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The double lives of phosphatases of regenerating liver: A structural view of their catalytic and noncatalytic activities

Open AccessPublished:December 07, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101471
      Phosphatases of regenerating liver (PRLs) are protein phosphatases involved in the control of cell growth and migration. They are known to promote cancer metastasis but, despite over 20 years of study, there is still no consensus about their mechanism of action. Recent work has revealed that PRLs lead double lives, acting both as catalytically active enzymes and as pseudophosphatases. The three known PRLs belong to the large family of cysteine phosphatases that form a phosphocysteine intermediate during catalysis. Uniquely to PRLs, this intermediate is stable, with a lifetime measured in hours. As a consequence, PRLs have very little phosphatase activity. Independently, PRLs also act as pseudophosphatases by binding CNNM membrane proteins to regulate magnesium homeostasis. In this function, an aspartic acid from CNNM inserts into the phosphatase catalytic site of PRLs, mimicking a substrate–enzyme interaction. The delineation of PRL pseudophosphatase and phosphatase activities in vivo was impossible until the recent identification of PRL mutants defective in one activity or the other. These mutants showed that CNNM binding was sufficient for PRL oncogenicity in one model of metastasis, but left unresolved its role in other contexts. As the presence of phosphocysteine prevents CNNM binding and CNNM-binding blocks catalytic activity, these two activities are inherently linked. Additional studies are needed to untangle the intertwined catalytic and noncatalytic functions of PRLs. Here, we review the current understanding of the structure and biophysical properties of PRL phosphatases.

      Keywords

      Abbreviations:

      CBP (cysteine-based phosphatase), CBS (cystathionine-β-synthase), CDC14 (cell division cycle 14), DUSP (dual-specificity phosphatase), PRL (Phosphatase of regenerating liver), PTP (protein tyrosine phosphatase)
      Phosphatases and kinases regulate the phosphorylation state of molecules involved in cell proliferation, adhesion, migration, differentiation, survival, and apoptosis, etc (
      • Alonso A.
      • Sasin J.
      • Bottini N.
      • Friedberg I.
      • Friedberg I.
      • Osterman A.
      • Godzik A.
      • Hunter T.
      • Dixon J.
      • Mustelin T.
      Protein tyrosine phosphatases in the human genome.
      ). Cysteine-based phosphatases (CBPs) constitute the largest family of phosphatases and include protein tyrosine phosphatases (PTPs), dual-specificity phosphatases (DUSPs) as well as lipid phosphatases such as PTEN (
      • Chen M.J.
      • Dixon J.E.
      • Manning G.
      Genomics and evolution of protein phosphatases.
      ,
      • Tonks N.K.
      Protein tyrosine phosphatases: From genes, to function, to disease.
      ). Structurally, the catalytic portion of CBPs consists of a domain of roughly 150 residues present either alone or with other domains. The first CBPs identified were in the PTP family, which has led to the incongruous use of the term to refer to all members despite widely differing substrate specificities. The roughly 100 human CBPs also include a small number of pseudophosphatases, which have lost their catalytic activity through evolution and act through the direct binding of proteins.
      Here, we review structural aspects of a unique family of protein phosphatases that have both catalytic and noncatalytic functions. Termed phosphatases of regenerating liver (PRLs) due to their initial discovery in the liver (
      • Diamond R.H.
      • Cressman D.E.
      • Laz T.M.
      • Abrams C.S.
      • Taub R.
      PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth.
      ), their biological function has remained incompletely understood for over 25 years. Biochemically, PRLs exhibit very unusual enzyme kinetics and tightly bind to a family of membrane ion transporters. The interpretation of the catalytic and noncatalytic functions has divided the research literature on PRLs. We present different hypotheses to bridge the gap with perspectives for future studies.

