these two phosphorylation sites do not fulfill the minimum requirements for ERK phosphorylation (proline in the +1 position to the site of phosphorylation) and thus are highly unlikely to be direct ERK substrates (
      • Songyang Z.
      • Lu K.P.
      • Kwon Y.T.
      • Tsai L.H.
      • Filhol O.
      • Cochet C.
      • Brickey D.A.
      • Soderling T.R.
      • Bartleson C.
      • Graves D.J.
      • DeMaggio A.J.
      • Hoekstra M.F.
      • Blenis J.
      • Hunter T.
      • Cantley L.C.
      A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.
      ,
      • Carlson S.M.
      • Chouinard C.R.
      • Labadorf A.
      • Lam C.J.
      • Schmelzle K.
      • Fraenkel E.
      • White F.M.
      Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3.
      ,
      • Sheridan D.L.
      • Kong Y.
      • Parker S.A.
      • Dalby K.N.
      • Turk B.E.
      Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs.
      ). Interestingly, Ser-400 lies next to a putative NLS on the Hsp70 sequence, and the S400A mutation prevents its nuclear export (
      • Miyata Y.
      • Rauch J.N.
      • Jinwal U.K.
      • Thompson A.D.
      • Srinivasan S.
      • Dickey C.A.
      • Gestwicki J.E.
      Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.
      ). Going forward, it will be interesting to identify the specific kinase for these sites, the impact these sites have on co-chaperone binding, and the specific clients impacted that result in the increased cellular proliferation. Taken together, it is clear that several sites on Hsp70 and Hsc70 are regulated by multiple kinases throughout the cell cycle, in turn leading to the targeted stabilization/destabilization of key effectors of the cell cycle.

      Hsp70 phosphorylation in cancer

      Many of the driver mutations involved in cancer alter writer and eraser enzymes in signal transduction pathways (
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      ). It is a likely conclusion that chaperone PTMs will be altered to varying degrees in different cancers. These changes may impact the stability of oncoproteins, activity of key pathways required for tumorigenesis, and anticancer drug resistance. A known example of this is alteration of methotrexate resistance in leukemia cells upon Hsc70 tyrosine phosphorylation. The interaction between Hsc70 and reduced folate carrier protein (RFC), the primary transporter of folate and antifolate drugs, regulates cellular methotrexate uptake (
      • Matherly L.H.
      • Hou Z.
      • Deng Y.
      Human reduced folate carrier: translation of basic biology to cancer etiology and therapy.
      ). Tyr-288 phosphorylation is required for the binding of Hsc70 to methotrexate. A phosphoinhibitory mutant of Tyr-288 disrupted the interaction between Hsc70-RFC and methotrexate, which affects its transport into the cells, rendering the cells resistant to this drug (
      • Liu T.
      • Singh R.
      • Rios Z.
      • Bhushan A.
      • Li M.
      • Sheridan P.P.
      • Bearden S.E.
      • Lai J.C.K.
      • Agbenowu S.
      • Cao S.
      • Daniels C.K.
      Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.
      ). Decreases in tyrosine phosphorylation of Hsc70 further lead to increased methotrexate resistance in these cells (
      • Liu T.
      • Dean A.
      • Ashwini S.
      • Sheridan P.P.
      • Bhushan A.
      • Lai J.C.
      • Cao S.
      • Daniels C.K.
      Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.
      ).
      Hsp70 phosphorylation exerts anti-apoptotic properties upon serum starvation in hepatocellular carcinoma. Retinoic acid–induced 16 (RAI16) is a protein kinase A–anchoring protein, which gets activated in response to serum starvation and drives protein kinase A–mediated phosphorylation of Ser-486 on Hsp70, thus preventing caspase-3 cleavage and apoptosis (
      • Ding C.L.
      • Xu G.
      • Tang H.L.
      • Zhu S.Y.
      • Zhao L.J.
      • Ren H.
      • Zhao P.
      • Qi Z.T.
      • Wang W.
      Anchoring of both PKA-RIIα and 14-3-3θ regulates retinoic acid induced 16 mediated phosphorylation of heat shock protein 70.
      ). Further studies are required to identify the key players that mediate these phosphorylation and dephosphorylation events so that the properties of PTM-based Hsp70 and Hsc70 regulation in cancer can be utilized as a novel chemotherapeutic option.

      Hsp70 phosphorylation as regulator of Hsp70 client triaging

      Although Hsp70 is required for the folding of both new synthesized and misfolded proteins, it is also able to target damaged proteins for degradation via the ubiquitin-mediated proteasomal system (
      • Fernández-Fernández M.R.
      • Gragera M.
      • Ochoa-Ibarrola L.
      • Quintana-Gallardo L.
      • Valpuesta J.M.
      Hsp70—a master regulator in protein degradation.
      ). The strategy that Hsp70 uses to decide whether to fold or degrade a client still remains unclear. The protein-folding and degradation activities of Hsp70 are mediated by its co-chaperones Hop and CHIP, respectively. The co-chaperone binding to C-terminal of Hsp70 is facilitated by the interaction between the tetratricopeptide repeat region of both Hop and CHIP with the C-terminal domain of Hsp70 (
      • Ballinger C.A.
      • Connell P.
      • Wu Y.
      • Hu Z.
      • Thompson L.J.
      • Yin L.Y.
      • Patterson C.
      Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.
      ). Phosphorylation of Hsp70 at Thr-636 leads to an increase in Hsp70-HOP binding and a corresponding decrease in Hsp70-CHIP binding, leading to increased client stability. Loss of this phosphorylation has the converse effect, triggering enhanced client degradation resulting from Hsp70-CHIP interaction (
      • Muller P.
      • Ruckova E.
      • Halada P.
      • Coates P.J.
      • Hrstka R.
      • Lane D.P.
      • Vojtesek B.
      C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.
      ). Similarly, another study demonstrated that the SBD of Hsp70 is phosphorylated by Akt1 at Ser-631, which decreases Hsp70-CHIP interaction (
      • Zemanovic S.
      • Ivanov M.V.
      • Ivanova L.V.
      • Bhatnagar A.
      • Michalkiewicz T.
      • Teng R.J.
      • Kumar S.
      • Rathore R.
      • Pritchard K.A.
      • Konduri G.G.
      • Afolayan A.J.
      Dynamic phosphorylation of the C terminus of Hsp70 regulates the mitochondrial import of SOD2 and redox balance.
      ). In this report, the downstream effect observed was to prevent degradation of superoxide dismutase-2 (SOD2) and promote the import of SOD2 into the mitochondria. In this way, Hsp70 regulates SOD2 activity and is a dynamic regulator of mitochondrial redox homeostasis.

      Hsp70 phosphorylation in host-pathogen interactions

      Hsp70 folds a large proportion of cellular proteins and thus supports the activity of a wide range of signaling pathways. It is thus unsurprising that Hsp70 phosphorylation appears to be altered in both hosts and pathogens in response to infection. Legionella pneumophila secretes over 300 bacterial effector proteins into target host cells, several of which are eukaryotic-like Ser/Thr protein kinases (
      • Finsel I.
      • Hilbi H.
      Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones.
      ). One of these kinases, LegK4, phosphorylates the host cytosolic Hsp70 and Hsc70 at Thr-495 in the SBD. Phosphorylation of this well-conserved site reduces the ability of the HDJ1 co-chaperone to stimulate the ATPase activity of Hsc70. At a cellular level, the knock-on effect of this phosphorylation is a reduction in the unfolded protein response and protein synthesis. Overall, this study reveals a fascinating mechanism by which Legionella is able to directly manipulate chaperone function to increase its probability of survival in host cells.
      Hsp70 phosphorylation in pathogens themselves may also be important for infectivity. In the opportunistic pathogenic yeast Candida albicans, Ser-361, Tyr-370, and Thr-576 are constitutively phosphorylated. Upon transition to the pathogenic hyphal form, an additional eight sites (Thr-11, Thr-136, Thr-161, Thr-175, Ser-328, Thr-387, Thr-494, and Ser-578) become activated (
      • Weissman Z.
      • Pinsky M.
      • Wolfgeher D.J.
      • Kron S.J.
      • Truman A.W.
      • Kornitzer D.
      Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis.
      ). Mutation of these sites produces cells with growth defects, including loss of the ability to form hyphae.
      In a similar manner, the malarial parasite Plasmodium berghei displays increased phosphorylation of Hsp70 at the gametocyte stage. This phosphorylation occurs on Ser-106, Ser-585, Thr-587, and Ser-588 (
      • Alonso-Morales A.
      • González-López L.
      • Cázares-Raga F.E.
      • Cortés-Martínez L.
      • Torres-Monzón J.A.
      • Gallegos-Pérez J.L.
      • Rodríguez M.H.
      • James A.A.
      • Hernández-Hernández F.e L.
      Protein phosphorylation during Plasmodium berghei gametogenesis.
      ). Although more mechanistic studies are required, these phosphorylation sites may have a possible role in conferring protection to the parasite in its host. It is interesting to note that some of the activated sites seen in host-pathogen interactions are conserved in other organisms under differing conditions. The functional changes brought about by these phosphorylations may have evolved to be utilized in organism-specific manners.

