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Post-translational modifications of Hsp90 and translating the chaperone code

  • Sarah J. Backe
    Footnotes
    Affiliations
    Department of Urology, SUNY Upstate Medical University, Syracuse, New York, USA

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA

    Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, New York, USA
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  • Rebecca A. Sager
    Footnotes
    Affiliations
    Department of Urology, SUNY Upstate Medical University, Syracuse, New York, USA

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA

    Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, New York, USA

    College of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA
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  • Mark R. Woodford
    Affiliations
    Department of Urology, SUNY Upstate Medical University, Syracuse, New York, USA

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA

    Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, New York, USA
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  • Alan M. Makedon
    Affiliations
    Department of Urology, SUNY Upstate Medical University, Syracuse, New York, USA

    Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, New York, USA
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  • Mehdi Mollapour
    Correspondence
    For correspondence: Mehdi Mollapour
    Affiliations
    Department of Urology, SUNY Upstate Medical University, Syracuse, New York, USA

    Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA

    Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, New York, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
Open AccessPublished:June 11, 2020DOI:https://doi.org/10.1074/jbc.REV120.011833
      Cells have a remarkable ability to synthesize large amounts of protein in a very short period of time. Under these conditions, many hydrophobic surfaces on proteins may be transiently exposed, and the likelihood of deleterious interactions is quite high. To counter this threat to cell viability, molecular chaperones have evolved to help nascent polypeptides fold correctly and multimeric protein complexes assemble productively, while minimizing the danger of protein aggregation. Heat shock protein 90 (Hsp90) is an evolutionarily conserved molecular chaperone that is involved in the stability and activation of at least 300 proteins, also known as clients, under normal cellular conditions. The Hsp90 clients participate in the full breadth of cellular processes, including cell growth and cell cycle control, signal transduction, DNA repair, transcription, and many others. Hsp90 chaperone function is coupled to its ability to bind and hydrolyze ATP, which is tightly regulated both by co-chaperone proteins and post-translational modifications (PTMs). Many reported PTMs of Hsp90 alter chaperone function and consequently affect myriad cellular processes. Here, we review the contributions of PTMs, such as phosphorylation, acetylation, SUMOylation, methylation, O-GlcNAcylation, ubiquitination, and others, toward regulation of Hsp90 function. We also discuss how the Hsp90 modification state affects cellular sensitivity to Hsp90-targeted therapeutics that specifically bind and inhibit its chaperone activity. The ultimate challenge is to decipher the comprehensive and combinatorial array of PTMs that modulate Hsp90 chaperone function, a phenomenon termed the “chaperone code.”
      Molecular chaperones are necessary for the stability, folding, and activation of a wide array of “client proteins” (
      • Schopf F.H.
      • Biebl M.M.
      • Buchner J.
      The HSP90 chaperone machinery.
      ). One such molecular chaperone, the 90-kDa heat shock protein 90 (Hsp90), has over 300 clients, including protein kinases, transcription factors, oncoproteins, and tumor suppressors. Tight regulation of Hsp90 chaperone function and the downstream activities of its client proteins is essential for the maintenance of proteostasis, execution of the full spectrum of normal cellular processes, and preservation of tissue and organismal health (
      • O'Brien D.
      • van Oosten-Hawle P.
      Regulation of cell-non-autonomous proteostasis in metazoans.
      ,
      • O'Brien D.
      • Jones L.M.
      • Good S.
      • Miles J.
      • Vijayabaskar M.S.
      • Aston R.
      • Smith C.E.
      • Westhead D.R.
      • van Oosten-Hawle P.
      A PQM-1-mediated response triggers transcellular chaperone signaling and regulates organismal proteostasis.
      ). Hsp90 possesses an ATPase activity that is coupled to its chaperone function along with a series of Hsp90 conformational changes collectively known as the chaperone cycle (
      • Schopf F.H.
      • Biebl M.M.
      • Buchner J.
      The HSP90 chaperone machinery.
      ). A group of proteins, called co-chaperones, and post-translational modifications (PTMs) of Hsp90 together regulate Hsp90 chaperone activity and fine-tune it to the needs of the client proteins and the cell (
      • Cox M.B.
      • Johnson J.L.
      Evidence for Hsp90 co-chaperones in regulating Hsp90 function and promoting client protein folding.
      ,
      • Sahasrabudhe P.
      • Rohrberg J.
      • Biebl M.M.
      • Rutz D.A.
      • Buchner J.
      The plasticity of the Hsp90 co-chaperone system.
      ,
      • Zierer B.K.
      • Rübbelke M.
      • Tippel F.
      • Madl T.
      • Schopf F.H.
      • Rutz D.A.
      • Richter K.
      • Sattler M.
      • Buchner J.
      Importance of cycle timing for the function of the molecular chaperone Hsp90.
      ,
      • Röhl A.
      • Rohrberg J.
      • Buchner J.
      The chaperone Hsp90: changing partners for demanding clients.
      ). Cancer cells often use Hsp90 function to promote tumor growth and metastasis, proffering Hsp90 as an attractive therapeutic target (
      • Neckers L.
      • Workman P.
      Hsp90 molecular chaperone inhibitors: are we there yet?.
      ,
      • Barrott J.J.
      • Haystead T.A.
      Hsp90, an unlikely ally in the war on cancer.
      ,
      • Rodina A.
      • Wang T.
      • Yan P.
      • Gomes E.D.
      • Dunphy M.P.
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      • Koren J.
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      • Taldone T.
      • Zong H.
      • Caldas-Lopes E.
      • Alpaugh M.
      • Corben A.
      • Riolo M.
      • Beattie B.
      • et al.
      The epichaperome is an integrated chaperome network that facilitates tumour survival.
      ,
      • Wang H.
      • Lu M.
      • Yao M.
      • Zhu W.
      Effects of treatment with an Hsp90 inhibitor in tumors based on 15 phase II clinical trials.
      ). Specific Hsp90-targeted therapeutics can simultaneously affect a myriad of cellular processes through chaperone function inhibition. Despite strong preclinical evidence of Hsp90 inhibitor efficacy, several drugs have had only limited success in phase 3 clinical trials, and none has achieved FDA approval. Co-chaperone dynamics and Hsp90 PTM status contribute to the complex variables that determine Hsp90 inhibitor sensitivity (
      • Walton-Diaz A.
      • Khan S.
      • Bourboulia D.
      • Trepel J.B.
      • Neckers L.
      • Mollapour M.
      Contributions of co-chaperones and post-translational modifications towards Hsp90 drug sensitivity.
      ,
      • Woodford M.R.
      • Dunn D.
      • Miller J.B.
      • Jamal S.
      • Neckers L.
      • Mollapour M.
      Impact of posttranslational modifications on the anticancer activity of Hsp90 inhibitors.
      ,
      • Soroka J.
      • Buchner J.
      Mechanistic aspects of the Hsp90 phosphoregulation.
      ). Effort toward elucidating the impact of post-translational modifications of Hsp90 on its chaperone function has found that these modification states alter Hsp90 ATPase activity, co-chaperone and client binding, client maturation, Hsp90 subcellular localization and degradation, and Hsp90 inhibitor sensitivity (
      • Cloutier P.
      • Coulombe B.
      Regulation of molecular chaperones through post-translational modifications: decrypting the chaperone code.
      ,
      • 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.
      ,
      • Nitika
      • Truman A.W.
      Cracking the chaperone code: cellular roles for Hsp70 phosphorylation.
      ). Dozens of modification sites, including phosphorylation, acetylation, SUMOylation, methylation, and others, have been functionally and mechanistically studied, as will be reviewed here. Taken together, the literature suggests that we need to develop a holistic understanding of the combinatorial array of these PTMs that target Hsp90 and modulate its chaperone activity. This phenomenon is also known as the “chaperone code.”