      PRLs are members of CBP superfamily

      The three mammalian PRLs are highly related in their amino acid sequences. PRL-1 and PRL-2 are the most similar with 88% identity while PRL-1 and PRL-3 share 79% identity. The proteins are classified as class IVa protein tyrosine phosphatases, leading to their gene designations: PTP4A1, PTP4A2, and PTP4A3. The phosphatases are often considered to be DUSPs, which are phosphatases that act on both phosphotyrosine and phosphothreonine/serine. Among CBPs, the PRL phosphatases are most similar to cell division cycle 14 (CDC14) phosphatases and share approximately 30% sequence identity. Structurally, they consist of a single catalytic domain followed by an unfolded C-terminal tail of ∼20-residues capped with a CAAX farnesylation motif (Fig. 1A). Among protein phosphatases, the C-terminal farnesylation is unique to PRLs and serves to localize them to cellular membranes including the plasma membrane (
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      Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases.
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      Mouse PRL-2 and PRL-3, two potentially prenylated protein tyrosine phosphatases homologous to PRL-1.
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      Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome.
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      Subcellular localization of intracellular protein tyrosine phosphatases in T cells.
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      Phosphatase activity, trimerization, and the C-terminal polybasic region are all required for PRL1-mediated cell growth and migration.
      ).
      Figure thumbnail gr1
      Figure 1Structure and sequence of PRL phosphatases. A, conserved elements include catalytic residues present in all CBPs (yellow) and features unique to PRLs (cyan). B, secondary structure of PRLs and key residues in the catalytic site, numbered according to PRL-1/3. C, catalytic mechanism of CBPs. Cysteine in its ionized form attacks the phosphorus atom to form a phosphocysteine intermediate, which is hydrolyzed to regenerate the active enzyme (
      • Zhang Z.Y.
      • Wang Y.
      • Dixon J.E.
      Dissecting the catalytic mechanism of protein-tyrosine phosphatases.
      ,
      • Denu J.M.
      • Dixon J.E.
      Protein tyrosine phosphatases: Mechanisms of catalysis and regulation.
      ). The serine/threonine residue present in other CBPs is replaced by an alanine in PRLs (
      • Kozlov G.
      • Cheng J.
      • Ziomek E.
      • Banville D.
      • Gehring K.
      • Ekiel I.
      Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.
      ). D, phylogenetic tree of PRLs.