      BiP phosphorylation

      Early studies identified BiP as being serine/threonine-phosphorylated and that this may represent an inactive, noncomplexed form of BiP (
      • Hendershot L.M.
      • Ting J.
      • Lee A.S.
      Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.
      ). Follow-up studies revealed that BiP phosphorylation is associated with a dimeric form and that its phosphorylation decreases upon cellular exposure to ER stressors such as tunicamycin (
      • Freiden P.J.
      • Gaut J.R.
      • Hendershot L.M.
      Interconversion of three differentially modified and assembled forms of BiP.
      ,
      • Satoh M.
      • Nakai A.
      • Sokawa Y.
      • Hirayoshi K.
      • Nagata K.
      Modulation of the phosphorylation of glucose-regulated protein, GRP78, by transformation and inhibition of glycosylation.
      ). Taken together, the current model suggests that phosphorylation may promote a dimeric, inactive form of BiP. Upon ER stress, BiP phosphorylation decreases, allowing dimer dissociation and formation of BiP-client complexes. Whereas many sites of phosphorylation have been detected on BiP (Figure 1, Figure 2, Figure 3), their individual functions remain unclear.
      Figure thumbnail gr3
      Figure 3Conservation of Hsp70 PTMs and surrounding sequence between Hsp70 isoforms. Alignment was build using human Hsp70, Hsc70, and BiP as well as S. cerevisiae Ssa1, Ssa2, Ssa3, and Ssa4 sequences. Sequences were aligned using ClustalX (
      • Larkin M.A.
      • Blackshields G.
      • Brown N.P.
      • Chenna R.
      • McGettigan P.A.
      • McWilliam H.
      • Valentin F.
      • Wallace I.M.
      • Wilm A.
      • Lopez R.
      • Thompson J.D.
      • Gibson T.J.
      • Higgins D.G.
      Clustal W and Clustal X version 2.0.
      ), and sites of PTM were labeled as in .

      Hsp70 AMPylation (adenylylation, adenylation)