      Structure and chaperone function of Hsp90

      The two cytosolic isoforms of Hsp90, Hsp90α and Hsp90β, share 85% sequence identity (
      • Csermely P.
      • Schnaider T.
      • Sőti C.
      • Prohaszka Z.
      • Nardai G.
      The 90-kDa molecular chaperone family: structure, function, and clinical applications: a comprehensive review.
      ,
      • Johnson J.L.
      Evolution and function of diverse Hsp90 homologs and cochaperone proteins.
      ,
      • Zuehlke A.D.
      • Beebe K.
      • Neckers L.
      • Prince T.
      Regulation and function of the human HSP90AA1 gene.
      ). Hsp90β is constitutively expressed under normal physiological conditions. Hsp90α, however, is stress-inducible, and increased levels of Hsp90α have been associated with poor prognosis in cancer (
      • Zuehlke A.D.
      • Beebe K.
      • Neckers L.
      • Prince T.
      Regulation and function of the human HSP90AA1 gene.
      ). The chaperone function of Hsp90 encompasses an ordered series of conformational changes coupled to its ATPase activity, collectively known as the “chaperone cycle” (Fig. 1) (
      • Zierer B.K.
      • Rübbelke M.
      • Tippel F.
      • Madl T.
      • Schopf F.H.
      • Rutz D.A.
      • Richter K.
      • Sattler M.
      • Buchner J.
      Importance of cycle timing for the function of the molecular chaperone Hsp90.
      ,
      • Panaretou B.
      • Prodromou C.
      • Roe S.M.
      • O'Brien R.
      • Ladbury J.E.
      • Piper P.W.
      • Pearl L.H.
      ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.
      ,
      • Obermann W.M.
      • Sondermann H.
      • Russo A.A.
      • Pavletich N.P.
      • Hartl F.U.
      In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.
      ,
      • Graf C.
      • Stankiewicz M.
      • Kramer G.
      • Mayer M.P.
      Spatially and kinetically resolved changes in the conformational dynamics of the Hsp90 chaperone machine.
      ,
      • Mickler M.
      • Hessling M.
      • Ratzke C.
      • Buchner J.
      • Hugel T.
      The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis.
      ). The functional unit of Hsp90 is a dimer, and each protomer consists of three structural domains (
      • Wayne N.
      • Bolon D.N.
      Dimerization of Hsp90 is required for in vivo function: design and analysis of monomers and dimers.
      ,
      • Ali M.M.
      • Roe S.M.
      • Vaughan C.K.
      • Meyer P.
      • Panaretou B.
      • Piper P.W.
      • Prodromou C.
      • Pearl L.H.
      Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex.
      ,
      • Verba K.A.
      • Wang R.Y.
      • Arakawa A.
      • Liu Y.
      • Shirouzu M.
      • Yokoyama S.
      • Agard D.A.
      Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.
      ). The amino-terminal domain, or “N-domain,” contains the nucleotide-binding pocket, which also serves as the binding site for most Hsp90 inhibitors (
      • Prodromou C.
      • Roe S.M.
      • O'Brien R.
      • Ladbury J.E.
      • Piper P.W.
      • Pearl L.H.
      Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.
      ,
      • Prodromou C.
      • Roe S.M.
      • Piper P.W.
      • Pearl L.H.
      A molecular clamp in the crystal structure of the N-terminal domain of the yeast Hsp90 chaperone.
      ,
      • Tsutsumi S.
      • Mollapour M.
      • Prodromou C.
      • Lee C.T.
      • Panaretou B.
      • Yoshida S.
      • Mayer M.P.
      • Neckers L.M.
      Charged linker sequence modulates eukaryotic heat shock protein 90 (Hsp90) chaperone activity.
      ). The middle domain is connected to the N-domain by a flexible and highly charged linker region (
      • Hainzl O.
      • Lapina M.C.
      • Buchner J.
      • Richter K.
      The charged linker region is an important regulator of Hsp90 function.
      ,
      • Jahn M.
      • Rehn A.
      • Pelz B.
      • Hellenkamp B.
      • Richter K.
      • Rief M.
      • Buchner J.
      • Hugel T.
      The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function.
      ). The middle domain harbors the catalytic loop that is necessary for ATP hydrolysis and also serves as the binding site for the majority of client proteins and many co-chaperones (
      • Schopf F.H.
      • Biebl M.M.
      • Buchner J.
      The HSP90 chaperone machinery.
      ,
      • Meyer P.
      • Prodromou C.
      • Hu B.
      • Vaughan C.
      • Roe S.M.
      • Panaretou B.
      • Piper P.W.
      • Pearl L.H.
      Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions.
      ,
      • Biebl M.M.
      • Buchner J.
      Structure, Function, and Regulation of the Hsp90 Machinery.
      ). The carboxyl-terminal domain, or “C-domain,” is the site of constitutive dimerization of the Hsp90 protomers (
      • Harris S.F.
      • Shiau A.K.
      • Agard D.A.
      The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site.
      ,
      • Prodromou C.
      • Pearl L.H.
      Structure and functional relationships of Hsp90.
      ) and contains the extreme C-terminal MEEVD sequence that serves as an interaction site for tetratricopeptide repeat (TPR)-domain–containing co-chaperones (
      • Schopf F.H.
      • Biebl M.M.
      • Buchner J.
      The HSP90 chaperone machinery.
      ). The Hsp90 chaperone cycle is considered to begin with an “open” dimer, which is only dimerized at the C-domain. ATP binding to the nucleotide pocket of the N-domain contributes to the large-scale conformational rearrangements that lead to transient dimerization of the Hsp90 N-domains. This transiently N-terminally dimerized state is referred to as the “closed” conformation. Upon ATP hydrolysis, Hsp90 returns to its “open” V-shaped conformation and is ready to begin another cycle (Fig. 1) (
      • Zierer B.K.
      • Rübbelke M.
      • Tippel F.
      • Madl T.
      • Schopf F.H.
      • Rutz D.A.
      • Richter K.
      • Sattler M.
      • Buchner J.
      Importance of cycle timing for the function of the molecular chaperone Hsp90.
      ,
      • Mickler M.
      • Hessling M.
      • Ratzke C.
      • Buchner J.
      • Hugel T.
      The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis.
      ,
      • Prodromou C.
      • Pearl L.H.
      Structure and functional relationships of Hsp90.
      ,
      • Hessling M.
      • Richter K.
      • Buchner J.
      Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90.
      ,
      • Neckers L.
      • Mollapour M.
      • Tsutsumi S.
      The complex dance of the molecular chaperone Hsp90.
      ,
      • Shiau A.K.
      • Harris S.F.
      • Southworth D.R.
      • Agard D.A.
      Structural analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements.
      ). There is also a great deal of interdomain connectivity and communication across the Hsp90 protein (
      • Stetz G.
      • Tse A.
      • Verkhivker G.M.
      Dissecting structure-encoded determinants of allosteric cross-talk between post-translational modification sites in the Hsp90 chaperones.
      ,
      • Cunningham C.N.
      • Krukenberg K.A.
      • Agard D.A.
      Intra- and intermonomer interactions are required to synergistically facilitate ATP hydrolysis in Hsp90.
      ,
      • Xu W.
      • Beebe K.
      • Chavez J.D.
      • Boysen M.
      • Lu Y.
      • Zuehlke A.D.
      • Keramisanou D.
      • Trepel J.B.
      • Prodromou C.
      • Mayer M.P.
      • Bruce J.E.
      • Gelis I.
      • Neckers L.
      Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1.
      ). Progression through the chaperone cycle is required for client chaperoning and is tailored to individual client needs. In general, clients require chaperoning to either achieve a final active conformation, assemble into multiprotein complexes, or promote and stabilize a ligand-competent state that is awaiting activation (
      • Schopf F.H.
      • Biebl M.M.
      • Buchner J.
      The HSP90 chaperone machinery.
      ).
      Figure thumbnail gr1
      Figure 1The Hsp90 chaperone cycle. Hsp90 begins its chaperone cycle in an open conformation that is dimerized only at the C-domain. ATP binding and an ordered series of conformational changes allow it to adopt a closed conformation, which is N-terminally dimerized. Upon ATP hydrolysis, Hsp90 returns back to the open conformation and is ready to begin another chaperone cycle. This allows for the activation of client proteins. This cycle is tightly regulated by co-chaperone proteins as well as PTMs, and Hsp90 inhibitors can also modulate the chaperone cycle.
      Many factors contribute to Hsp90 chaperone cycle regulation and subsequent client chaperoning, including interacting co-chaperone proteins and post-translational modifications, and Hsp90 inhibitors can also affect the cycle. Co-chaperones, unlike client proteins, are not themselves dependent on Hsp90 chaperone function for their stability or activity. Generally, co-chaperones alter the progression of Hsp90 through the chaperone cycle by stabilizing different Hsp90 states and conformational intermediates. The TPR-containing co-chaperone Hsp70-Hsp90–organizing protein (HOP) binds to the open conformation of Hsp90, slows down its ATPase activity, and also helps transfer client proteins to Hsp90 (
      • Wegele H.
      • Wandinger S.K.
      • Schmid A.B.
      • Reinstein J.
      • Buchner J.
      Substrate transfer from the chaperone Hsp70 to Hsp90.
      ,
      • Li J.
      • Richter K.
      • Buchner J.
      Mixed Hsp90-cochaperone complexes are important for the progression of the reaction cycle.
      ,
      • Prodromou C.
      • Siligardi G.
      • O'Brien R.
      • Woolfson D.N.
      • Regan L.
      • Panaretou B.
      • Ladbury J.E.
      • Piper P.W.
      • Pearl L.H.
      Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones.
      ). Client scaffolding or loading is another common attribute of Hsp90 co-chaperones. The co-chaperone cell division cycle 37 (Cdc37) specifically recruits kinase clients to Hsp90. The Cdc37-mediated chaperoning of kinases requires coordinated phosphorylation and subsequent dephosphorylation of Cdc37 by the phosphatase co-chaperone protein phosphatase 5 (PP5) (
      • Miyata Y.
      • Nishida E.
      CK2 binds, phosphorylates, and regulates its pivotal substrate Cdc37, an Hsp90-cochaperone.
      ,
      • Vaughan C.K.
      • Mollapour M.
      • Smith J.R.
      • Truman A.
      • Hu B.
      • Good V.M.
      • Panaretou B.
      • Neckers L.
      • Clarke P.A.
      • Workman P.
      • Piper P.W.
      • Prodromou C.
      • Pearl L.H.
      Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37.
      ,
      • Keramisanou D.
      • Aboalroub A.
      • Zhang Z.
      • Liu W.
      • Marshall D.
      • Diviney A.
      • Larsen R.W.
      • Landgraf R.
      • Gelis I.
      Molecular mechanism of protein kinase recognition and sorting by the Hsp90 kinome-specific cochaperone Cdc37.
      ). The co-chaperone activator of Hsp90 ATPase (Aha1), on the other hand, displaces HOP and helps to mediate Hsp90 N-domain dimerization and enhances the ATPase activity of Hsp90 (
      • Panaretou B.
      • Siligardi G.
      • Meyer P.
      • Maloney A.
      • Sullivan J.K.
      • Singh S.
      • Millson S.H.
      • Clarke P.A.
      • Naaby-Hansen S.
      • Stein R.
      • Cramer R.
      • Mollapour M.
      • Workman P.
      • Piper P.W.
      • Pearl L.H.
      • et al.
      Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1.
      ,
      • Meyer P.
      • Prodromou C.
      • Liao C.
      • Hu B.
      • Mark Roe S.
      • Vaughan C.K.
      • Vlasic I.
      • Panaretou B.
      • Piper P.W.
      • Pearl L.H.
      Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery.
      ,
      • Retzlaff M.
      • Hagn F.
      • Mitschke L.
      • Hessling M.
      • Gugel F.
      • Kessler H.
      • Richter K.
      • Buchner J.
      Asymmetric activation of the hsp90 dimer by its cochaperone aha1.
      ,
      • Mercier R.
      • Wolmarans A.
      • Schubert J.
      • Neuweiler H.
      • Johnson J.L.
      • LaPointe P.
      The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation.
      ). The co-chaperone prostaglandin E synthase 3 (p23) preferentially binds to and stabilizes Hsp90 in the closed conformation, slowing ATP hydrolysis and aiding client activation (
      • Ali M.M.
      • Roe S.M.
      • Vaughan C.K.
      • Meyer P.
      • Panaretou B.
      • Piper P.W.
      • Prodromou C.
      • Pearl L.H.
      Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex.
      ,
      • Richter K.
      • Walter S.
      • Buchner J.
      The co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle.
      ). Co-chaperone dynamics, as well as several other processes, including Hsp90 ATPase activity, client activation, and Hsp90 inhibitor binding, are further altered by Hsp90 PTMs, as will be highlighted and discussed in the subsequent sections.