      PRL function

      PRLs have been implicated in a wide variety of cellular functions. In mammals, PRLs appear to be primarily involved in regulation of cell proliferation and migration. PRL-1 and -2 have broad tissue distributions while PRL3 is normally largely restricted to muscle (
      • Hardy S.
      • Kostantin E.
      • Hatzihristidis T.
      • Zolotarov Y.
      • Uetani N.
      • Tremblay M.L.
      Physiological and oncogenic roles of the PRL phosphatases.
      ). Much of the interest in PRLs arises from their overexpression in cancer. PRL-3 is highly upregulated in metastatic colon cancer and is associated with poor prognosis in many types of cancer (
      • Saha S.
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      • Velculescu V.E.
      • Rago C.
      • St Croix B.
      • Romans K.E.
      • Choti M.A.
      • Lengauer C.
      • Kinzler K.W.
      • Vogelstein B.
      A phosphatase associated with metastasis of colorectal cancer.
      ). PRL-1 and PRL-2 overexpression is less prevalent in human cancers, but they have been shown to be oncogenic in cell and animal model systems (reviewed in (
      • Hardy S.
      • Kostantin E.
      • Hatzihristidis T.
      • Zolotarov Y.
      • Uetani N.
      • Tremblay M.L.
      Physiological and oncogenic roles of the PRL phosphatases.
      ,
      • Campbell A.M.
      • Zhang Z.Y.
      Phosphatase of regenerating liver: A novel target for cancer therapy.
      )).
      The genetic knockout of the PRLs is viable in mice (
      • Dong Y.
      • Zhang L.
      • Zhang S.
      • Bai Y.
      • Chen H.
      • Sun X.
      • Yong W.
      • Li W.
      • Colvin S.C.
      • Rhodes S.J.
      • Shou W.
      • Zhang Z.Y.
      Phosphatase of regenerating liver 2 (PRL2) is essential for placental development by down-regulating PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) and activating Akt protein.
      ,
      • Bai Y.
      • Zhou H.M.
      • Zhang L.
      • Dong Y.
      • Zeng Q.
      • Shou W.
      • Zhang Z.Y.
      Role of phosphatase of regenerating liver 1 (PRL1) in spermatogenesis.
      ,
      • Yan H.
      • Kong D.
      • Ge X.
      • Gao X.
      • Han X.
      Generation of conditional knockout alleles for PRL-3.
      ,
      • Zimmerman M.W.
      • Homanics G.E.
      • Lazo J.S.
      Targeted deletion of the metastasis-associated phosphatase Ptp4a3 (PRL-3) suppresses murine colon cancer.
      ,
      • Sacchetti C.
      • Bai Y.
      • Stanford S.M.
      • Di Benedetto P.
      • Cipriani P.
      • Santelli E.
      • Piera-Velazquez S.
      • Chernitskiy V.
      • Kiosses W.B.
      • Ceponis A.
      • Kaestner K.H.
      • Boin F.
      • Jimenez S.A.
      • Giacomelli R.
      • Zhang Z.Y.
      • et al.
      PTP4A1 promotes TGFbeta signaling and fibrosis in systemic sclerosis.
      ). This is likely due to complementation between the highly related genes as the double knockout of PRL-1 and PRL-2 is embryonic lethal (
      • Dong Y.
      • Zhang L.
      • Bai Y.
      • Zhou H.M.
      • Campbell A.M.
      • Chen H.
      • Yong W.
      • Zhang W.
      • Zeng Q.
      • Shou W.
      • Zhang Z.Y.
      Phosphatase of regenerating liver 2 (PRL2) deficiency impairs kit signaling and spermatogenesis.
      ). PRL-2−/− mice show defects in placental development and spermatogenesis, which are accentuated with partial (heterozygotic) loss of PRL-1. In zebrafish, PRL-3 expression affects melanocyte stem cell differentiation (