      AMPylation (also known as adenylylation and adenylation) involves the covalent addition of AMP to serine, tyrosine, or threonine residues (
      • Woolery A.R.
      • Luong P.
      • Broberg C.A.
      • Orth K.
      AMPylation: something old is new again.
      ). Enzymes responsible for AMPylation (AMPylases) have been identified in all three domains of life (
      • Khater S.
      • Mohanty D.
      In silico identification of AMPylating enzymes and study of their divergent evolution.
      ). Characterized AMPylases belong to four groups: Fic (filamentation induced by cAMP) domain-containing proteins (
      • Yarbrough M.L.
      • Li Y.
      • Kinch L.N.
      • Grishin N.V.
      • Ball H.L.
      • Orth K.
      AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling.
      ), GS-ATase (glutamine synthetase adenylyltransferase), SelO (
      • Sreelatha A.
      • Yee S.S.
      • Lopez V.A.
      • Park B.C.
      • Kinch L.N.
      • Pilch S.
      • Servage K.A.
      • Zhang J.
      • Jiou J.
      • Karasiewicz-Urbańska M.
      • Łobocka M.
      • Grishin N.V.
      • Orth K.
      • Kucharczyk R.
      • Pawłowski K.
      • et al.
      Protein AMPylation by an evolutionarily conserved pseudokinase.
      ), and DrrA (Legionella pneumophila effector protein) (
      • Kingdon H.S.
      • Shapiro B.M.
      • Stadtman E.R.
      Regulation of glutamine synthetase. 8. ATP: glutamine synthetase adenylyltransferase, an enzyme that catalyzes alterations in the regulatory properties of glutamine synthetase.
      ). Fic-domain–containing proteins are found in Archaea, Bacteria, and Eukarya, whereas SelO is found in Bacteria and Eukarya. GS-ATase is only present in Bacteria, and DrrA is restricted to strains of Legionella pneumophila (
      • Khater S.
      • Mohanty D.
      In silico identification of AMPylating enzymes and study of their divergent evolution.
      ,
      • Dudkiewicz M.
      • Szczepińska T.
      • Grynberg M.
      • Pawłowski K.
      A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.
      ). Most metazoans express a single fic type AMPylase, all of which share a highly conserved catalytic site architecture characterized by the fic motif HXFX(D/E)GNGRXXR (
      • Truttmann M.C.
      • Ploegh H.L.
      rAMPing up stress signaling: protein AMPylation in metazoans.
      ,
      • Engel P.
      • Goepfert A.
      • Stanger F.V.
      • Harms A.
      • Schmidt A.
      • Schirmer T.
      • Dehio C.
      Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins.
      ). Metazoan AMPylases and their functions, particularly their roles in the regulation of Hsp70 family chaperones, have gathered increasing attention in recent years. AMPylation is considered to inhibit the activity of Hsp70 family chaperones, conserving a pool of chaperones in a “stand-by,” or “primed,” state, which can rapidly respond to stress when de-AMPylated (
      • Casey A.K.
      • Moehlman A.T.
      • Zhang J.
      • Servage K.A.
      • Krämer H.
      • Orth K.
      Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.
      ,
      • Preissler S.
      • Rato C.
      • Perera L.A.
      • Saudek V.
      • Ron D.
      FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.
      ). The AMPylase FICD functions as a bidirectional enzyme, catalyzing both AMPylation and de-AMPylation involving a single active site (
      • Preissler S.
      • Rato C.
      • Perera L.A.
      • Saudek V.
      • Ron D.
      FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.
      ,
      • Sanyal A.
      • Ben E.A.Z.
      • Watson G.
      • Christoffer C.
      • Ma J.
      • Kihara D.
      • Mattoo S.
      Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.
      ). This bidirectionality is regulated by the oligomeric state of FICD, with monomeric FICD acting as an AMPylator and the dimeric form de-AMPylating (
      • Casey A.K.
      • Moehlman A.T.
      • Zhang J.
      • Servage K.A.
      • Krämer H.
      • Orth K.
      Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.
      ,
      • Perera L.A.
      • R C.
      • Yan Y.
      • Neidhardt L.
      • McLaughlin S.H.
      • Read R.J.
      • Preissler S.
      • Ron D.
      An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP.
      ).
      AMPylation of Hsp70s has been investigated in Homo sapiens (humans), Cricetulus griseus (Chinese hamsters), Drosophila melanogaster (fruit flies), and Caenorhabditis elegans (worms). In each of these species, at least one member of the Hsp70 family is regulated by AMPylation. In Chinese hamster ovary (CHO-K1) cells, FICD AMPylates the ER-resident Hsp70 BiP on Thr-518 (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). AMPylation levels increase and decrease inversely to unfolded protein burden (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). When AMPylated, BiP's substrate “on” and “off” rate is elevated, basal ATPase activity is decreased, and J-protein co-chaperone–stimulated ATP hydrolysis activity is attenuated, demonstrating that AMPylation is an inactivating modification in CHO-K1 cells (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). However, BiP AMPylation does not prevent the binding of ATP to BiP's active site nor the recruitment of J-type co-chaperones. AMPylated BiP is thus proposed to be trapped in an ATP- and J-domain co-chaperone–bound conformation that will engage in protein-refolding activities immediately following its de-AMPylation (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). Even when BiP is AMPylated in its ADP-bound state, it adopts an ATP-like state, with impaired oligomerization (
      • Preissler S.
      • Rohland L.
      • Yan Y.
      • Chen R.
      • Read R.J.
      • Ron D.
      AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.
      ). Increasing BiP AMPylation by the expression of FICD (E234G), the constitutively AMPylating mutant of FICD, induces the UPRER in CHO-K1 cells, whereas FICD-inactivated cells show a delayed UPRER in rat pancreatic acinar AR42j cells, suggesting a regulatory role for BiP AMPylation in UPRER induction (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). WT FICD, but not the E234G or the non-AMPylating H363A FICD mutants, is able to reverse AMPylation in vitro. In fact, when WT FICD is co-expressed with FICD (E234G) in CHO-K1 cells, the AMPylation levels and UPR effects of FICD (E234G) are mitigated (
      • Preissler S.
      • Rato C.
      • Perera L.A.
      • Saudek V.
      • Ron D.
      FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.
      ,
      • Sanyal A.
      • Ben E.A.Z.
      • Watson G.
      • Christoffer C.
      • Ma J.
      • Kihara D.
      • Mattoo S.
      Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.
      ).
      The Drosophila ortholog of BiP is AMPylated and deAMPylated by dFic at Thr-366 and Thr-518 (
      • Casey A.K.
      • Moehlman A.T.
      • Zhang J.
      • Servage K.A.
      • Krämer H.
      • Orth K.
      Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.
      ,
      • Ham H.
      • Woolery A.R.
      • Tracy C.
      • Stenesen D.
      • Krämer H.
      • Orth K.
      Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.
      ). AMPylation occurs in a Ca2+-dependent manner, and preferentially on the inactive conformation of BiP (
      • Ham H.
      • Woolery A.R.
      • Tracy C.
      • Stenesen D.
      • Krämer H.
      • Orth K.
      Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.
      ). During homeostasis, BiP is highly AMPylated, whereas AMPylated BiP levels decrease when unfolded proteins accumulate in the ER, suggesting that AMPylation also plays an inactivating role in Drosophila. Further, transcription of both BiP and dFic is increased in response to ER stress (
      • Ham H.
      • Woolery A.R.
      • Tracy C.
      • Stenesen D.
      • Krämer H.
      • Orth K.
      Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.
      ). dFic can also be secreted and locate to the surface of capitate projections (glial projections that insert into the axons of photoreceptors in the eye of flies), and loss of dFic leads to blindness in flies, due to loss of postsynaptic neuron activation (
      • Ham H.
      • Woolery A.R.
      • Tracy C.
      • Stenesen D.
      • Krämer H.
      • Orth K.
      Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.
      ). BiP(T366A) is resistant to AMPylation, and BiP(T366A) flies also exhibit loss of synaptic function, indicating that the effects of dFic loss are caused by deregulation of BiP (
      • Rahman M.
      • Ham H.
      • Liu X.
      • Sugiura Y.
      • Orth K.
      • Krämer H.
      Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections.
      ). Expression of a constitutively active dFic mutant (FicE247G) in a dFic knockout background is lethal but tolerated in the presence of WT dFic or BiP(T366A), further demonstrating the importance of residue Thr-366 in AMPylation-mediated BiP regulation in Drosophila (
      • Ham H.
      • Woolery A.R.
      • Tracy C.
      • Stenesen D.
      • Krämer H.
      • Orth K.
      Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.
      ,
      • Moehlman A.T.
      • Casey A.K.
      • Servage K.
      • Orth K.
      • Krämer H.
      Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.
      ).
      In C. elegans, the BiP/GRP78 orthologs HSP3 and HSP4 and the cytosolic Hsc70 ortholog HSP1 are AMPylated (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ,
      • Truttmann M.C.
      • Cruz V.E.
      • Guo X.
      • Engert C.
      • Schwartz T.U.
      • Ploegh H.L.
      The Caenorhabditis elegans protein FIC-1 is an AMPylase that covalently modifies heat-shock 70 family proteins, translation elongation factors and histones.
      ). Illustrating the role of AMPylation in regulation of Hsp70s, in strains expressing aggregating amyloid β or α-synuclein, hyper-AMPylation increases the formation of large protein aggregates while decreasing their cytotoxicity (
      • Evans C.G.
      • Wisén S.
      • Gestwicki J.E.
      Heat shock proteins 70 and 90 inhibit early stages of amyloid β-(1-42) aggregation in vitro.
      ,
      • Fonte V.
      • Kapulkin W.J.
      • Kapulkin V.
      • Taft A.
      • Fluet A.
      • Friedman D.
      • Link C.D.
      Interaction of intracellular β amyloid peptide with chaperone proteins.
      ,
      • Truttmann M.C.
      • Pincus D.
      • Ploegh H.L.
      Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.
      ,
      • Klucken J.
      • Shin Y.
      • Masliah E.
      • Hyman B.T.
      • McLean P.J.
      Hsp70 reduces α-synuclein aggregation and toxicity.
      ). RNAi-mediated knockdown of Hsp70 family chaperones HSP1 as well as HSP3 and HSP4 phenocopies this reduction in aggregate toxicity observed in strains with increased AMPylation levels, suggesting that the effects of hyper-AMPylation are caused by AMPylation-mediated inactivation of Hsp70s in these worms (
      • Truttmann M.C.
      • Pincus D.
      • Ploegh H.L.
      Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.
      ). The site of AMPylation on HSP3 is Thr-176, but the AMPylation site(s) of HSP-1 and HSP-4 remain to be mapped (
      • Truttmann M.C.
      • Pincus D.
      • Ploegh H.L.
      Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.
      ). Expression of the constitutively active C. elegans AMPylase Fic-1(E274G) in S. cerevisiae induces up-regulation of the heat shock response and HSPs expression, as well as growth arrest and increased protein aggregation (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ). These phenotypes are partially rescued by increasing the expression of the cytosolic Hsp70 family member Ssa2, suggesting that Fic-1(E274G) targets Hsp70 family proteins in budding yeast as well (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ).
      Human BiP is AMPylated at Thr-366 and Thr-518 (
      • Sanyal A.
      • Ben E.A.Z.
      • Watson G.
      • Christoffer C.
      • Ma J.
      • Kihara D.
      • Mattoo S.
      Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.
      ,
      • Sanyal A.
      • Chen A.J.
      • Nakayasu E.S.
      • Lazar C.S.
      • Zbornik E.A.
      • Worby C.A.
      • Koller A.
      • Mattoo S.
      A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.
      ). These sites play slightly different roles in regulation of the Hsp70 ATPase cycle. Whereas AMPylation of Thr-366 increases basal ATPase activity with no effect on J-protein–stimulated ATPase activity, AMPylation of Thr-518 decreases J-protein–stimulated ATPase activity with no effect on basal ATPase activity (
      • Sanyal A.
      • Ben E.A.Z.
      • Watson G.
      • Christoffer C.
      • Ma J.
      • Kihara D.
      • Mattoo S.
      Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.
      ,
      • Sanyal A.
      • Chen A.J.
      • Nakayasu E.S.
      • Lazar C.S.
      • Zbornik E.A.
      • Worby C.A.
      • Koller A.
      • Mattoo S.
      A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.
      ). In addition to BiP, human FICD also AMPylates other major chaperones, including Hsp70, Hsp40, and Hsp90 (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ). In vitro, Hsp70 is AMPylated on five threonine residues in the ATPase domain (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ). In HEK293T cells, expression of FICD(E234G) induces higher expression of BiP, presumably to compensate for the BiP inactivated by AMPylation (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). Transfection of HeLa cells with the constitutively AMPylating FICD(E234G) leads to up-regulation of the heat shock response and activation of heat shock factor 1 (HSF1), a key transcription factor negatively regulated by Hsp70 that promotes the transcription of chaperones and thereby regulates the heat shock response (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ,
      • Krakowiak J.
      • Zheng X.
      • Patel N.
      • Feder Z.A.
      • Anandhakumar J.
      • Valerius K.
      • Gross D.S.
      • Khalil A.S.
      • Pincus D.
      Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response.
      ). FICD(E234G) also inhibits translocation of Hsp70 to the nucleus (
      • Truttmann M.C.
      • Zheng X.
      • Hanke L.
      • Damon J.R.
      • Grootveld M.
      • Krakowiak J.
      • Pincus D.
      • Ploegh H.L.
      Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.
      ).
      A recent screen for AMPylated proteins in eight different human cell lines using a novel N6pA probe further revealed that HSPA2, HSPA4, BiP, Hsc70, and mortalin are AMPylated by endogenous FICD in a cell type–specific pattern (
      • Kielkowski P.
      • B I.
      • Kirsch V.K.
      • Bach N.
      • Drukker M.
      • Cappello S.
      • Sieber S.A.
      FICD activity and AMPylation remodelling modulate human neurogenesis.
      ): whereas BiP was modified in all cell lines, HSPA2 (fibroblasts), HSPA4 (fibroblasts), Hsc70 (fibroblasts, SH-SY5Ys), and HSPA9 (fibroblasts, IPSCs) were AMPylated in a subset of the tested cell types. This supports the claim that AMPylation elicits regulatory functions on distinct Hsp70 family members.
      Taken as a whole, these studies show that AMPylation occurs on threonine and serine residues of several Hsp70 family proteins and is particularly important in regulating BiP function. Whether BiP is simultaneously AMPylated on multiple residues or Thr-365 and Thr-518 are uniquely modified in response to distinct cues remains to be defined. The majority of studies have shown that this modification inactivates or inhibits the activity of Hsp70 proteins, at least with regard to its chaperone activities. This suggests a model in which AMPylation acts as a “holding” mechanism, in which a steady level of Hsp70s can be maintained in the cell inactive during times of low stress, but ready to rapidly respond to stress events without the need to wait for transcription and translation. The homeostasis/stress AMPylation cycle has not yet been worked out for all Hsp70 family members. In the case of BiP, however, under basal nonstressed conditions, BiP appears to be largely AMPylated, and when stress is induced, BiP is de-AMPylated in response. AMPylation can have both advantageous and deleterious effects on cells, depending on context. Whereas constitutive AMPylation leads to activation of the UPR in cell models, no AMPylation leads to delayed UPR activation (
      • Preissler S.
      • Rato C.
      • Perera L.A.
      • Saudek V.
      • Ron D.
      FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.
      ,
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ). In flies, constitutive AMPylation is lethal, but lack of AMPylation leads to blindness (
      • Rahman M.
      • Ham H.
      • Liu X.
      • Sugiura Y.
      • Orth K.
      • Krämer H.
      Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections.
      ,
      • Moehlman A.T.
      • Casey A.K.
      • Servage K.
      • Orth K.
      • Krämer H.
      Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.
      ). In C. elegans protein aggregation models, increased AMPylation leads to increased protein aggregates but reduces the toxicity of those aggregates (
      • Truttmann M.C.
      • Pincus D.
      • Ploegh H.L.
      Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.
      ). This balance reflects the tight balance needed to maintain homeostasis and illustrates the crucial role of AMPylation.