      Post-translational modifications and Hsp90 function

      Phosphorylation

      Hsp90 is subject to various PTMs, as will be considered in the sections to follow. Phosphorylation of serine, threonine, and tyrosine residues is the most well-studied of these Hsp90 modifications. The earliest functional work demonstrated that treatment with the nonselective serine/threonine phosphatase inhibitor okadaic acid resulted in hyperphosphorylated Hsp90 and compromised chaperoning of the classic kinase client v-Src (
      • Mimnaugh E.G.
      • Worland P.J.
      • Whitesell L.
      • Neckers L.M.
      Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase.
      ). As will be discussed, much of the functional work focuses on how different PTMs impact chaperone function via examination of ATPase activity of Hsp90, co-chaperone binding, and client stability. Additional works further detail how Hsp90 modification affects downstream cellular processes such as cell cycle control, DNA repair, and steroid hormone signaling as well as many others via effects on the stability and activity of Hsp90 client proteins (Fig. 2). Of note, a large number of potential Hsp90 modification sites in addition to those discussed below have been identified by high-throughput screening studies (RRID:SCR_001837). These studies will not be discussed here, as these sites have not been validated or linked to functional consequences; however, continued evaluation of these sites is necessary to further our understanding and application of the chaperone code.
      Figure thumbnail gr2
      Figure 2Modification by varied enzymes regulates Hsp90 function in biological processes. Enzymes known to modify Hsp90 regulate its PTM state, and this in turn influences various cellular functions as indicated. Arrows are color-coded to match the cellular functions. Yellow arrows indicate cell cycle and proliferation, purple indicates cytoskeleton remodeling and migration, light blue indicates transcription, red indicates angiogenesis and tumor formation, and navy blue indicates DNA repair, apoptosis, and metabolism. Enzymes that are also Hsp90 clients are shaded in green.