      • Johansson J.A.
      • Marie K.L.
      • Lu Y.
      • Brombin A.
      • Santoriello C.
      • Zeng Z.
      • Zich J.
      • Gautier P.
      • von Kriegsheim A.
      • Brunsdon H.
      • Wheeler A.P.
      • Dreger M.
      • Houston D.R.
      • Dooley C.M.
      • Sims A.H.
      • et al.
      PRL3-DDX21 transcriptional control of endolysosomal genes restricts melanocyte stem cell differentiation.
      ).
      PRLs have been implicated in many signaling pathways, reviewed in (
      • Hardy S.
      • Kostantin E.
      • Hatzihristidis T.
      • Zolotarov Y.
      • Uetani N.
      • Tremblay M.L.
      Physiological and oncogenic roles of the PRL phosphatases.
      ,
      • Campbell A.M.
      • Zhang Z.Y.
      Phosphatase of regenerating liver: A novel target for cancer therapy.
      ,
      • Rubio T.
      • Kohn M.
      Regulatory mechanisms of phosphatase of regenerating liver (PRL)-3.
      ,
      • Duciel L.
      • Monraz Gomez L.C.
      • Kondratova M.
      • Kuperstein I.
      • Saule S.
      The phosphatase PRL-3 is involved in key steps of cancer metastasis.
      ,
      • Abdollahi P.
      • Vandsemb E.N.
      • Børset M.
      Phosphatases of regenerating liver are key regulators of metabolism in cancer cells - role of Serine/Glycine metabolism.
      ). The PTEN/PI3K/Akt cancer pathway is the most frequently mentioned although there are papers suggesting the involvement of the Rho-family GTPase, Src, p53, STAT3/5, cKit/Notch, and Ras/MAPK pathways, among others. In the majority of studies, PRLs were proposed to act via dephosphorylation with the substrates identified from coimmunoprecipitation experiments and changes in protein phosphorylation in cells. Confirmation of the substrates has been difficult due to the low activity of PRLs in vitro. As an example, Li et al. (
      • Li Q.
      • Bai Y.
      • Lyle L.T.
      • Yu G.
      • Amarasinghe O.
      • Nguele Meke F.
      • Carlock C.
      • Zhang Z.Y.
      Mechanism of PRL2 phosphatase-mediated PTEN degradation and tumorigenesis.
      ) identified phosphorylated PTEN as a substrate of PRL-2 based on coimmunoprecipitation assays and decreased phosphorylation in cells when PRL-2 is overexpressed. However, direct measurements of phosphatase activity with purified components measured very low turnover numbers, suggesting that additional factors, such as membrane localization or unidentified cofactors, are required for full phosphatase activity (
      • Li Q.
      • Bai Y.
      • Lyle L.T.
      • Yu G.
      • Amarasinghe O.
      • Nguele Meke F.
      • Carlock C.
      • Zhang Z.Y.
      Mechanism of PRL2 phosphatase-mediated PTEN degradation and tumorigenesis.
      ). PRLs have also been suggested to be lipid phosphatases. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was dephosphorylated by PRL-3 in two different biochemical assays (
      • McParland V.
      • Varsano G.
      • Li X.
      • Thornton J.
      • Baby J.
      • Aravind A.
      • Meyer C.
      • Pavic K.
      • Rios P.
      • Kohn M.
      The metastasis-promoting phosphatase PRL-3 shows activity toward phosphoinositides.
      ). Given the membrane localization and involvement of PRLs in the PTEN/PI3K/Akt pathway, PTEN and PI(4,5)P2 are compelling substrates from a biological standpoint, but their identification requires further confirmation.