      Hsp70 ADP-ribosylation

      Another post translational modification observed on Hsp70s is ADP-ribosylation (
      • Hendershot L.M.
      • Ting J.
      • Lee A.S.
      Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.
      ,
      • Lüscher B.
      • Bütepage M.
      • Eckei L.
      • Krieg S.
      • Verheugd P.
      • Shilton B.H.
      ADP-ribosylation, a multifaceted posttranslational modification involved in the control of cell physiology in health and disease.
      ,
      • Cohen M.S.
      • Chang P.
      Insights into the biogenesis, function, and regulation of ADP-ribosylation.
      ,
      • Carlsson L.
      • Lazarides E.
      ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation.
      ,
      • Ledford B.E.
      • Leno G.H.
      ADP-ribosylation of the molecular chaperone GRP78/BiP.
      ,
      • Ledford B.E.
      • Jacobs D.F.
      Translational control of ADP-ribosylation in eucaryotic cells.
      ,
      • Dani N.
      • Stilla A.
      • Marchegiani A.
      • Tamburro A.
      • Till S.
      • Ladurner A.G.
      • Corda D.
      • Di Girolamo M.
      Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome.
      ). ADP-ribosylation, a process catalyzed by ADP-ribosyltransferases (ARTs), involves the transfer of an ADP-ribose from NADH (NAD+) onto a target protein with a nucleophilic oxygen, nitrogen, or sulfur (
      • Lüscher B.
      • Bütepage M.
      • Eckei L.
      • Krieg S.
      • Verheugd P.
      • Shilton B.H.
      ADP-ribosylation, a multifaceted posttranslational modification involved in the control of cell physiology in health and disease.
      ,
      • Cohen M.S.
      • Chang P.
      Insights into the biogenesis, function, and regulation of ADP-ribosylation.
      ). ARTs fall into three families of proteins: arginine-specific ADP-ribosyltransferases (ART/ARTC), poly(ADP-ribose) polymerases (PARP/ARTD), and sirtuins. Proteins can be either mono- or poly-ADP-ribosylated (
      • Lüscher B.
      • Bütepage M.
      • Eckei L.
      • Krieg S.
      • Verheugd P.
      • Shilton B.H.
      ADP-ribosylation, a multifaceted posttranslational modification involved in the control of cell physiology in health and disease.
      ,
      • Cohen M.S.
      • Chang P.
      Insights into the biogenesis, function, and regulation of ADP-ribosylation.
      ). ADP-ribosylation can be removed by hydrolysis by ADP-ribosyl hydrolases or hydrolases of the macrodomain family (
      • Rack J.G.M.
      • Palazzo L.
      • Ahel I.
      (ADP-ribosyl)hydrolases: structure, function, and biology.
      ). In CHO and human HEK293T and HeLa cells, BiP is ADP-ribosylated by hamster ARTC2.1 and by human ART1, respectively.
      Early studies identified several conditions that induced BiP ADP-ribosylation, including suspension of protein translation and ER stress (
      • Ledford B.E.
      • Jacobs D.F.
      Translational control of ADP-ribosylation in eucaryotic cells.
      ,
      • Leno G.H.
      • Ledford B.E.
      Reversible ADP-ribosylation of the 78 kDa glucose-regulated protein.
      ,
      • Laitusis A.L.
      • Brostrom M.A.
      • Brostrom C.O.
      The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing.
      ,
      • Chambers J.E.
      • Petrova K.
      • Tomba G.
      • Vendruscolo M.
      • Ron D.
      ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load.
      ). In mice, BiP is less ADP-ribosylated in B cells making γ1-heavy chains than in those making neither heavy nor light chains (
      • Hendershot L.M.
      • Ting J.
      • Lee A.S.
      Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.
      ). In quiescent Swiss 3T3, Rat-1 cells, and mouse embryonic fibroblasts, BiP is ADP-ribosylated; however, when proliferation is induced, ADP-ribosylation is reduced (
      • Staddon J.M.
      • Bouzyk M.M.
      • Rozengurt E.
      Interconversion of GRP78/BiP: a novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.
      ). When mice are fasted overnight or administered cycloheximide to halt protein production, their pancreases have increased levels of ADP-ribosylated BiP, which is reversed by feeding (
      • Chambers J.E.
      • Petrova K.
      • Tomba G.
      • Vendruscolo M.
      • Ron D.
      ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load.
      ).
      The specific sites and exact function of ADP-ribosylation have not been well-validated. MS-based studies have identified ADP-ribosylation at residues Asp-78 and Lys-81 of BiP, as well as Asp-53 of Hsc70 in HeLa cells, and residue Arg-50 of BiP and Arg-346 of HSPA13 in murine skeletal muscle (
      • Leutert M.
      • Menzel S.
      • Braren R.
      • Rissiek B.
      • Hopp A.K.
      • Nowak K.
      • Bisceglie L.
      • Gehrig P.
      • Li H.
      • Zolkiewska A.
      • Koch-Nolte F.
      • Hottiger M.O.
      Proteomic characterization of the heart and skeletal muscle reveals widespread arginine ADP-ribosylation by the ARTC1 ectoenzyme.
      ). Additionally, mutation of either Arg-470 or Arg-492 to lysine in hamster BiP substantially decreases ADP-ribosylation, and whereas ribosylation-mimicking mutations decrease client binding, they do not appear to alter intrinsic ATPase activity. It should be noted that the authors did not definitively identify Arg-470 or Arg-492 as a site of ribosylation (by MS or other related technologies), and advances in the field suggest that these sites may also be modified by AMPylation (see below). On size-exclusion chromatography columns, ADP-ribosylated BiP is present in lower-molecular weight fractions, indicating that ADP-ribosylation prevents BiP participating in multichaperone complexes (
      • Chambers J.E.
      • Petrova K.
      • Tomba G.
      • Vendruscolo M.
      • Ron D.
      ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load.
      ).
      Additionally, ADP-ribosylation is only found on the oligomeric form of BiP, which is the predominant form under low protein-folding burden (
      • Freiden P.J.
      • Gaut J.R.
      • Hendershot L.M.
      Interconversion of three differentially modified and assembled forms of BiP.
      ). Combined, these studies indicate that higher levels of ADP-ribosylated BiP are found during low protein production/low unprocessed protein burden (
      • Hendershot L.M.
      • Ting J.
      • Lee A.S.
      Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.
      ,
      • Ledford B.E.
      • Jacobs D.F.
      Translational control of ADP-ribosylation in eucaryotic cells.
      ,
      • Leno G.H.
      • Ledford B.E.
      Reversible ADP-ribosylation of the 78 kDa glucose-regulated protein.
      ,
      • Laitusis A.L.
      • Brostrom M.A.
      • Brostrom C.O.
      The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing.
      ,
      • Chambers J.E.
      • Petrova K.
      • Tomba G.
      • Vendruscolo M.
      • Ron D.
      ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load.
      ). On the other hand, BiP ADP-ribosylation is decreased when the unprocessed protein burden is higher, as when more proteins are being produced and when protein glycosylation is hindered (
      • Hendershot L.M.
      • Ting J.
      • Lee A.S.
      Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.
      ,
      • Staddon J.M.
      • Bouzyk M.M.
      • Rozengurt E.
      Interconversion of GRP78/BiP: a novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.
      ,
      • Leno G.H.
      • Ledford B.E.
      ADP-ribosylation of the 78-kDa glucose-regulated protein during nutritional stress.
      ). Taken together, this suggests that ADP-ribosylation may play a role in regulation of BiP's chaperoning activities.
      Overall, studies on BiP ADP-ribosylation describe a modification that is most present under conditions in which BiP's folding activities are not needed and which may be inhibitory to BiP's chaperoning functions. Inversely, when unfolded protein burden is high, or when BiP is actively bound to a client protein, ADP-ribosylation levels are low. This suggests that ADP-ribosylation may act as a temporary “off” switch for BiP.
      Studies on ADP-ribosylation and AMPylation have been complicated by the fact that early studies on ADP-ribosylation used radiolabeled adenosine, which can also label AMPylated sites. Cleavage by an ADP-ribosyl hydrolase was not used to confirm ADP-ribosylation in the above studies. Chambers et al. (
      • Chambers J.E.
      • Petrova K.
      • Tomba G.
      • Vendruscolo M.
      • Ron D.
      ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load.
      ), investigating BiP arginine modification, point out that the identified mammalian arginine hydrolase, ARH1 (
      • Rack J.G.M.
      • Palazzo L.
      • Ahel I.
      (ADP-ribosyl)hydrolases: structure, function, and biology.
      ), is a cytoplasmic protein and unlikely to act on BiP. In some of these studies, ADP-ribosylation was confirmed by digestion with snake venom phosphodiesterase, which cleaves phosphodiester bonds (
      • Ouyang C.
      • Huang T.F.
      Inhibition of platelet aggregation by 5′-nucleotidase purified from Trimeresurus gramineus snake venom.
      ), and by blocking ADP-ribosylation using nicotinamide, which inhibits ARTs as well as PARPs (
      • Rankin P.W.
      • Jacobson E.L.
      • Benjamin R.C.
      • Moss J.
      • Jacobson M.K.
      Quantitative studies of inhibitors of ADP-ribosylation in vitro in vivo.
      ). Importantly, snake venom phosphodiesterase is also able to cleave the phosphodiester bond of an AMPylation modification. Furthermore, nicotinamide treatment has many effects on the cell other than blockage of ADP-ribosylation, including stimulation of DNA damage repair and blocking hexose uptake, which could in turn have effects on cellular metabolism leading to decreases in AMPylation, not ADP-ribosylation (
      • Grunfeld C.
      • Shigenaga J.K.
      Nicotinamide and other inhibitors of ADP-ribosylation block deoxyglucose uptake in cultured cells.
      ,
      • Banasik M.
      • Ueda K.
      Inhibitors and activators of ADP-ribosylation reactions.
      ). In fact, Preissler et al. (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ), the same group that published Chambers et al. (
      • Chambers J.E.
      • Petrova K.
      • Tomba G.
      • Vendruscolo M.
      • Ron D.
      ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load.
      ), themselves postulate that previous studies of BiP ADP-ribosylation may have been mistakenly characterizing BiP AMPylation (
      • Preissler S.
      • Rato C.
      • Chen R.
      • Antrobus R.
      • Ding S.
      • Fearnley I.M.
      • Ron D.
      AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
      ).