      Serine/threonine phosphorylation

      Chaperone cycle

      As discussed above, Hsp90 chaperone function toward client maturation and downstream processes depends on the dynamics of the chaperone cycle, including Hsp90 ATPase activity and co-chaperone binding. The Hsp90 chaperone cycle is in part regulated by phosphorylation mediated by casein kinase 2 (CK2), a ubiquitously expressed and constitutively active protein kinase (
      • Lees-Miller S.P.
      • Anderson C.W.
      Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II.
      ,
      • Franchin C.
      • Borgo C.
      • Zaramella S.
      • Cesaro L.
      • Arrigoni G.
      • Salvi M.
      • Pinna L.A.
      Exploring the CK2 paradox: restless, dangerous, dispensable.
      ). Phosphorylation of yeast Hsp90-T22 (human Hsp90α-T36) or Hsp90β-S365 by CK2 disrupts binding of Hsp90 with the kinase-specific co-chaperone Cdc37 (Table 1 and Fig. 3) (
      • Mollapour M.
      • Tsutsumi S.
      • Truman A.W.
      • Xu W.
      • Vaughan C.K.
      • Beebe K.
      • Konstantinova A.
      • Vourganti S.
      • Panaretou B.
      • Piper P.W.
      • Trepel J.B.
      • Prodromou C.
      • Pearl L.H.
      • Neckers L.
      Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity.
      ,
      • Nguyen M.T.N.
      • Knieß R.A.
      • Daturpalli S.
      • Le Breton L.
      • Ke X.
      • Chen X.
      • Mayer M.P.
      Isoform-specific phosphorylation in human Hsp90β affects interaction with clients and the cochaperone Cdc37.
      ). Additionally, yHsp90-T22 phosphorylation compromises Hsp90 interaction with the activating co-chaperone Aha1 and subsequently results in decreased Hsp90 ATPase activity (
      • Mollapour M.
      • Tsutsumi S.
      • Truman A.W.
      • Xu W.
      • Vaughan C.K.
      • Beebe K.
      • Konstantinova A.
      • Vourganti S.
      • Panaretou B.
      • Piper P.W.
      • Trepel J.B.
      • Prodromou C.
      • Pearl L.H.
      • Neckers L.
      Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity.
      ). Further, ATP binding is impacted by CK2 phosphorylation of the Hsp90β charged linker, which has been suggested to precede Hsp90:Cdc37 complex dissociation when ATP levels are low (
      • Olesen S.H.
      • Ingles D.J.
      • Zhu J.Y.
      • Martin M.P.
      • Betzi S.
      • Georg G.I.
      • Tash J.S.
      • Schönbrunn E.
      Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.
      ). Interestingly, CK2 phosphorylation of Cdc37-S13 promotes its association with kinases and recruitment of Hsp90 to kinase:Cdc37:Hsp90 ternary complexes for the chaperoning of kinase clients (
      • Miyata Y.
      • Nishida E.
      CK2 binds, phosphorylates, and regulates its pivotal substrate Cdc37, an Hsp90-cochaperone.
      ,
      • Vaughan C.K.
      • Mollapour M.
      • Smith J.R.
      • Truman A.
      • Hu B.
      • Good V.M.
      • Panaretou B.
      • Neckers L.
      • Clarke P.A.
      • Workman P.
      • Piper P.W.
      • Prodromou C.
      • Pearl L.H.
      Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37.
      ,
      • Keramisanou D.
      • Aboalroub A.
      • Zhang Z.
      • Liu W.
      • Marshall D.
      • Diviney A.
      • Larsen R.W.
      • Landgraf R.
      • Gelis I.
      Molecular mechanism of protein kinase recognition and sorting by the Hsp90 kinome-specific cochaperone Cdc37.
      ,
      • Stetz G.
      • Astl L.
      • Verkhivker G.M.
      Exploring mechanisms of communication switching in the Hsp90-Cdc37 regulatory complexes with client kinases through allosteric coupling of phosphorylation sites: perturbation-based modeling and hierarchical community analysis of residue interaction networks.
      ). Furthermore, sequential and ordered phosphorylation of the co-chaperone folliculin-interacting protein 1 (FNIP1) on several adjacent serine residues promotes its binding to Hsp90 and facilitates kinase and nonkinase client chaperoning (
      • Sager R.A.
      • Woodford M.R.
      • Backe S.J.
      • Makedon A.M.
      • Baker-Williams A.J.
      • DiGregorio B.T.
      • Loiselle D.R.
      • Haystead T.A.
      • Zachara N.E.
      • Prodromou C.
      • Bourboulia D.
      • Schmidt L.S.
      • Linehan W.M.
      • Bratslavsky G.
      • Mollapour M.
      Post-translational regulation of FNIP1 creates a rheostat for the molecular chaperone Hsp90.
      ). CK2-mediated phosphorylation of both Cdc37 and FNIP1 is specifically reversed by the phosphatase co-chaperone PP5, highlighting the complex interplay of PTMs in the chaperone machinery (
      • Vaughan C.K.
      • Mollapour M.
      • Smith J.R.
      • Truman A.
      • Hu B.
      • Good V.M.
      • Panaretou B.
      • Neckers L.
      • Clarke P.A.
      • Workman P.
      • Piper P.W.
      • Prodromou C.
      • Pearl L.H.
      Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37.
      ,
      • Sager R.A.
      • Woodford M.R.
      • Backe S.J.
      • Makedon A.M.
      • Baker-Williams A.J.
      • DiGregorio B.T.
      • Loiselle D.R.
      • Haystead T.A.
      • Zachara N.E.
      • Prodromou C.
      • Bourboulia D.
      • Schmidt L.S.
      • Linehan W.M.
      • Bratslavsky G.
      • Mollapour M.
      Post-translational regulation of FNIP1 creates a rheostat for the molecular chaperone Hsp90.
      ).
      Table 1Hsp90 post-translational modifications, identified modifying enzymes, and functional consequences
      PTMResidueEnzymeSmall-molecule bindingReferences
      Hsp90αHsp90βCompoundATPInhibitors
      Serine/threonine phosphorylationT5NAATM, DNA-PK
      • Solier S.
      • Kohn K.W.
      • Scroggins B.
      • Xu W.
      • Trepel J.
      • Neckers L.
      • Pommier Y.
      Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response.
      • Park S.J.
      • Gavrilova O.
      • Brown A.L.
      • Soto J.E.
      • Bremner S.
      • Kim J.
      • Xu X.
      • Yang S.
      • Um J.H.
      • Koch L.G.
      • Britton S.L.
      • Lieber R.L.
      • Philp A.
      • Baar K.
      • Kohama S.G.
      • et al.
      DNA-PK promotes the mitochondrial, metabolic, and physical decline that occurs during aging.
      ,
      • Elaimy A.L.
      • Ahsan A.
      • Marsh K.
      • Pratt W.B.
      • Ray D.
      • Lawrence T.S.
      • Nyati M.K.
      ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.
      ,
      • Pennisi R.
      • Antoccia A.
      • Leone S.
      • Ascenzi P.
      • di Masi A.
      Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.
      ,
      • Hasan M.
      • Hama S.
      • Kogure K.
      Low electric treatment activates Rho GTPase via heat shock protein 90 and protein kinase C for intracellular delivery of siRNA.
      • Lees-Miller S.P.
      • Anderson C.W.
      The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 α at two NH2-terminal threonine residues.
      T7NAATM, DNA-PK
      • Solier S.
      • Kohn K.W.
      • Scroggins B.
      • Xu W.
      • Trepel J.
      • Neckers L.
      • Pommier Y.
      Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response.
      • Park S.J.
      • Gavrilova O.
      • Brown A.L.
      • Soto J.E.
      • Bremner S.
      • Kim J.
      • Xu X.
      • Yang S.
      • Um J.H.
      • Koch L.G.
      • Britton S.L.
      • Lieber R.L.
      • Philp A.
      • Baar K.
      • Kohama S.G.
      • et al.
      DNA-PK promotes the mitochondrial, metabolic, and physical decline that occurs during aging.
      ,
      • Elaimy A.L.
      • Ahsan A.
      • Marsh K.
      • Pratt W.B.
      • Ray D.
      • Lawrence T.S.
      • Nyati M.K.
      ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.
      ,
      • Pennisi R.
      • Antoccia A.
      • Leone S.
      • Ascenzi P.
      • di Masi A.
      Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.
      ,
      • Hasan M.
      • Hama S.
      • Kogure K.
      Low electric treatment activates Rho GTPase via heat shock protein 90 and protein kinase C for intracellular delivery of siRNA.
      ,
      • Lees-Miller S.P.
      • Anderson C.W.
      The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 α at two NH2-terminal threonine residues.
      • Quanz M.
      • Herbette A.
      • Sayarath M.
      • de Koning L.
      • Dubois T.
      • Sun J.S.
      • Dutreix M.
      Heat shock protein 90α (Hsp90α) is phosphorylated in response to DNA damage and accumulates in repair foci.
      T36 (*,†)T31CK2
      • Mollapour M.
      • Tsutsumi S.
      • Truman A.W.
      • Xu W.
      • Vaughan C.K.
      • Beebe K.
      • Konstantinova A.
      • Vourganti S.
      • Panaretou B.
      • Piper P.W.
      • Trepel J.B.
      • Prodromou C.
      • Pearl L.H.
      • Neckers L.
      Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity.
      • He Q.
      • Liu K.
      • Tian Z.
      • Du S.J.
      The effects of Hsp90α1 mutations on myosin thick filament organization.
      • Mollapour M.
      • Tsutsumi S.
      • Kim Y.S.
      • Trepel J.
      • Neckers L.
      Casein kinase 2 phosphorylation of Hsp90 threonine 22 modulates chaperone function and drug sensitivity.
      S63S58CK2
      • Huttlin E.L.
      • Jedrychowski M.P.
      • Elias J.E.
      • Goswami T.
      • Rad R.
      • Beausoleil S.A.
      • Villén J.
      • Haas W.
      • Sowa M.E.
      • Gygi S.P.
      A tissue-specific atlas of mouse protein phosphorylation and expression.
      • Rose D.W.
      • Wettenhall R.E.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      The 90-kilodalton peptide of the heme-regulated eIF-2 α kinase has sequence similarity with the 90-kilodalton heat shock protein.
      • Kettenbach A.N.
      • Schweppe D.K.
      • Faherty B.K.
      • Pechenick D.
      • Pletnev A.A.
      • Gerber S.A.
      Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.
      T65T60CK2
      • Rose D.W.
      • Wettenhall R.E.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      The 90-kilodalton peptide of the heme-regulated eIF-2 α kinase has sequence similarity with the 90-kilodalton heat shock protein.
      ,
      • Sharma K.
      • D'Souza R.C.J.
      • Tyanova S.
      • Schaab C.
      • Wiśniewski J.R.
      • Cox J.
      • Mann M.
      Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.
      S68S63CK2
      • Rose D.W.
      • Wettenhall R.E.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      The 90-kilodalton peptide of the heme-regulated eIF-2 α kinase has sequence similarity with the 90-kilodalton heat shock protein.
      S72S67CK2
      • Rose D.W.
      • Wettenhall R.E.
      • Kudlicki W.
      • Kramer G.
      • Hardesty B.
      The 90-kilodalton peptide of the heme-regulated eIF-2 α kinase has sequence similarity with the 90-kilodalton heat shock protein.
      T88T83PKA
      • Kettenbach A.N.
      • Schweppe D.K.
      • Faherty B.K.
      • Pechenick D.
      • Pletnev A.A.
      • Gerber S.A.
      Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.
      ,
      • Lei H.
      • Venkatakrishnan A.
      • Yu S.
      • Kazlauskas A.
      Protein kinase A-dependent translocation of Hsp90 α impairs endothelial nitric-oxide synthase activity in high glucose and diabetes.
      ,
      • Molina H.
      • Horn D.M.
      • Tang N.
      • Mathivanan S.
      • Pandey A.
      Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry.
      T90T85PKA
      • Molina H.
      • Horn D.M.
      • Tang N.
      • Mathivanan S.
      • Pandey A.
      Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry.
      • Wang X.
      • Lu X.A.
      • Song X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Luo Y.
      Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.
      ,
      • Dagar M.
      • Singh J.P.
      • Dagar G.
      • Tyagi R.K.
      • Bagchi G.
      Phosphorylation of HSP90 by protein kinase A is essential for the nuclear translocation of androgen receptor.
      ,
      • Wang X.
      • Song X.
      • Zhuo W.
      • Fu Y.
      • Shi H.
      • Liang Y.
      • Tong M.
      • Chang G.
      • Luo Y.
      The regulatory mechanism of Hsp90α secretion and its function in tumor malignancy.
      • Schweppe D.K.
      • Rigas J.R.
      • Gerber S.A.