      PRL structure

      The first atomic structure of a PRL phosphatase (PRL-3) was solved by solution NMR in 2004 (
      • Kozlov G.
      • Cheng J.
      • Ziomek E.
      • Banville D.
      • Gehring K.
      • Ekiel I.
      Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.
      ,
      • Kim K.A.
      • Song J.S.
      • Jee J.
      • Sheen M.R.
      • Lee C.
      • Lee T.G.
      • Ro S.
      • Cho J.M.
      • Lee W.
      • Yamazaki T.
      • Jeon Y.H.
      • Cheong C.
      Structure of human PRL-3, the phosphatase associated with cancer metastasis.
      ) followed by X-ray crystal structures of PRL-1 in 2005 (
      • Sun J.P.
      • Wang W.Q.
      • Yang H.
      • Liu S.
      • Liang F.
      • Fedorov A.A.
      • Almo S.C.
      • Zhang Z.Y.
      Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion.
      ,
      • Jeong D.G.
      • Kim S.J.
      • Kim J.H.
      • Son J.H.
      • Park M.R.
      • Lim S.M.
      • Yoon T.S.
      • Ryu S.E.
      Trimeric structure of PRL-1 phosphatase reveals an active enzyme conformation and regulation mechanisms.
      ). At present, almost 20 structures are known, either alone or in complexes (Table 1). PRL phosphatases have the typical CBP-fold consisting of a five-stranded central β-sheet covered by two α-helices on one side and a four-helical bundle on the other (Fig. 1B). The β-sheet is mostly parallel with only one antiparallel strand (β1).
      Table 1Structures of PRL phosphatases
      Protein/complexSpeciesPDB codeResolution (Å)Comments
      PRL-1
       PRL-1 (1–173)H. sapiens1XM22.7C104S mutant; trimeric
       PRL-1 (7–160)R. norvegicus1ZCK1.9oxidized form; trimeric
       PRL-1 (1–160)R. norvegicus1ZCL2.9C104S mutant; trimeric
       PRL-1 (1–160)R. norvegicus1X243.2oxidized form; trimeric
       PRL-1 (1–160)/peptideR. norvegicus3RZ22.8oxidized form; trimeric
       PRL-1 (8–160)/compound 3H. sapiens5BX11.9oxidized form
       PRL-1 (1–173)/CBS CNNM2M. musculus5LXQ3.33oxidized form
       PRL-1 (1–173)/CBS CNNM2M. musculus5MMZ2.4oxidized form
       PRL-1 (7–160)/CBS CNNM2H. sapiens6WUS2.76C104D mutant
      PRL-2
       PRL-2 (1–163)/CBS CNNM3H. sapiens5K223.0reduced form
       PRL-2 (1–167)/CBS CNNM3H. sapiens5K232.96oxidized form
       PRL-2 (1–163)/CBS CNNM3M. musculus5K243.1oxidized form
       PRL-2 (1–167)/CBS CNNM3/ADPH. sapiens5K253.05oxidized form
       PRL-2 (1–167)/CBS CNNM3H. sapiens6WUR2.88C101D mutant
      PRL-3
       PRL-3 (1–169)H. sapiens1R6Hn/aNMR structure
       PRL-3 (1–173)H. sapiens1V3An/aNMR structure
       PRL-3 (1–162)H. sapiens2MBCn/aNMR structure; vanadate complex
       PRL-3 (1–169)/CBS CNNM3H. sapiens5TSR3.19C104A mutant
      Nonmammalian PRL
       PRL (1–163)Leishmania3S4O2.3oxidized form
      The first structures of PRL-1 showed the protein crystallized as a trimer (
      • Sun J.P.
      • Wang W.Q.
      • Yang H.
      • Liu S.
      • Liang F.
      • Fedorov A.A.
      • Almo S.C.
      • Zhang Z.Y.
      Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion.
      ,
      • Jeong D.G.
      • Kim S.J.
      • Kim J.H.
      • Son J.H.
      • Park M.R.
      • Lim S.M.
      • Yoon T.S.
      • Ryu S.E.
      Trimeric structure of PRL-1 phosphatase reveals an active enzyme conformation and regulation mechanisms.
      ). This led to the hypothesis that PRLs exist as trimers in vivo. Although oligomers have been detected in cells by chemical crosslinking, their physiological relevance remains unclear. Trimers have not been observed in structures of other PRLs (Table 1).
      The PRL catalytic site is highly conserved and formed by residues from two loops. The phosphate-binding loop (P-loop) connecting strand β5 and helix α4 contains an arginine that coordinates phosphate binding and a cysteine (C104 in PRL-3) that forms a phosphocysteine intermediate as part of the catalytic cycle. In the acidic loop between strand β4 and helix α3, an aspartic acid acts both as a general acid and a base during the two-step catalytic cycle (Fig. 1C). At physiological pH, the catalytic cysteine is deprotonated due to the spatial alignment of the sulfur with the dipole of the following α4 helix. Ionization of the cysteine is required for catalysis but also increases the propensity of the cysteine to form a disulfide with an adjacent cysteine (C49 in PRL-3). PRLs readily crystallize as disulfides and, in fact, only one PRL crystal structure has the catalytic cysteine in its reduced form.
      To initiate catalysis, the aspartic acid acts as a general acid to prime the phosphorus atom for nucleophilic attack by the cysteine and release of the dephosphorylated product. In the second step, the aspartic acid, now acting as a general base, activates a water molecule for hydrolysis of the phosphocysteine intermediate. In CBPs other than PRLs, attack of the water is facilitated by a serine or threonine residue from the P-loop. In PRLs, this residue is always alanine (A111 in PRL-3). This serves to stabilize the phosphocysteine intermediate and is a unique feature of the PRL family. The phosphocysteine intermediate has been visualized by X-ray crystallography in the CBP family phosphatase PTP1B (
      • Pannifer A.D.
      • Flint A.J.
      • Tonks N.K.
      • Barford D.
      Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by x-ray crystallography.
      ).