      Hsp70 acetylation

      Acetylation involves the transfer of an acetyl group onto a lysine residue, the source of which is the metabolite acetyl-CoA (
      • Narita T.
      • Weinert B.T.
      • Choudhary C.
      Functions and mechanisms of non-histone protein acetylation.
      ). Acetylation is catalyzed by three related families of lysine acetyltransferases, GCN5, p300, and MYST, whereas removal of acetyl groups is processed by Sirtuin NAD-dependent lysine deacetylases (
      • Narita T.
      • Weinert B.T.
      • Choudhary C.
      Functions and mechanisms of non-histone protein acetylation.
      ). The Hsp70 family of proteins are heavily modified by acetylation; a total of 50, 58, and 40 acetylated sites have been identified so far on Hsc70, BiP, and yeast Ssa1, respectively (Figure 1, Figure 2, Figure 3). As with phosphorylation, acetylation fine-tunes Hsp70 function in a wide range of cellular processes.

      Hsp70 acetylation control of the heat shock response

      The historical paradigm for the heat shock response is that during heat shock, Hsp70 levels are induced through increased expression to compensate for the increase in unfolded proteins (
      • Verghese J.
      • Abrams J.
      • Wang Y.
      • Morano K.A.
      Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system.
      ,
      • Zheng X.
      • Krakowiak J.
      • Patel N.
      • Beyzavi A.
      • Ezike J.
      • Khalil A.S.
      • Pincus D.
      Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation.
      ). A recent study in yeast has revealed that in addition, Ssa1 is rapidly deacetylated at four lysine residues, Lys-86, Lys-185, Lys-354, and Lys-562, in response to heat shock (
      • Xu L.
      • Nitika
      • Hasin N.
      • Cuskelly D.D.
      • Wolfgeher D.
      • Doyle S.
      • Moynagh P.
      • Perrett S.
      • Jones G.W.
      • Truman A.W.
      Rapid deacetylation of yeast Hsp70 mediates the cellular response to heat stress.
      ). These deacetylation events are required for interaction with key co-chaperones such as Ydj1, Zuo1, Sgt2, and Hsp26 during heat shock. Fascinatingly, the inducible Hsp70s (Ssa3 and Ssa4) have an alanine at position 562 and thus cannot undergo acetylation at this site. It is interesting to speculate that this naturally occurring mutation in Ssa3 and Ssa4 makes them immediately prepared for action when expressed during heat shock (for more on isoform differences in the code, please see below).

      Hsp70 acetylation in client triaging and cell survival

      In response to oxidative stress, the ARD1 acetyltransferase acetylates Hsp70 at Lys-77, allowing Hsp70 to bind to Hop and allowing refolding of denatured clients. After longer periods of stress, Hsp70 becomes deacetylated, promoting interaction with CHIP to degrade damaged proteins. This switch from protein refolding to degradation is required for the maintenance of protein homoeostasis and protects the cells from stress-induced cell death. Thus, in a similar manner to phosphorylation-mediated triaging reported by Muller et al. (
      • Muller P.
      • Ruckova E.
      • Halada P.
      • Coates P.J.
      • Hrstka R.
      • Lane D.P.
      • Vojtesek B.
      C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.
      ), ARD1-mediated Hsp70 acetylation is a regulatory mechanism that balances protein refolding/degradation in response to stress (
      • Seo J.H.
      • Park J.H.
      • Lee E.J.
      • Vo T.T.
      • Choi H.
      • Kim J.Y.
      • Jang J.K.
      • Wee H.J.
      • Lee H.S.
      • Jang S.H.
      • Park Z.Y.
      • Jeong J.
      • Lee K.J.
      • Seok S.H.
      • Park J.Y.
      • et al.
      ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation.
      ). In addition to altered co-chaperone binding, Hsp70 acetylation on Lys-77 facilitates its binding with pro-apoptotic proteins Apaf-1 and AIF and inhibits Apaf-1– and AIF–dependent apoptosis. Hsp70 acetylation also attenuates autophagy by Atg12-Atg5 complex formation, Beclin-1 expression, and perinuclear LC3 puncta formation, resulting in the inhibition of autophagic cell death. It is worth noting that only the inducible Hsp70 has Lys-77; the corresponding site in the constitutive Hsc70 is a nonacetylatable arginine, suggesting isoform-specific functionality.
      Aside from Lys-77, other Hsp70 acetylation sites can impact autophagy. Post-amino acid starvation, Hsp70 Lys-159 acetylation is up-regulated. Acetylated Hsp70 displays enhanced binding affinity to KAP1 (SUMO E3 ligase), which in turn increases the SUMOylation of autophagy protein Vps34. This newly formed Hsp70-KAP1-Vps34 complex binds to Beclin 1 (complexed to autophagy proteins ATG14L, Ambra1, Bif1, or UVRAG) and promotes phagophore formation. Subsequently, the ULK kinase complex (ULK1/2, ATG13, FIP200) proteins are recruited to the phagophore. Finally, the membrane encloses the cytosolic cargos, resulting in the formation of an autophagosome. Thus, under nutrient starvation, acetylation of Hsp70 is a key step for activating autophagy (
      • Yang Y.
      • Fiskus W.
      • Yong B.
      • Atadja P.
      • Takahashi Y.
      • Pandita T.K.
      • Wang H.G.
      • Bhalla K.N.
      Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy.
      ). Interestingly, in contrast to Hsp70 Lys-77 acetylation, Lys-126 acetylation weakens its binding to Hop and Hip yet strengthens the interaction between CHIP and Bag1. Lys-126 acetylation also inhibits Hsp70-mediated tumor cell invasion and migration and the binding of Hsp70 to AIF1 and Apaf1 for promoting mitochondria-mediated apoptosis (
      • Sun F.
      • Jiang X.
      • Wang X.
      • Bao Y.
      • Feng G.
      • Liu H.
      • Kou X.
      • Zhu Q.
      • Jiang L.
      • Yang Y.
      Vincristine ablation of Sirt2 induces cell apoptosis and mitophagy via Hsp70 acetylation in MDA-MB-231 cells.
      ). Going forward, it will be interesting to underpin how activation and function of Lys-77, Lys-126, and Lys-159 acetylation relate to one another.

      Hsp70 methylation

      Methylation represents a highly abundant PTM found on all Hsp70 isoforms (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ,
      • Guo A.
      • Gu H.
      • Zhou J.
      • Mulhern D.
      • Wang Y.
      • Lee K.A.
      • Yang V.
      • Aguiar M.
      • Kornhauser J.
      • Jia X.
      • Ren J.
      • Beausoleil S.A.
      • Silva J.C.
      • Vemulapalli V.
      • Bedford M.T.
      • et al.
      Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
      ,
      • Larsen S.C.
      • Sylvestersen K.B.
      • Mund A.
      • Lyon D.
      • Mullari M.
      • Madsen M.V.
      • Daniel J.A.
      • Jensen L.J.
      • Nielsen M.L.
      Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.
      ). This modification is conferred by three enzyme families: methyltransferase-like proteins (METTL) (
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ,
      • Cloutier P.
      • Lavallée-Adam M.
      • Faubert D.
      • Blanchette M.
      • Coulombe B.
      A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity.
      ), protein arginine methyltransferases (PRMTs) (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ,
      • Larsen S.C.
      • Sylvestersen K.B.
      • Mund A.
      • Lyon D.
      • Mullari M.
      • Madsen M.V.
      • Daniel J.A.
      • Jensen L.J.
      • Nielsen M.L.
      Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.
      ), and SET domain–containing histone methyltransferases (SETD) (
      • Cho H.S.
      • Shimazu T.
      • Toyokawa G.
      • Daigo Y.
      • Maehara Y.
      • Hayami S.
      • Ito A.
      • Masuda K.
      • Ikawa N.
      • Field H.I.
      • Tsuchiya E.
      • Ohnuma S.
      • Ponder B.A.
      • Yoshida M.
      • Nakamura Y.
      • et al.
      Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.
      ). Modifications occur on surface-exposed lysine residues, which can be mono- (me1), di- (me2), or trimethylated (me3) (
      • Biggar K.K.
      • Li S.S.
      Non-histone protein methylation as a regulator of cellular signalling and function.
      ), and arginine residues, which are monomethylated, asymmetrically dimethylated, or symmetrically dimethylated (
      • Biggar K.K.
      • Li S.S.
      Non-histone protein methylation as a regulator of cellular signalling and function.
      ,
      • Blanc R.S.
      • Richard S.
      Arginine methylation: the coming of age.
      ). Most sites of methylation were found in proteome-wide MS studies (
      • Guo A.
      • Gu H.
      • Zhou J.
      • Mulhern D.
      • Wang Y.
      • Lee K.A.
      • Yang V.
      • Aguiar M.
      • Kornhauser J.
      • Jia X.
      • Ren J.
      • Beausoleil S.A.
      • Silva J.C.
      • Vemulapalli V.
      • Bedford M.T.
      • et al.
      Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
      ,
      • Larsen S.C.
      • Sylvestersen K.B.
      • Mund A.
      • Lyon D.
      • Mullari M.
      • Madsen M.V.
      • Daniel J.A.
      • Jensen L.J.
      • Nielsen M.L.
      Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.
      ,
      • Cloutier P.
      • Lavallée-Adam M.
      • Faubert D.
      • Blanchette M.
      • Coulombe B.
      A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity.
      ,
      • Shimazu T.
      • Barjau J.
      • Sohtome Y.
      • Sodeoka M.
      • Shinkai Y.
      Selenium-based S-adenosylmethionine analog reveals the mammalian seven-β-strand methyltransferase METTL10 to be an EF1A1 lysine methyltransferase.
      ). The functional consequences of Hsp70 methylation, with a few exceptions discussed below, remain poorly understood.