      Quantitative phosphoproteomic profiling of human non-small cell lung cancer tumors.
      S113S108 (*)HopBF1
      • Lopez V.A.
      • Park B.C.
      • Nowak D.
      • Sreelatha A.
      • Zembek P.
      • Fernandez J.
      • Servage K.A.
      • Gradowski M.
      • Hennig J.
      • Tomchick D.R.
      • Pawłowski K.
      • Krzymowska M.
      • Tagliabracci V.S.
      A bacterial effector mimics a host HSP90 client to undermine immunity.
      T115T110Mps1, Cdc14, PKCγ
      • Woodford M.R.
      • Truman A.W.
      • Dunn D.M.
      • Jensen S.M.
      • Cotran R.
      • Bullard R.
      • Abouelleil M.
      • Beebe K.
      • Wolfgeher D.
      • Wierzbicki S.
      • Post D.E.
      • Caza T.
      • Tsutsumi S.
      • Panaretou B.
      • Kron S.J.
      • et al.
      Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors.
      ,
      • Lu X.A.
      • Wang X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Fu Y.
      • Luo Y.
      The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.
      S164S159Cdc7-Dbf4
      • Cheng A.N.
      • Fan C.C.
      • Lo Y.K.
      • Kuo C.L.
      • Wang H.C.
      • Lien I.H.
      • Lin S.Y.
      • Chen C.H.
      • Jiang S.S.
      • Chang I.S.
      • Juan H.F.
      • Lyu P.C.
      • Lee A.Y.
      Cdc7-Dbf4-mediated phosphorylation of HSP90-S164 stabilizes HSP90-HCLK2-MRN complex to enhance ATR/ATM signaling that overcomes replication stress in cancer.
      ,
      • Tsai C.F.
      • Wang Y.T.
      • Yen H.Y.
      • Tsou C.C.
      • Ku W.C.
      • Lin P.Y.
      • Chen H.Y.
      • Nesvizhskii A.I.
      • Ishihama Y.
      • Chen Y.J.
      Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.
      S211S206PKA/PKG
      • Huang S.Y.
      • Tsai M.L.
      • Chen G.Y.
      • Wu C.J.
      • Chen S.H.
      A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri.
      ,
      • Klammer M.
      • Kaminski M.
      • Zedler A.
      • Oppermann F.
      • Blencke S.
      • Marx S.
      • Müller S.
      • Tebbe A.
      • Godl K.
      • Schaab C.
      Phosphosignature predicts dasatinib response in non-small cell lung cancer.
      S231S226CK2, PP5
      • Lees-Miller S.P.
      • Anderson C.W.
      Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II.
      ,
      • Olesen S.H.
      • Ingles D.J.
      • Zhu J.Y.
      • Martin M.P.
      • Betzi S.
      • Georg G.I.
      • Tash J.S.
      • Schönbrunn E.
      Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.
      • Woo S.H.
      • An S.
      • Lee H.C.
      • Jin H.O.
      • Seo S.K.
      • Yoo D.H.
      • Lee K.H.
      • Rhee C.H.
      • Choi E.J.
      • Hong S.I.
      • Park I.C.
      A truncated form of p23 down-regulates telomerase activity via disruption of Hsp90 function.
      ,
      • Kurokawa M.
      • Zhao C.
      • Reya T.
      • Kornbluth S.
      Inhibition of apoptosome formation by suppression of Hsp90β phosphorylation in tyrosine kinase-induced leukemias.
      ,
      • Ogiso H.
      • Kagi N.
      • Matsumoto E.
      • Nishimoto M.
      • Arai R.
      • Shirouzu M.
      • Mimura J.
      • Fujii-Kuriyama Y.
      • Yokoyama S.
      Phosphorylation analysis of 90 kDa heat shock protein within the cytosolic arylhydrocarbon receptor complex.
      ,
      • Wang Z.
      • Gucek M.
      • Hart G.W.
      Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc.
      • Kim S.W.
      • Hasanuzzaman M.
      • Cho M.
      • Heo Y.R.
      • Ryu M.J.
      • Ha N.Y.
      • Park H.J.
      • Park H.Y.
      • Shin J.G.
      Casein kinase 2 (CK2)-mediated phosphorylation of Hsp90β as a novel mechanism of rifampin-induced MDR1 expression.
      S263S255CK2, B-Raf, PP5
      • Lees-Miller S.P.
      • Anderson C.W.
      Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II.
      ,
      • Olesen S.H.
      • Ingles D.J.
      • Zhu J.Y.
      • Martin M.P.
      • Betzi S.
      • Georg G.I.
      • Tash J.S.
      • Schönbrunn E.
      Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.
      • Woo S.H.
      • An S.
      • Lee H.C.
      • Jin H.O.
      • Seo S.K.
      • Yoo D.H.
      • Lee K.H.
      • Rhee C.H.
      • Choi E.J.
      • Hong S.I.
      • Park I.C.
      A truncated form of p23 down-regulates telomerase activity via disruption of Hsp90 function.
      ,
      • Kurokawa M.
      • Zhao C.
      • Reya T.
      • Kornbluth S.
      Inhibition of apoptosome formation by suppression of Hsp90β phosphorylation in tyrosine kinase-induced leukemias.
      ,
      • Ogiso H.
      • Kagi N.
      • Matsumoto E.
      • Nishimoto M.
      • Arai R.
      • Shirouzu M.
      • Mimura J.
      • Fujii-Kuriyama Y.
      • Yokoyama S.
      Phosphorylation analysis of 90 kDa heat shock protein within the cytosolic arylhydrocarbon receptor complex.
      ,
      • Wang Z.
      • Gucek M.
      • Hart G.W.
      Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc.
      ,
      • Kim S.W.
      • Hasanuzzaman M.
      • Cho M.
      • Heo Y.R.
      • Ryu M.J.
      • Ha N.Y.
      • Park H.J.
      • Park H.Y.
      • Shin J.G.
      Casein kinase 2 (CK2)-mediated phosphorylation of Hsp90β as a novel mechanism of rifampin-induced MDR1 expression.
      ,
      • Negroni L.
      • Samson M.
      • Guigonis J.M.
      • Rossi B.
      • Pierrefite-Carle V.
      • Baudoin C.
      Treatment of colon cancer cells using the cytosine deaminase/5-fluorocytosine suicide system induces apoptosis, modulation of the proteome, and Hsp90β phosphorylation.
      ,
      • Beranova-Giorgianni S.
      • Zhao Y.
      • Desiderio D.M.
      • Giorgianni F.
      Phosphoproteomic analysis of the human pituitary.
      • Old W.M.
      • Shabb J.B.
      • Houel S.
      • Wang H.
      • Couts K.L.
      • Yen C.Y.
      • Litman E.S.
      • Croy C.H.
      • Meyer-Arendt K.
      • Miranda J.G.
      • Brown R.A.
      • Witze E.S.
      • Schweppe R.E.
      • Resing K.A.
      • Ahn N.G.
      Functional proteomics identifies targets of phosphorylation by B-Raf signaling in melanoma.
      N373S365CK2
      • Klammer M.
      • Kaminski M.
      • Zedler A.
      • Oppermann F.
      • Blencke S.
      • Marx S.
      • Müller S.
      • Tebbe A.
      • Godl K.
      • Schaab C.
      Phosphosignature predicts dasatinib response in non-small cell lung cancer.
      ,
      • Nguyen M.T.N.
      • Knieß R.A.
      • Daturpalli S.
      • Le Breton L.
      • Ke X.
      • Chen X.
      • Mayer M.P.
      Isoform-specific phosphorylation in human Hsp90β affects interaction with clients and the cochaperone Cdc37.
      S399 (*)S391
      • Huttlin E.L.
      • Jedrychowski M.P.
      • Elias J.E.
      • Goswami T.
      • Rad R.
      • Beausoleil S.A.
      • Villén J.
      • Haas W.
      • Sowa M.E.
      • Gygi S.P.
      A tissue-specific atlas of mouse protein phosphorylation and expression.
      ,
      • Soroka J.
      • Wandinger S.K.
      • Mäusbacher N.
      • Schreiber T.
      • Richter K.
      • Daub H.
      • Buchner J.
      Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.
      • Mayya V.
      • Lundgren D.H.
      • Hwang S.I.
      • Rezaul K.
      • Wu L.
      • Eng J.K.
      • Rodionov V.
      • Han D.K.
      Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.
      • Wiśniewski J.R.
      • Nagaraj N.
      • Zougman A.
      • Gnad F.
      • Mann M.
      Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology.
      T425S417PKCγ
      • Lu X.A.
      • Wang X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Fu Y.
      • Luo Y.
      The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.
      S460 (o)S452 (o)PKA
      • Huang S.Y.
      • Tsai M.L.
      • Chen G.Y.
      • Wu C.J.
      • Chen S.H.
      A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri.
      S505 (*)S497
      • Kettenbach A.N.
      • Schweppe D.K.
      • Faherty B.K.
      • Pechenick D.
      • Pletnev A.A.
      • Gerber S.A.
      Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.
      ,
      • Soroka J.
      • Wandinger S.K.
      • Mäusbacher N.
      • Schreiber T.
      • Richter K.
      • Daub H.
      • Buchner J.
      Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.
      S595S587Mitogen-activated protein kinase 12 (p38γ)
      • Mertins P.
      • Qiao J.W.
      • Patel J.
      • Udeshi N.D.
      • Clauser K.R.
      • Mani D.R.
      • Burgess M.W.
      • Gillette M.A.
      • Jaffe J.D.
      • Carr S.A.
      Integrated proteomic analysis of post-translational modifications by serial enrichment.
      • Qi X.
      • Xie C.
      • Hou S.
      • Li G.
      • Yin N.
      • Dong L.
      • Lepp A.
      • Chesnik M.A.
      • Mirza S.P.
      • Szabo A.
      • Tsai S.
      • Basir Z.
      • Wu S.
      • Chen G.
      Identification of a ternary protein-complex as a therapeutic target for K-Ras-dependent colon cancer.
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      T603T595PKCγnc
      • Lu X.A.
      • Wang X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Fu Y.
      • Luo Y.
      The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.
      ,
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      S623 (*)S615
      • Soroka J.
      • Wandinger S.K.
      • Mäusbacher N.
      • Schreiber T.
      • Richter K.
      • Daub H.
      • Buchner J.
      Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.
      ,
      • Zhou H.
      • Di Palma S.
      • Preisinger C.
      • Peng M.
      • Polat A.N.
      • Heck A.J.
      • Mohammed S.
      Toward a comprehensive characterization of a human cancer cell phosphoproteome.
      ,
      • Beli P.
      • Lukashchuk N.
      • Wagner S.A.
      • Weinert B.T.
      • Olsen J.V.
      • Baskcomb L.
      • Mann M.
      • Jackson S.P.
      • Choudhary C.
      Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response.
      T624T616
      • Deb T.B.
      • Zuo A.H.
      • Wang Y.
      • Barndt R.J.
      • Cheema A.K.
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      Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex.
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      M625 (*)M617
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      Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.
      T725A717CK2, CK1, GSK3β
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      • Lane D.P.
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      A quantitative atlas of mitotic phosphorylation.
      S726S718CK2, CK1, GSK3β
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      • Lane D.P.
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      C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.
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      • Dephoure N.
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      A quantitative atlas of mitotic phosphorylation.
      Tyrosine phosphorylationY38 (*,n)Y33 (n)Swe1
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ,
      • Mollapour M.
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      • Donnelly A.C.
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      Swe1Wee1-dependent tyrosine phosphorylation of Hsp90 regulates distinct facets of chaperone function.
      Y197Y192v-Src, Yes
      • Xu W.
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      • Wang S.
      • Scroggins B.T.
      • Palchick Z.
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      • Miyata Y.
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      Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.
      • Bachman A.B.
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      • Beebe K.
      • Moses M.A.
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      Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation.
      ,
      • Walter R.
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      HSP90 promotes Burkitt lymphoma cell survival by maintaining tonic B-cell receptor signaling.
      ,
      • Beebe K.
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      • Taldone T.
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      • Bolon D.