      Defining features and phylogeny

      Within the CBP family of phosphatases, PRLs are distinguished by their farnesylation site and two features of the catalytic site: the alanine in the P-loop and the second cysteine residue (also present in DUSPs). PRLs from different species show the greatest divergence in the length of the loop between helix α3 and strand β5, which is far from the active site and unlikely to affect the distinctive catalytic properties of PRLs.
      PRL phosphatases are absent in prokaryotes but occur widely in eukaryotes from unicellular organisms to mammals (
      • Lin M.D.
      • Lee H.T.
      • Wang S.C.
      • Li H.R.
      • Hsien H.L.
      • Cheng K.W.
      • Chang Y.D.
      • Huang M.L.
      • Yu J.K.
      • Chen Y.H.
      Expression of phosphatase of regenerating liver family genes during embryogenesis: An evolutionary developmental analysis among Drosophila, amphioxus, and zebrafish.
      ) (Fig. 1D). While many fungi species (such as S. cerevisiae and C. albicans) do not contain PRLs, their presence in algae, unicellular parasites, and some fungi points to their development early in evolution. Most vertebrates contain three PRLs. Phylogenetic analysis suggests that gene duplication occurred early as the isoforms from different vertebrates are evolutionarily more related to each other than to isoforms in the same species. The proliferation of forms also occurred independently in Leishmania and Trypanosoma protozoan parasites, which contain two or three PRL proteins, respectively.
      Invertebrates generally contain only a single PRL gene. The PRL paralog in flies has received attention with recent reports that it plays developmental and protective roles in the nervous system (
      • Guo P.
      • Xu X.
      • Wang F.
      • Yuan X.
      • Tu Y.
      • Zhang B.
      • Zheng H.
      • Yu D.
      • Ge W.
      • Gong Z.
      • Yang X.
      • Xi Y.
      A novel neuroprotective role of phosphatase of regenerating liver-1 against CO2 stimulation in Drosophila.
      ,
      • Urwyler O.
      • Izadifar A.
      • Vandenbogaerde S.
      • Sachse S.
      • Misbaer A.
      • Schmucker D.
      Branch-restricted localization of phosphatase Prl-1 specifies axonal synaptogenesis domains.
      ,
      • Kula-Eversole E.
      • Lee D.H.
      • Samba I.
      • Yildirim E.
      • Levine D.C.
      • Hong H.K.
      • Lear B.C.
      • Bass J.
      • Rosbash M.
      • Allada R.
      Phosphatase of regenerating liver-1 selectively times circadian behavior in darkness via function in PDF neurons and dephosphorylation of TIMELESS.
      ). In worms, PRL was shown to signal through a lysosomal Ca2+ channel, TRPML (
      • Funato Y.
      • Yoshida A.
      • Hirata Y.
      • Hashizume O.
      • Yamazaki D.
      • Miki H.
      The oncogenic PRL protein causes acid addiction of cells by stimulating lysosomal exocytosis.
      ). Leishmania and trypanosoma have multiple PRL genes, which have been suggested to contribute to the intracellular survival of the parasites (
      • Cuevas I.C.
      • Rohloff P.
      • Sanchez D.O.
      • Docampo R.
      Characterization of farnesylated protein tyrosine phosphatase TcPRL-1 from Trypanosoma cruzi.
      • Leitherer S.
      • Clos J.
      • Liebler-Tenorio E.M.
      • Schleicher U.
      • Bogdan C.
      • Soulat D.
      Characterization of the protein tyrosine phosphatase LmPRL-1 secreted by Leishmania major via the exosome pathway.
      ).