      Hsp70 methylation in the regulation of gene expression

      Histone and DNA methylation are well-known regulatory traits that control gene transcription. Recent advances in our understanding of histone methylases and demethylases highlight that some of these enzymes are also capable of modifying nonhistone substrates, including Hsp70 family chaperones. Upon methylation of the conserved Arg-469 residue, Hsp70s associate with chromatin and regulate retinoid acid–dependent retinoid acid receptor β2 (RARβ2) gene expression (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ). Whereas both the methylation-incompetent R469A mutant and WT Hsp70 bind to the promoter region of RARβ2, only methylated Hsp70 recruits TFIIH to the preinitiation complex during RARβ2 transcription initiation (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ). Arg-469 methylation is conferred by PRMT4, which monomethylates the conserved Arg-469 residue across Hsp70 isoforms (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ). This methylation is at least partly removed from Hsp70s by the JmjC-domain–containing demethylase JMJD6 (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ). Together, PRMT4 and JMJD6 constitute a classic “writer-eraser” pair that regulates transcriptional events through Hsp70, rather than histone methylation.

      Hsp70 methylation in direct regulation of chaperoning function

      The protein-folding and -refolding activities of Hsp70 chaperones are in part regulated by lysine methylation events. Hsc70 Lys-561 and the orthogonal residues in BiP, Hsp70, HSPA2/Hsp70-2, and HSPA6/Hsp70B′ are trimethylated by the nonhistone methylase METTL21A (
      • Guo A.
      • Gu H.
      • Zhou J.
      • Mulhern D.
      • Wang Y.
      • Lee K.A.
      • Yang V.
      • Aguiar M.
      • Kornhauser J.
      • Jia X.
      • Ren J.
      • Beausoleil S.A.
      • Silva J.C.
      • Vemulapalli V.
      • Bedford M.T.
      • et al.
      Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
      ,
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ,
      • Cloutier P.
      • Lavallée-Adam M.
      • Faubert D.
      • Blanchette M.
      • Coulombe B.
      A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity.
      ,
      • Jakobsson M.E.
      • Moen A.
      • Falnes P.
      Correspondence: on the enzymology and significance of HSPA1 lysine methylation.
      ). In the presence of ER stress, BiP K586me3 is degraded by the lysosome and replaced with methyl-free de novo translated BiP, implicating a role for K586me3 in the regulation of BiP's chaperoning function (
      • Sieber J.
      • Wieder N.
      • Ostrosky-Frid M.
      • Dvela-Levitt M.
      • Aygün O.
      • Udeshi N.D.
      • Carr S.A.
      • Greka A.
      Lysine trimethylation regulates 78-kDa glucose-regulated protein proteostasis during endoplasmic reticulum stress.
      ). Hsc70 Lys-561 trimethylation affects its ability to bind to substrates, such as α-synuclein (
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ). Conversely to BiP, methylation-mimetic Hsc70 K561R is more stable than a K561A nonmethylatable protein, suggesting that Hsc70 K561me3 regulates Hsc70 turnover and degradation (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ). There are several possible explanations for the apparent opposite effects of trimethylation on BiP and Hsc70. One is that they are separate proteins in separate subcellular compartments, and so this same modification may play different roles in specific contexts. Additionally, BiP K586me3 is degraded following ER stress, as are several other ER-resident chaperones (
      • Sieber J.
      • Wieder N.
      • Ostrosky-Frid M.
      • Dvela-Levitt M.
      • Aygün O.
      • Udeshi N.D.
      • Carr S.A.
      • Greka A.
      Lysine trimethylation regulates 78-kDa glucose-regulated protein proteostasis during endoplasmic reticulum stress.
      ). Finally, the stabilizing effects of Hsc70 K561me3 were studied using a lysine-to-arginine mutation to mimic lysine methylation (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ). However, another group used a Hsp70 K561R mutant as nonmethylatable mutant (
      • Cho H.S.
      • Shimazu T.
      • Toyokawa G.
      • Daigo Y.
      • Maehara Y.
      • Hayami S.
      • Ito A.
      • Masuda K.
      • Ikawa N.
      • Field H.I.
      • Tsuchiya E.
      • Ohnuma S.
      • Ponder B.A.
      • Yoshida M.
      • Nakamura Y.
      • et al.
      Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.
      ), illustrating the complications of interpreting mutants. In addition, as others have pointed out, this is a conserved residue, and so mutants may investigate the importance of an important residue rather than methylation (
      • Jakobsson M.E.
      • Moen A.
      • Falnes P.
      Correspondence: on the enzymology and significance of HSPA1 lysine methylation.
      ). A possible mechanism was proposed by Zhang et al. (
      • Zhang H.
      • Amick J.
      • Chakravarti R.
      • Santarriaga S.
      • Schlanger S.
      • McGlone C.
      • Dare M.
      • Nix J.C.
      • Scaglione K.M.
      • Stuehr D.J.
      • Misra S.
      • Page R.C.
      A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.
      ), showing that Hsc70 Lys-561 trimethylation interferes with CHIP-mediated Hsc70 ubiquitination (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ). Interestingly, Lys-561 methylation is not required for Hsc70-dependent chaperone-mediated autophagy; nor is Lys-561 trimethylation inhibitory to Hsc70's chaperoning function (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ). Knockout of METTL21C, a close paralogue of METTL21A, leads to increased Hsc70 Lys-561 dimethylation, indicating possible competing roles of di- and trimethylators (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ). In vitro, METTL21A predominantly catalyzes mono- and dimethylation of Hsp70, BiP and Hsc70, with trimethylation only occurring at high METTL21A concentrations (
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ). METTL21A knock-out cells are deficient in Hsp70 methylation, suggesting that this enzyme is strictly required for Hsp70 Lys-561 methylation (
      • Jakobsson M.E.
      • Moen A.
      • Falnes P.
      Correspondence: on the enzymology and significance of HSPA1 lysine methylation.
      ). Localization studies are conflicting. Cho et al. (
      • Cho H.S.
      • Shimazu T.
      • Toyokawa G.
      • Daigo Y.
      • Maehara Y.
      • Hayami S.
      • Ito A.
      • Masuda K.
      • Ikawa N.
      • Field H.I.
      • Tsuchiya E.
      • Ohnuma S.
      • Ponder B.A.
      • Yoshida M.
      • Nakamura Y.
      • et al.
      Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.
      ) found Hsp70 K561me2 predominantly located in the nucleus of cancer cells, whereas all other HSP70 predominantly localized to the cytosol. In contrast, Jakobsson et al. (
      • Jakobsson M.E.
      • Moen A.
      • Falnes P.
      Correspondence: on the enzymology and significance of HSPA1 lysine methylation.
      ) found me0, 1, 2, and 3 HSP70 Lys-561 in both the cytosol and nuclei in HeLa and HEK293 cells and found HSP70 K561me3 to be the most prevalent form in either compartment, with the relative amounts of each methylation status the same between both compartments. Gao et al. (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ) also found Hsp70 R469me1 in both cytosolic and nuclear compartments. Cloutier et al. (
      • Cloutier P.
      • Lavallée-Adam M.
      • Faubert D.
      • Blanchette M.
      • Coulombe B.
      A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity.
      ) found that most METTL21A and HSC70 in cells overexpressing METTL21A localizes to the cytosol. Further studies are needed to clarify how methylation of different Hsp70s affects localization, and which additional methylases might be critically involved in Hsp70 Lys-561 modification remains to be defined in detail (
      • Guo A.
      • Gu H.
      • Zhou J.
      • Mulhern D.
      • Wang Y.
      • Lee K.A.
      • Yang V.
      • Aguiar M.
      • Kornhauser J.
      • Jia X.
      • Ren J.
      • Beausoleil S.A.
      • Silva J.C.
      • Vemulapalli V.
      • Bedford M.T.
      • et al.
      Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
      ,
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ,
      • Cloutier P.
      • Lavallée-Adam M.
      • Faubert D.
      • Blanchette M.
      • Coulombe B.
      A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity.
      ,
      • Jakobsson M.E.
      • Moen A.
      • Falnes P.
      Correspondence: on the enzymology and significance of HSPA1 lysine methylation.
      ).
      Given that methylated Hsp70-R469me1 promotes RARβ2 transcription (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ) and is more stable (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ), whereas Hsc70 K561me3 has impaired substrate-binding abilities (
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ), it is likely that methylated and nonmethylated Hsp70s play different physiologic roles. Just as Hsc70 K561me3 has reduced substrate affinity (
      • Jakobsson M.E.
      • Moen A.
      • Bousset L.
      • Egge-Jacobsen W.
      • Kernstock S.
      • Melki R.
      • Falnes P.O.
      Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.
      ), this may be true for other trimethylated Hsp70s. As such, trimethylated forms of these chaperones would be expected to be lower in the case of high unfolded protein burden. Meanwhile, other functions of Hsp70s, such as induction of target protein degradation (
      • Wang C.
      • Arrington J.
      • Ratliff A.C.
      • Chen J.
      • Horton H.E.
      • Nie Y.
      • Yue F.
      • Hrycyna C.A.
      • Tao W.A.
      • Kuang S.
      Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.
      ) and transcript initiation (
      • Gao W.W.
      • Xiao R.Q.
      • Peng B.L.
      • Xu H.T.
      • Shen H.F.
      • Huang M.F.
      • Shi T.T.
      • Yi J.
      • Zhang W.J.
      • Wu X.N.
      • Gao X.
      • Lin X.Z.
      • Dorrestein P.C.
      • Rosenfeld M.G.
      • Liu W.
      Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation.
      ), appear to rely on trimethylation of Hsp70s. These diverse roles might occur in different cell compartments, which would explain why some methylated Hsp70 forms and methylators are restricted to or enriched in specific cellular compartments.
      Hsp70 family methylation has been identified in other organisms, yet at a lower frequency. In yeast, Hsp70 isoforms Ssa2 and Ssa4 are monomethylated on Lys-421/422 or Glu-423/424, respectively, the consequence of which remains unknown (
      • Caslavka Zempel K.E.
      • Vashisht A.A.
      • Barshop W.D.
      • Wohlschlegel J.A.
      • Clarke S.G.
      Determining the mitochondrial methyl proteome in Saccharomyces cerevisiae using heavy methyl SILAC.
      ).