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      Posttranslational modification and conformational state of heat shock protein 90 differentially affect binding of chemically diverse small molecule inhibitors.
      ,
      • Gu T.L.
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      Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia.
      • Rikova K.
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      Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.
      Y309Y301c-Src
      • Beebe K.
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      • Scroggins B.
      • Prodromou C.
      • Xu W.
      • Tokita M.
      • Taldone T.
      • Pullen L.
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      • Bolon D.
      • Chiosis G.
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      Posttranslational modification and conformational state of heat shock protein 90 differentially affect binding of chemically diverse small molecule inhibitors.
      ,
      • Duval M.
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      Src-mediated phosphorylation of Hsp90 in response to vascular endothelial growth factor (VEGF) is required for VEGF receptor-2 signaling to endothelial NO synthase.
      ,
      • Desjardins F.
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      Modulation of the cochaperone AHA1 regulates heat-shock protein 90 and endothelial NO synthase activation by vascular endothelial growth factor.
      Y313Y305
      • Xu W.
      • Beebe K.
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      • Mayer M.P.
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      • Gelis I.
      • Neckers L.
      Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1.
      ,
      • Kettenbach A.N.
      • Schweppe D.K.
      • Faherty B.K.
      • Pechenick D.
      • Pletnev A.A.
      • Gerber S.A.
      Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.
      ,
      • Xu W.
      • Mollapour M.
      • Prodromou C.
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      • Scroggins B.T.
      • Palchick Z.
      • Beebe K.
      • Siderius M.
      • Lee M.J.
      • Couvillon A.
      • Trepel J.B.
      • Miyata Y.
      • Matts R.
      • Neckers L.
      Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.
      ,
      • Ballif B.A.
      • Carey G.R.
      • Sunyaev S.R.
      • Gygi S.P.
      Large-scale identification and evolution indexing of tyrosine phosphorylation sites from murine brain.
      Y627 (*)Y619
      • Kettenbach A.N.
      • Schweppe D.K.
      • Faherty B.K.
      • Pechenick D.
      • Pletnev A.A.
      • Gerber S.A.
      Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.
      ,
      • Xu W.
      • Mollapour M.
      • Prodromou C.
      • Wang S.
      • Scroggins B.T.
      • Palchick Z.
      • Beebe K.
      • Siderius M.
      • Lee M.J.
      • Couvillon A.
      • Trepel J.B.
      • Miyata Y.
      • Matts R.
      • Neckers L.
      Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.
      ,
      • Zuehlke A.D.
      • Reidy M.
      • Lin C.
      • LaPointe P.
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      • Lee D.J.
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      • Beebe K.
      • Prince T.
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      • Xu W.
      • Johnson J.
      • Masison D.
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      An Hsp90 co-chaperone protein in yeast is functionally replaced by site-specific posttranslational modification in humans.
      ,
      • Li J.
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      • Fang B.
      • Bai Y.
      • Edwards A.
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      • Superti-Furga G.
      • Koomen J.
      • Haura E.B.
      A chemical and phosphoproteomic characterization of dasatinib action in lung cancer.
      AcetylationK41 (§,u)K36 (u)
      • Li X.
      • Robbins N.
      • O'Meara T.R.
      • Cowen L.E.
      Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31.
      • Lamoth F.
      • Juvvadi P.R.
      • Soderblom E.J.
      • Moseley M.A.
      • Asfaw Y.G.
      • Steinbach W.J.
      Identification of a key lysine residue in heat shock protein 90 required for azole and echinocandin resistance in Aspergillus fumigatus.
      • Wu D.
      • Gu Q.
      • Zhao N.
      • Xia F.
      • Li Z.
      Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis.
      K69 (u)K64 (u)HAT p300
      • Beli P.
      • Lukashchuk N.
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      • Weinert B.T.
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      • Baskcomb L.
      • Mann M.
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      Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response.
      ,
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      • Fiskus W.
      • Rao R.
      • Fernandez P.
      • Herger B.
      • Yang Y.
      • Chen J.
      • Kolhe R.
      • Mandawat A.
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      • Joshi R.
      • Eaton K.
      • Lee P.
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      • Peiper S.
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      Molecular and biologic characterization and drug sensitivity of pan-histone deacetylase inhibitor-resistant acute myeloid leukemia cells.
      ,
      • Wang Y.
      • Fiskus W.
      • Chong D.G.
      • Buckley K.M.
      • Natarajan K.
      • Rao R.
      • Joshi A.
      • Balusu R.
      • Koul S.
      • Chen J.
      • Savoie A.
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      • Jillella A.P.
      • Atadja P.
      • Levine R.L.
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      Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells.
      • Choudhary C.
      • Kumar C.
      • Gnad F.
      • Nielsen M.L.
      • Rehman M.
      • Walther T.C.
      • Olsen J.V.
      • Mann M.
      Lysine acetylation targets protein complexes and co-regulates major cellular functions.
      K74 (u,g)K69 (u)
      • Mertins P.
      • Qiao J.W.
      • Patel J.
      • Udeshi N.D.
      • Clauser K.R.
      • Mani D.R.
      • Burgess M.W.
      • Gillette M.A.
      • Jaffe J.D.
      • Carr S.A.
      Integrated proteomic analysis of post-translational modifications by serial enrichment.
      ,
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      K100 (u,g)K95HAT p300
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      K292 (u)K284 (u)HAT p300
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      ,
      • Choudhary C.
      • Kumar C.
      • Gnad F.
      • Nielsen M.L.
      • Rehman M.
      • Walther T.C.
      • Olsen J.V.
      • Mann M.
      Lysine acetylation targets protein complexes and co-regulates major cellular functions.
      K294 (†,§,u)K286 (u)HDAC6
      • He Q.
      • Liu K.
      • Tian Z.
      • Du S.J.
      The effects of Hsp90α1 mutations on myosin thick filament organization.
      ,
      • Li X.
      • Robbins N.
      • O'Meara T.R.
      • Cowen L.E.
      Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31.
      ,
      • Lamoth F.
      • Juvvadi P.R.
      • Soderblom E.J.
      • Moseley M.A.
      • Asfaw Y.G.
      • Steinbach W.J.
      Identification of a key lysine residue in heat shock protein 90 required for azole and echinocandin resistance in Aspergillus fumigatus.
      ,
      • Scroggins B.T.
      • Robzyk K.
      • Wang D.
      • Marcu M.G.
      • Tsutsumi S.
      • Beebe K.
      • Cotter R.J.
      • Felts S.
      • Toft D.
      • Karnitz L.
      • Rosen N.
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      An acetylation site in the middle domain of Hsp90 regulates chaperone function.
      • Yang C.
      • Rahimpour S.
      • Lu J.
      • Pacak K.
      • Ikejiri B.
      • Brady R.O.
      • Zhuang Z.
      Histone deacetylase inhibitors increase glucocerebrosidase activity in Gaucher disease by modulation of molecular chaperones.
      ,
      • Jochems J.
      • Teegarden S.L.
      • Chen Y.
      • Boulden J.
      • Challis C.
      • Ben-Dor G.A.
      • Kim S.F.
      • Berton O.
      Enhancement of stress resilience through histone deacetylase 6-mediated regulation of glucocorticoid receptor chaperone dynamics.
      ,
      • Jiménez-Canino R.
      • Lorenzo-Diaz F.
      • Jaisser F.
      • Farman N.
      • Giraldez T.
      • Alvarez de la Rosa D.
      Histone deacetylase 6-controlled Hsp90 acetylation significantly alters mineralocorticoid receptor subcellular dynamics but not its transcriptional activity.
      • Deskin B.
      • Lasky J.
      • Zhuang Y.
      • Shan B.
      Requirement of HDAC6 for activation of Notch1 by TGF-β1.
      K327 (u)K319 (u)HAT p300
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ,
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      K407 (u)K399 (u)
      • Choudhary C.
      • Kumar C.
      • Gnad F.
      • Nielsen M.L.
      • Rehman M.
      • Walther T.C.
      • Olsen J.V.
      • Mann M.
      Lysine acetylation targets protein complexes and co-regulates major cellular functions.
      ,
      • Woodford M.R.
      • Hughes M.
      • Sager R.A.
      • Backe S.J.
      • Baker-Williams A.J.
      • Bratslavsky M.S.
      • Jacob J.M.
      • Shapiro O.
      • Wong M.
      • Bratslavsky G.
      • Bourboulia D.
      • Mollapour M.
      Mutation of the co-chaperone Tsc1 in bladder cancer diminishes Hsp90 acetylation and reduces drug sensitivity and selectivity.
      K419 (u)K411
      • Woodford M.R.
      • Hughes M.
      • Sager R.A.
      • Backe S.J.
      • Baker-Williams A.J.
      • Bratslavsky M.S.
      • Jacob J.M.
      • Shapiro O.
      • Wong M.
      • Bratslavsky G.
      • Bourboulia D.
      • Mollapour M.
      Mutation of the co-chaperone Tsc1 in bladder cancer diminishes Hsp90 acetylation and reduces drug sensitivity and selectivity.
      ,
      • Weinert B.T.
      • Schölz C.
      • Wagner S.A.
      • Iesmantavicius V.
      • Su D.
      • Daniel J.A.
      • Choudhary C.
      Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.
      K478 (u,g)S470HAT p300
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      K546 (u,g,sc)K538 (u,sc)HAT p300
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      ,
      • Weinert B.T.
      • Schölz C.
      • Wagner S.A.
      • Iesmantavicius V.
      • Su D.
      • Daniel J.A.
      • Choudhary C.
      Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.
      K558 (u)K550HAT p300
      • Yang Y.
      • Rao R.
      • Shen J.
      • Tang Y.
      • Fiskus W.
      • Nechtman J.
      • Atadja P.
      • Bhalla K.
      Role of acetylation and extracellular location of heat shock protein 90α in tumor cell invasion.
      MonomethylationK209 (†,u)K204SMYD2
      • He Q.
      • Liu K.
      • Tian Z.
      • Du S.J.
      The effects of Hsp90α1 mutations on myosin thick filament organization.
      ,
      • Abu-Farha M.
      • Lanouette S.
      • Elisma F.
      • Tremblay V.
      • Butson J.
      • Figeys D.
      • Couture J.F.
      Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2.
      K539K531 (a,u)SMYD2
      • Hamamoto R.
      • Toyokawa G.
      • Nakakido M.
      • Ueda K.
      • Nakamura Y.
      SMYD2-dependent HSP90 methylation promotes cancer cell proliferation by regulating the chaperone complex formation.
      K582K574 (a,u)SMYD2
      • Hamamoto R.
      • Toyokawa G.
      • Nakakido M.
      • Ueda K.
      • Nakamura Y.
      SMYD2-dependent HSP90 methylation promotes cancer cell proliferation by regulating the chaperone complex formation.
      K615 (*,†,a,u)K607SMYD2
      • He Q.
      • Liu K.
      • Tian Z.
      • Du S.J.
      The effects of Hsp90α1 mutations on myosin thick filament organization.
      ,
      • Abu-Farha M.
      • Lanouette S.
      • Elisma F.
      • Tremblay V.
      • Butson J.
      • Figeys D.
      • Couture J.F.
      Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2.
      ,
      • Rehn A.
      • Lawatscheck J.
      • Jokisch M.L.
      • Mader S.L.
      • Luo Q.
      • Tippel F.
      • Blank B.
      • Richter K.
      • Lang K.