      Ubiquitination

      In contrast to small chemical modifications on amino acids, ubiquitination refers to the covalent addition of an 8-kDa ubiquitin (Ub) protein to substrate proteins. Ubiquitination is an ATP-dependent process and is catalyzed by the sequential activity of three enzymes. First, ubiquitin-activating enzymes (E1) adenylate Ub and load this primed Ub unit to one of ∼40 ubiquitin-conjugating enzymes (E2). The Ub-containing E2 enzyme forms a complex with one of ∼600 ubiquitin ligases (E3) (
      • Kerscher O.
      • Felberbaum R.
      • Hochstrasser M.
      Modification of proteins by ubiquitin and ubiquitin-like proteins.
      ,
      • Zheng N.
      • Shabek N.
      Ubiquitin ligases: structure, function, and regulation.
      ). Directed by the E3 ligase, the E2-E3 complex finally interacts with its substrate and promotes the formation of an isopeptide bond connecting the carboxyl-terminal glycine residue of Ub with an accessible lysine residue. The presence of seven lysines in Ub itself (Lys-6, -11, -27, -29, -33, -48, and -63) enables the assembly of branched poly-Ub chains, which have different implications for substrate fates (
      • Xu P.
      • Duong D.M.
      • Seyfried N.T.
      • Cheng D.
      • Xie Y.
      • Robert J.
      • Rush J.
      • Hochstrasser M.
      • Finley D.
      • Peng J.
      Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.
      ). Whereas Lys-48–linked Ub chains label substrates for degradation by the 26S proteasome, Lys-63–linked ubiquitin chains contribute to signaling (
      • Iwai K.
      Diverse roles of the ubiquitin system in NF-κB activation.
      ,
      • Iwai K.
      Diverse ubiquitin signaling in NF-κB activation.
      ,
      • Thrower J.S.
      • Hoffman L.
      • Rechsteiner M.
      • Pickart C.M.
      Recognition of the polyubiquitin proteolytic signal.
      ).
      Hsp70s are modified by several undisclosed E3 Ub ligases at multiple residues (Figure 1, Figure 2, Figure 3). Most of these sites were identified in high-throughput Ub-proteomics studies, and the functional implications of these modifications remain largely unclear (
      • Kim W.
      • Bennett E.J.
      • Huttlin E.L.
      • Guo A.
      • Li J.
      • Possemato A.
      • Sowa M.E.
      • Rad R.
      • Rush J.
      • Comb M.J.
      • Harper J.W.
      • Gygi S.P.
      Systematic and quantitative assessment of the ubiquitin-modified proteome.
      ,
      • Shi Y.
      • Chan D.W.
      • Jung S.Y.
      • Malovannaya A.
      • Wang Y.
      • Qin J.
      A data set of human endogenous protein ubiquitination sites.
      ,
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Nielsen M.L.
      • Cox J.
      • Mann M.
      • Choudhary C.
      A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.
      ,
      • Gendron J.M.
      • Webb K.
      • Yang B.
      • Rising L.
      • Zuzow N.
      • Bennett E.J.
      Using the ubiquitin-modified proteome to monitor distinct and spatially restricted protein homeostasis dysfunction.
      ). The best-understood Ub ligase-Hsp70 interactions are CHIP-Hsp70/Hsc70 and Parkin-Hsp70/Hsc70. CHIP binds to Hsp70 and Hsc70 and modifies their client proteins as well as Hsp70/Hsc70 itself. Whereas most studies suggest that polyubiquitination of Hsp70 and Hsc70 promotes their proteasomal degradation, other work did not find supporting evidence for this process (
      • Jiang J.
      • Ballinger C.A.
      • Wu Y.
      • Dai Q.
      • Cyr D.M.
      • Höhfeld J.
      • Patterson C.
      CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • Morales J.L.
      • Perdew G.H.
      Carboxyl terminus of hsc70-interacting protein (CHIP) can remodel mature aryl hydrocarbon receptor (AhR) complexes and mediate ubiquitination of both the AhR and the 90 kDa heat-shock protein (hsp90) in vitro.
      ,
      • Qian S.B.
      • McDonough H.
      • Boellmann F.
      • Cyr D.M.
      • Patterson C.
      CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70.
      ,
      • Soss S.E.
      • Rose K.L.
      • Hill S.
      • Jouan S.
      • Chazin W.J.
      Biochemical and proteomic analysis of ubiquitination of Hsc70 and Hsp70 by the E3 ligase CHIP.
      ). CHIP-conferred Ub arrays on Hsp70 and Hsc70, while substantially overlapping, differ in the modification of specific sites (e.g. Lys-159 in Hsc70 and its orthologous residue in Hsp70) and the length and branching of the added Ub chains (
      • Soss S.E.
      • Rose K.L.
      • Hill S.
      • Jouan S.
      • Chazin W.J.
      Biochemical and proteomic analysis of ubiquitination of Hsc70 and Hsp70 by the E3 ligase CHIP.
      ), indicating that a single E3 ligase can imprint different Ub patterns on distinct substrates. Parkin is a Parkinson's disease–associated E3 Ub ligase that monoubiquitinylates Hsp70 and Hsc70 on multiple sites (
      • Moore D.J.
      • West A.B.
      • Dikeman D.A.
      • Dawson V.L.
      • Dawson T.M.
      Parkin mediates the degradation-independent ubiquitination of Hsp70.
      ). Interestingly, monoubiquitination of Hsp70 and Hsc70 by Parkin does not alter their turnover, steady-state levels, or proteasomal degradation, suggesting a role for ubiquitination in signaling transduction or chaperone regulation beyond degradation.
      The ubiquitin-mediated degradation of several important proteins is regulated by other nearby PTMs. For example, phosphorylation of Thr-286 on cyclin D1 promotes its degradation and correct cell cycle progression (
      • Lin D.I.
      • Barbash O.
      • Kumar K.G.
      • Weber J.D.
      • Harper J.W.
      • Klein-Szanto A.J.
      • Rustgi A.
      • Fuchs S.Y.
      • Diehl J.A.
      Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-αB crystallin) complex.
      ). It is interesting to note that several PTMs exist in close proximity to identified sites of Hsp70 ubiquitination (see Figure 1, Figure 2, Figure 3). Given the number of PTMs on Hsp70, it is also possible that ubiquitination acts as a reset button for the chaperone code. While currently just speculation by the authors, it is feasible that once a certain number/combination of modifications has been reached, ubiquitination may promote chaperone destruction, with newly synthesized unmodified Hsp70 taking its place.

      Hsp70 thiol oxidation

      Thiol oxidation occurs on cysteines that are modified under conditions of oxidative stress or under exposure to thiol chelators or oxidizers, perhaps as an oxidative stress signal (
      • Wang Y.
      • Gibney P.A.
      • West J.D.
      • Morano K.A.
      The yeast Hsp70 Ssa1 is a sensor for activation of the heat shock response by thiol-reactive compounds.
      ,
      • Siegenthaler K.D.
      • Pareja K.A.
      • Wang J.
      • Sevier C.S.
      An unexpected role for the yeast nucleotide exchange factor Sil1 as a reductant acting on the molecular chaperone BiP.
      ,
      • Yang J.
      • Zhang H.
      • Gong W.
      • Liu Z.
      • Wu H.
      • Hu W.
      • Chen X.
      • Wang L.
      • Wu S.
      • Chen C.
      • Perrett S.
      Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid.
      ). This can lead to new disulfide linkages on the same protein or between proteins, formation of new moieties (including sulfenic acid and glutathionylation), and electrophilic adduction (
      • Wang Y.
      • Gibney P.A.
      • West J.D.
      • Morano K.A.
      The yeast Hsp70 Ssa1 is a sensor for activation of the heat shock response by thiol-reactive compounds.
      ,
      • Wang J.
      • Sevier C.S.
      Formation and reversibility of BiP protein cysteine oxidation facilitate cell survival during and post oxidative stress.
      ,
      • Poole L.B.
      The basics of thiols and cysteines in redox biology and chemistry.
      ). In yeast cells, Ssa1 and Ssa2 are subject to thiol oxidation on Cys-15, Cys-264, and Cys-303 when treated with thiol-reactive compounds (
      • Wei P.-C.
      • Hsieh Y.-H.
      • Su M.-I.
      • Jiang X.
      • Hsu P.-H.
      • Lo W.-T.
      • Weng J.-Y.
      • Jeng Y.-M.
      • Wang J.-M.
      • Chen P-L.
      • Chang Y.-C.
      • Lee K.-F.
      • Tsai M.-D.
      • Shew J.-Y.
      • Lee W.-H.
      Loss of the oxidative stress sensor NPGPx compromises GRP78 chaperone activity and induces systemic disease.
      ). Mutation of these sites prevents Hsf1 activation in response to oxidative agents. Several of these sites are conserved in mammalian cells and are also oxidized. Human Hsp70 (but not Hsc70) is also oxidized on Cys-267 and Cys-306, which inhibits its ATPase activity (
      • Miyata Y.
      • Rauch J.N.
      • Jinwal U.K.
      • Thompson A.D.
      • Srinivasan S.
      • Dickey C.A.
      • Gestwicki J.E.
      Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.
      ). This appears to be a primary mechanism of action for the anti-Tau drug methylene blue and suggests that going forward, compounds that manipulate Hsp70 oxidation status may be clinically relevant.
      The above studies suggest a fascinating mechanism by which Hsp70 is a direct sensor in the cellular response to oxidative stress (
      • Wang Y.
      • Gibney P.A.
      • West J.D.
      • Morano K.A.
      The yeast Hsp70 Ssa1 is a sensor for activation of the heat shock response by thiol-reactive compounds.
      ). It appears that other Hsp70 isoforms can also perform this function. The yeast BiP ortholog Kar2 is oxidized on Cys-63 under ER oxidative stress, which is reversed via one of Kar2's nucleotide exchange factors, Sil1, apparently via thiol-disulfide exchange between the oxidized Cys-63 of Kar2 and the Cys-52 and/or Cys-57 of Sil1 (
      • Siegenthaler K.D.
      • Pareja K.A.
      • Wang J.
      • Sevier C.S.
      An unexpected role for the yeast nucleotide exchange factor Sil1 as a reductant acting on the molecular chaperone BiP.
      ,
      • Wang J.
      • Sevier C.S.
      Formation and reversibility of BiP protein cysteine oxidation facilitate cell survival during and post oxidative stress.
      ,
      • Wang J.
      • Pareja K.A.
      • Kaiser C.A.
      • Sevier C.S.
      Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress.
      ). Sulfenylation and glutathionylation diminish Kar2's ATPase activities while leaving its peptide binding activities intact, converting Kar2 from an ATP-dependent foldase into an ATP-independent holdase (
      • Wang J.
      • Sevier C.S.
      Formation and reversibility of BiP protein cysteine oxidation facilitate cell survival during and post oxidative stress.
      ,
      • Wang J.
      • Pareja K.A.
      • Kaiser C.A.
      • Sevier C.S.
      Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress.
      ). This modification is cytoprotective during oxidative stress, but not during nonstress conditions (
      • Wang J.
      • Sevier C.S.
      Formation and reversibility of BiP protein cysteine oxidation facilitate cell survival during and post oxidative stress.
      ,
      • Wang J.
      • Pareja K.A.
      • Kaiser C.A.
      • Sevier C.S.
      Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress.
      ). Recent work suggests that Cys-574 and Cys-603 of HSP70 can also undergo a novel type of modification (glutathionylation) that promotes in vitro an increase in ATPase activity but decreased interaction with HSF1 in HeLa cells (
      • Yang J.
      • Zhang H.
      • Gong W.
      • Liu Z.
      • Wu H.
      • Hu W.
      • Chen X.
      • Wang L.
      • Wu S.
      • Chen C.
      • Perrett S.
      Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid.
      ). Although further studies are needed, this suggests that glutathionylation in response to oxidative stress may also contribute to the cellular response to oxidative stress though HSF1 activation. In human U2OS bone cells, the peroxidase GPx7/NPGPx interacts with BiP under oxidative stress, and in vitro mediates the formation of a disulfide bond between BiP Cys-41 and Cys-420 (
      • Wei P.-C.
      • Hsieh Y.-H.
      • Su M.-I.
      • Jiang X.
      • Hsu P.-H.
      • Lo W.-T.
      • Weng J.-Y.
      • Jeng Y.-M.
      • Wang J.-M.
      • Chen P-L.
      • Chang Y.-C.
      • Lee K.-F.
      • Tsai M.-D.
      • Shew J.-Y.
      • Lee W.-H.
      Loss of the oxidative stress sensor NPGPx compromises GRP78 chaperone activity and induces systemic disease.
      ). The formation of this bond promotes increased BiP activity, ER protein folding, and ER oxidative stress resistance (
      • Guo A.
      • Gu H.
      • Zhou J.
      • Mulhern D.
      • Wang Y.
      • Lee K.A.
      • Yang V.
      • Aguiar M.
      • Kornhauser J.
      • Jia X.
      • Ren J.
      • Beausoleil S.A.
      • Silva J.C.
      • Vemulapalli V.
      • Bedford M.T.
      • et al.
      Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.
      ). Although this disulfide bond formation was not shown in vivo, other proteins, including the peroxide-detoxifying enzyme PRDX-2, have been shown to form disulfide bonds in C. elegans in response to H2O2 (
      • Oláhová M.
      • Taylor S.R.
      • Khazaipoul S.
      • Wang J.
      • Morgan B.A.
      • Matsumoto K.
      • Blackwell T.K.
      • Veal E.A.
      A redox-sensitive peroxiredoxin that is important for longevity has tissue- and stress-specific roles in stress resistance.
      ,
      • Kumsta C.
      • Thamsen M.
      • Jakob U.
      Effects of oxidative stress on behavior, physiology, and the redox thiol proteome of Caenorhabditis elegans.
      ,
      • Knoefler D.
      • Thamsen M.
      • Koniczek M.
      • Niemuth N.J.
      • Diederich A.K.
      • Jakob U.
      Quantitative in vivo redox sensors uncover oxidative stress as an early event in life.
      ), demonstrating oxidized disulfide bond formation in vivo and suggesting the possibility that Hsp70 family members that form disulfide bonds in yeast cells during stress response may also do so in other eukaryotic organisms (
      • Wei P.-C.
      • Hsieh Y.-H.
      • Su M.-I.
      • Jiang X.
      • Hsu P.-H.
      • Lo W.-T.
      • Weng J.-Y.
      • Jeng Y.-M.
      • Wang J.-M.
      • Chen P-L.
      • Chang Y.-C.
      • Lee K.-F.
      • Tsai M.-D.
      • Shew J.-Y.
      • Lee W.-H.
      Loss of the oxidative stress sensor NPGPx compromises GRP78 chaperone activity and induces systemic disease.
      ). In C. elegans, Cys-307 of the cytosolic Hsc70 ortholog HSP1 is oxidized when H2O2 detoxification is impaired, during induced H2O2 stress, and during development (
      • Kumsta C.
      • Thamsen M.
      • Jakob U.
      Effects of oxidative stress on behavior, physiology, and the redox thiol proteome of Caenorhabditis elegans.
      ,
      • Knoefler D.
      • Thamsen M.
      • Koniczek M.
      • Niemuth N.J.
      • Diederich A.K.
      • Jakob U.
      Quantitative in vivo redox sensors uncover oxidative stress as an early event in life.