      • Kaila V.R.I.
      • Buchner J.
      A methylated lysine is a switch point for conformational communication in the chaperone Hsp90.
      • Donlin L.T.
      • Andresen C.
      • Just S.
      • Rudensky E.
      • Pappas C.T.
      • Kruger M.
      • Jacobs E.Y.
      • Unger A.
      • Zieseniss A.
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      • Voelkel T.
      • Chait B.T.
      • Gregorio C.C.
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      Smyd2 controls cytoplasmic lysine methylation of Hsp90 and myofilament organization.
      • Olsen J.B.
      • Cao X.J.
      • Han B.
      • Chen L.H.
      • Horvath A.
      • Richardson T.I.
      • Campbell R.M.
      • Garcia B.A.
      • Nguyen H.
      Quantitative profiling of the activity of protein lysine methyltransferase SMYD2 using SILAC-based proteomics.
      ThiocarbamylationC529C5216-HITC-ME
      • Shibata T.
      • Kimura Y.
      • Mukai A.
      • Mori H.
      • Ito S.
      • Asaka Y.
      • Oe S.
      • Tanaka H.
      • Takahashi T.
      • Uchida K.
      Transthiocarbamoylation of proteins by thiolated isothiocyanates.
      C597 (sn)C589 (sn)STCA
      • Zhang Y.
      • Dayalan Naidu S.
      • Samarasinghe K.
      • Van Hecke G.C.
      • Pheely A.
      • Boronina T.N.
      • Cole R.N.
      • Benjamin I.J.
      • Cole P.A.
      • Ahn Y.H.
      • Dinkova-Kostova A.T.
      Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation.
      S-NitrosylationC597 (t)C589 (t)Nitric oxide
      • Martínez-Ruiz A.
      • Villanueva L.
      • González de Orduña C.
      • López-Ferrer D.
      • Higueras M.A.
      • Tarín C.
      • Rodríguez-Crespo I.
      • Vázquez J.
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      S-Nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities.
      ,
      • Retzlaff M.
      • Stahl M.
      • Eberl H.C.
      • Lagleder S.
      • Beck J.
      • Kessler H.
      • Buchner J.
      Hsp90 is regulated by a switch point in the C-terminal domain.
      SUMOylationK191 (a,u)K186SUMO-1
      • Mollapour M.
      • Bourboulia D.
      • Beebe K.
      • Woodford M.R.
      • Polier S.
      • Hoang A.
      • Chelluri R.
      • Li Y.
      • Guo A.
      • Lee M.J.
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      • Khan S.
      • Prince T.
      • Miyajima N.
      • Yoshida S.
      • et al.
      Asymmetric Hsp90 N domain SUMOylation recruits Aha1 and ATP-competitive inhibitors.
      K559 (u)K551SUMO peptidase sentrin/SUMO-specific protease 2 (SENP2)
      • Preuss K.D.
      • Pfreundschuh M.
      • Weigert M.
      • Fadle N.
      • Regitz E.
      • Kubuschok B.
      Sumoylated HSP90 is a dominantly inherited plasma cell dyscrasias risk factor.
      UbiquitinationK112 (g)K107CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      • Povlsen L.K.
      • Beli P.
      • Wagner S.A.
      • Poulsen S.L.
      • Sylvestersen K.B.
      • Poulsen J.W.
      • Nielsen M.L.
      • Bekker-Jensen S.
      • Mailand N.
      • Choudhary C.
      Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass.
      K209 (m)K204 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • 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.
      K224 (a)K219 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • 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.
      K283 (a,g)K275 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • 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.
      K292 (a)K284 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • 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.
      R355K347 (a,m,sc)CHIP
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      K407 (a)K399 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • 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.
      K485 (a)K477 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      K489 (a,m,sc)K481 (a,sc)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • 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.
      K546 (a,g,sc)K538 (a,sc)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      • Povlsen L.K.
      • Beli P.
      • Wagner S.A.
      • Poulsen S.L.
      • Sylvestersen K.B.
      • Poulsen J.W.
      • Nielsen M.L.
      • Bekker-Jensen S.
      • Mailand N.
      • Choudhary C.
      Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass.
      K558 (a)K550CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      K615 (a,m)K607 (a)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • Danielsen J.M.
      • Sylvestersen K.B.
      • Bekker-Jensen S.
      • Szklarczyk D.
      • Poulsen J.W.
      • Horn H.
      • Jensen L.J.
      • Mailand N.
      • Nielsen M.L.
      Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level.
      K631 (a,sc)K623 (a,sc)CHIP
      • Akimov V.
      • Barrio-Hernandez I.
      • Hansen S.V.F.
      • Hallenborg P.
      • Pedersen A.K.
      • Bekker-Jensen D.B.
      • Puglia M.
      • Christensen S.D.K.
      • Vanselow J.T.
      • Nielsen M.M.
      • Kratchmarova I.
      • Kelstrup C.D.
      • Olsen J.V.
      • Blagoev B.
      UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.
      ,
      • Kundrat L.
      • Regan L.
      Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP.
      ,
      • Udeshi N.D.
      • Svinkina T.
      • Mertins P.
      • Kuhn E.
      • Mani D.R.
      • Qiao J.W.
      • Carr S.A.
      Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments.
      NitrationY38 (p)Y33 (p)
      • Franco M.C.
      • Ye Y.
      • Refakis C.A.
      • Feldman J.L.
      • Stokes A.L.
      • Basso M.
      • Melero Fernández de Mera R.M.
      • Sparrow N.A.
      • Calingasan N.Y.
      • Kiaei M.
      • Rhoads T.W.
      • Ma T.C.
      • Grumet M.
      • Barnes S.
      • Beal M.F.
      • et al.
      Nitration of Hsp90 induces cell death.
      ,
      • Franco M.C.
      • Ricart K.C.
      • Gonzalez A.S.
      • Dennys C.N.
      • Nelson P.A.
      • Janes M.S.
      • Mehl R.A.
      • Landar A.
      • Estévez A.G.
      Nitration of Hsp90 on tyrosine 33 regulates mitochondrial metabolism.
      Y61 (p)Y56 (p)
      • Franco M.C.
      • Ye Y.
      • Refakis C.A.
      • Feldman J.L.
      • Stokes A.L.
      • Basso M.
      • Melero Fernández de Mera R.M.
      • Sparrow N.A.
      • Calingasan N.Y.
      • Kiaei M.
      • Rhoads T.W.
      • Ma T.C.
      • Grumet M.
      • Barnes S.
      • Beal M.F.
      • et al.
      Nitration of Hsp90 induces cell death.
      O-GlcNAcylationS442 (p)S434 (p)
      • Overath T.
      • Kuckelkorn U.
      • Henklein P.
      • Strehl B.
      • Bonar D.
      • Kloss A.
      • Siele D.
      • Kloetzel P.M.
      • Janek K.
      Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.
      S460 (p)S452 (p)
      • Overath T.
      • Kuckelkorn U.
      • Henklein P.
      • Strehl B.
      • Bonar D.
      • Kloss A.
      • Siele D.
      • Kloetzel P.M.
      • Janek K.
      Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.
      Figure thumbnail gr3
      Figure 3Schematic representation of Hsp90α PTM sites. Residues that have been functionally studied and found to contribute to the chaperone code are shown in red with colored circles indicating effects on cellular function (blue), co-chaperone binding (orange), ATP binding (yellow), and Hsp90 inhibitor binding/sensitivity (green). Residues in black have been reported to be modified but have not yet been validated to affect chaperone function.
      Phosphorylation of Hsp90α-T90 by protein kinase A (PKA) also alters the complement of co-chaperones bound to Hsp90 and decreases Hsp90 affinity for ATP (
      • Wang X.
      • Lu X.A.
      • Song X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Luo Y.
      Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.
      ). Phosphomimetic mutation of Hsp90α-T90 amplified binding to the co-chaperones Aha1, p23, PP5, and C terminus of Hsp70-interacting protein (CHIP) but diminished binding to Hsp70, Cdc37, and HOP. In contrast, phosphorylation of residues in the Hsp90α C-domain by the kinases CK2, CK1, and glycogen synthase kinase 3β (GSK3β) increased interaction between Hsp90α and HOP while decreasing interaction with CHIP (
      • 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.
      ).
      Hsp90 phosphorylation also has a consequent impact on client binding and activation. Hsp90α-T90 phosphorylation abrogated binding to the client kinases Src, Akt, and PKCγ likely as a result of diminished interaction between Hsp90 and Cdc37 (
      • Wang X.
      • Lu X.A.
      • Song X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Luo Y.
      Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.
      ). Interestingly, phosphorylation of yHsp90-T101 (hHsp90-T115) by the dual-specificity kinase Mps1 promoted kinase client activation, whereas the nonphosphorylatable alanine mutation favored nonkinase clients, possibly due to altered co-chaperone interactions (
      • Woodford M.R.
      • Truman A.W.
      • Dunn D.M.
      • Jensen S.M.
      • Cotran R.
      • Bullard R.
      • Abouelleil M.
      • Beebe K.
      • Wolfgeher D.
      • Wierzbicki S.
      • Post D.E.
      • Caza T.
      • Tsutsumi S.
      • Panaretou B.
      • Kron S.J.
      • et al.
      Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors.
      ). Furthermore, phosphorylation of two residues in the charged linker, Hsp90α-S231 and -S263, was reduced in cells expressing a truncated form of the co-chaperone p23 (
      • Woo S.H.
      • An S.
      • Lee H.C.
      • Jin H.O.
      • Seo S.K.
      • Yoo D.H.
      • Lee K.H.
      • Rhee C.H.
      • Choi E.J.
      • Hong S.I.
      • Park I.C.
      A truncated form of p23 down-regulates telomerase activity via disruption of Hsp90 function.
      ). Phosphorylation at these residues was important for telomerase activity through stability of the hTERT telomerase catalytic subunit, which is an Hsp90 client (
      • Woo S.H.
      • An S.
      • Lee H.C.
      • Jin H.O.
      • Seo S.K.
      • Yoo D.H.
      • Lee K.H.
      • Rhee C.H.
      • Choi E.J.
      • Hong S.I.
      • Park I.C.
      A truncated form of p23 down-regulates telomerase activity via disruption of Hsp90 function.
      ).
      Phosphatases also play a critical role in modulating Hsp90 phosphorylation status and therefore the chaperone cycle. The co-chaperone Ppt1 (yeast ortholog of PP5) was shown to directly dephosphorylate Hsp90 and consequently affected its chaperone activity (
      • Wandinger S.K.
      • Suhre M.H.
      • Wegele H.
      • Buchner J.
      The phosphatase Ppt1 is a dedicated regulator of the molecular chaperone Hsp90.
      ). Another study found 10 residues of yHsp90 that are dephosphorylated by Ppt1 (
      • Soroka J.
      • Wandinger S.K.
      • Mäusbacher N.
      • Schreiber T.
      • Richter K.
      • Daub H.
      • Buchner J.
      Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.
      ). The phosphorylation status of these residues differentially influenced ATPase activity as well as client activity. Interestingly, Hsp90-S485E phosphomimetic mutant had the most robustly decreased ATPase activity despite being distant from the ATP-binding pocket, suggesting that phosphorylation of this residue may cause distant structural changes (
      • Soroka J.
      • Wandinger S.K.
      • Mäusbacher N.
      • Schreiber T.
      • Richter K.
      • Daub H.
      • Buchner J.
      Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.
      ).
      Taken together, phosphorylation is a dynamic regulatory mechanism on the Hsp90 chaperone cycle. Of note, when these sites are modified there is generally a resultant decrease in Hsp90 ATPase activity. Phosphorylation can additionally allosterically affect co-chaperone and client dynamics at binding sites far from the modified site, suggesting a complex interplay of communication across the chaperone protein. Furthermore, these allosteric effects on co-chaperone dynamics often have further consequences on Hsp90 ATPase activity, and the details of these regulatory mechanisms are not yet fully understood.

      Cell cycle control

      Hsp90 interacts with numerous cell cycle regulators, including the kinases CDK2, -4, and -6; Mps1 and Swe1 (yeast ortholog of human Wee1); and cyclin B (
      • Woodford M.R.
      • Truman A.W.
      • Dunn D.M.
      • Jensen S.M.
      • Cotran R.
      • Bullard R.
      • Abouelleil M.
      • Beebe K.
      • Wolfgeher D.
      • Wierzbicki S.
      • Post D.E.
      • Caza T.
      • Tsutsumi S.
      • Panaretou B.
      • Kron S.J.
      • et al.
      Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors.
      ,
      • Mollapour M.
      • Tsutsumi S.
      • Donnelly A.C.
      • Beebe K.
      • Tokita M.J.
      • Lee M.J.
      • Lee S.
      • Morra G.
      • Bourboulia D.
      • Scroggins B.T.
      • Colombo G.
      • Blagg B.S.
      • Panaretou B.
      • Stetler-Stevenson W.G.
      • Trepel J.B.
      • et al.
      Swe1Wee1-dependent tyrosine phosphorylation of Hsp90 regulates distinct facets of chaperone function.
      ,
      • Hallett S.T.
      • Pastok M.W.
      • Morgan R.M.L.
      • Wittner A.
      • Blundell K.
      • Felletar I.
      • Wedge S.R.
      • Prodromou C.
      • Noble M.E.M.
      • Pearl L.H.
      • Endicott J.A.
      Differential regulation of G1 CDK complexes by the Hsp90-Cdc37 chaperone system.
      ,
      • Prince T.
      • Sun L.
      • Matts R.L.
      Cdk2: a genuine protein kinase client of Hsp90 and Cdc37.
      ,
      • Basto R.
      • Gergely F.
      • Draviam V.M.
      • Ohkura H.
      • Liley K.
      • Raff J.W.
      Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle in Drosophila and humans.
      ). Hsp90α-T90 phosphorylation was found to be more abundant in actively proliferating cells (
      • Wang X.
      • Lu X.A.
      • Song X.
      • Zhuo W.
      • Jia L.
      • Jiang Y.
      • Luo Y.
      Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.
      ). Phosphorylation specifically by PKA on Hsp90-T90 also promoted prostate cancer cell proliferation (
      • Dagar M.
      • Singh J.P.
      • Dagar G.
      • Tyagi R.K.
      • Bagchi G.
      Phosphorylation of HSP90 by protein kinase A is essential for the nuclear translocation of androgen receptor.
      ). Additionally, Hsp90α-T725 and -S726 and Hsp90β-S718 phosphomimetic mutations decreased the doubling time of HEK293 cells (
      • 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.
      ). Furthermore, Hsp90 threonine phosphorylation levels fluctuate throughout the cell cycle (
      • Woodford M.R.
      • Truman A.W.
      • Dunn D.M.
      • Jensen S.M.
      • Cotran R.
      • Bullard R.
      • Abouelleil M.
      • Beebe K.
      • Wolfgeher D.
      • Wierzbicki S.
      • Post D.E.
      • Caza T.
      • Tsutsumi S.
      • Panaretou B.
      • Kron S.J.
      • et al.
      Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors.
      ). Yeast Hsp90-T101 (hHsp90α-T115) was phosphorylated throughout mitosis, but Hsp90-T101 phosphorylation was absent in G1 phase. The cell cycle mediators Mps1 and Cdc14 phosphorylate and dephosphorylate Hsp90-T101, respectively. Together, Mps1 and Cdc14 regulate mitotic arrest and exit from mitosis, at least in part, via Hsp90 phosphorylation (
      • Woodford M.R.
      • Truman A.W.
      • Dunn D.M.
      • Jensen S.M.
      • Cotran R.
      • Bullard R.
      • Abouelleil M.
      • Beebe K.
      • Wolfgeher D.
      • Wierzbicki S.
      • Post D.E.
      • Caza T.
      • Tsutsumi S.
      • Panaretou B.
      • Kron S.J.
      • et al.
      Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors.
      ).

      DNA repair and apoptosis

      DNA-dependent protein kinase (DNA-PK) is both a client and a regulator of Hsp90α that phosphorylates Hsp90α on Thr-5 and -7 (
      • Solier S.
      • Kohn K.W.
      • Scroggins B.
      • Xu W.
      • Trepel J.
      • Neckers L.
      • Pommier Y.
      Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response.
      ,
      • Park S.J.
      • Gavrilova O.
      • Brown A.L.
      • Soto J.E.
      • Bremner S.
      • Kim J.
      • Xu X.
      • Yang S.
      • Um J.H.
      • Koch L.G.
      • Britton S.L.
      • Lieber R.L.
      • Philp A.
      • Baar K.
      • Kohama S.G.
      • et al.
      DNA-PK promotes the mitochondrial, metabolic, and physical decline that occurs during aging.
      ,
      • Quanz M.
      • Herbette A.
      • Sayarath M.
      • de Koning L.
      • Dubois T.
      • Sun J.S.
      • Dutreix M.
      Heat shock protein 90α (Hsp90α) is phosphorylated in response to DNA damage and accumulates in repair foci.
      ). Phosphorylation of these residues occurred early in apoptosis and was critical for histone γH2AX formation at dsDNA breaks, DNA fragmentation, and apoptotic body formation (
      • Solier S.
      • Kohn K.W.
      • Scroggins B.
      • Xu W.
      • Trepel J.
      • Neckers L.
      • Pommier Y.
      Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response.
      ). Furthermore, Thr-7 phosphorylation in the cytosol was a prerequisite for Hsp90α accumulation at DNA double-strand breaks and subsequent formation of DNA repair foci. Hsp90α-T7 phosphorylation level was also found to correlate with the apoptotic marker pH2AX in tumors, suggesting that Hsp90α-T7 phosphorylation could serve as a potential marker for DNA damage (
      • Quanz M.
      • Herbette A.
      • Sayarath M.
      • de Koning L.
      • Dubois T.
      • Sun J.S.
      • Dutreix M.
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