      Kinetics of phosphatase activity

      CBPs, including PRLs, catalyze the dephosphorylation of substrates through a two-step reaction: (i) formation of a phosphoenzyme intermediate and release of the dephosphorylated substrate, and (ii) hydrolysis of phosphocysteine and release of free phosphate (Fig. 2A) (
      • Denu J.M.
      • Stuckey J.A.
      • Saper M.A.
      • Dixon J.E.
      Form and function in protein dephosphorylation.
      ). The two-step reaction leads to generation of a phosphoenzyme intermediate and a phenomenon known as burst kinetics. The existence of two steps has been observed in multiple CBPs but, in general, it is not detectable due to the rapid breakdown of the phosphoenzyme intermediate relative to formation (k3 >> k2). When the opposite occurs (k2 >> k3), there is an accumulation of the phosphoenzyme intermediate, which gives rise to burst kinetics: an initial burst of activity followed by a slow steady-state rate (
      • Johnson K.A.
      Transient-state kinetic analysis of enzyme reaction pathways.
      ). The size of the burst step is stoichiometric with the amount of enzyme while the steady-state rate depends on the rate of the second step in the catalytic cycle (Fig. 2C).
      Figure thumbnail gr2
      Figure 2Enzyme kinetics of PRLs. A, phosphatase activity proceeds in two steps with a phospho-enzyme intermediate (E∗). B, burst kinetics visualized at two different timescales. Stopped-flow traces of phosphatase activity of 3 μM PRL-2 measured with a fluorogenic substrate. Comparison of the slope of initial (inset) and steady-state curves reveals a roughly 400-fold difference in reaction rates. C, kinetic terms for a two-step enzyme mechanism (
      • Johnson K.A.
      Transient-state kinetic analysis of enzyme reaction pathways.
      ). When the k3 is larger than k2, the equations simplify to the usual Michael–Menton kinetics. When k3 is smaller, the turnover rate (kcat) reduces to k3 and the KM is decreased by the burst ratio (k2/k3). Paradoxically, the enzyme appears to have higher affinity for substrates. D, detection of the phosphoenzyme intermediate by SDS-PAGE with the Phostag reagent that slows the migration of phosphorylated proteins. PRLs become fully phosphorylated in the presence of the synthetic substrate OMFP. Phosphocysteine is hydrolyzed by boiling and dependent on the catalytic cysteine (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ). E, phosphorus NMR detection of phosphocysteine in PRL-2 confirms the hours-long lifetime (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ).
      PRLs are unusual in that the rate of hydrolysis of the phosphoenzyme intermediate (k3) is several orders of magnitude slower than for other CBPs. Typical values for k3 of protein phosphatases are in the range of 10 to 0.1 s−1 while, for PRLs, the rate is <1 h−1 (
      • Gottlin E.B.
      • Xu X.
      • Epstein D.M.
      • Burke S.P.
      • Eckstein J.W.
      • Ballou D.P.
      • Dixon J.E.
      Kinetic analysis of the catalytic domain of human cdc25B.
      ,
      • Zhang Z.Y.
      • VanEtten R.L.
      Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
      ,
      • Zhang Z.Y.
      • Zhou G.
      • Denu J.M.
      • Wu L.
      • Tang X.
      • Mondesert O.
      • Russell P.
      • Butch E.
      • Guan K.L.
      Purification and characterization of the low molecular weight protein tyrosine phosphatase, Stp1, from the fission yeast Schizosaccharomyces pombe.
      ,
      • Wang J.
      • Walsh C.T.
      Mechanistic studies on full length and the catalytic domain of the tandem SH2 domain-containing protein tyrosine phosphatase: Analysis of phosphoenzyme levels and Vmax stimulatory effects of glycerol and of a phosphotyrosyl peptide ligand.
      ,
      • Zhang Z.Y.
      Kinetic and mechanistic characterization of a mammalian protein-tyrosine phosphatase, PTP1.
      ,
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ). The ratio of k2 divided by k3 provides a measure of the burst-like character. For PRL-2, the ratio is roughly 400 compared with values of 10 or less for other CBPs. The stability of the PRL phosphoenzyme intermediate is, in part, due to the absence of serine or threonine residue in the P-loop (Fig. 1C). Replacement of the alanine (A111 in PRL-3) with serine increases the rate of steady-state catalysis, eliminating much of the burst effect (
      • Kozlov G.
      • Cheng J.
      • Ziomek E.
      • Banville D.
      • Gehring K.
      • Ekiel I.
      Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.
      ). Conversely, when Denu et al. replaced the naturally occurring serine with alanine in VHR (vaccinia H1-related) phosphatase, the mutant enzyme showed slowed hydrolysis, which allowed the detection of phosphocysteine by NMR spectroscopy (
      • Denu J.M.
      • Dixon J.E.
      A catalytic mechanism for the dual-specific phosphatases.
      • Denu J.M.
      • Lohse D.L.
      • Vijayalakshmi J.
      • Saper M.A.
      • Dixon J.E.
      Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.
      ). The strict conservation of alanine in PRLs in different species suggests that the stability of the phosphocysteine intermediate is a general feature of PRLs that has been maintained during evolution.
      Although the existence of PRL burst kinetics has been known for many years (
      • Kozlov G.
      • Cheng J.
      • Ziomek E.
      • Banville D.
      • Gehring K.
      • Ekiel I.
      Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.
      ,
      • Sun J.P.
      • Wang W.Q.
      • Yang H.
      • Liu S.
      • Liang F.
      • Fedorov A.A.
      • Almo S.C.
      • Zhang Z.Y.
      Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion.
      ), it is often overlooked. Typical phosphatase assays use a small amount of enzyme compared with substrate, so the initial burst of phosphatase activity is small and transient. However, the existence of the burst has major consequences for the interpretation of the kinetic constants. During the steady-state condition, PRLs exhibit classical Michaelis–Menten kinetics with the extra reaction rate (k3) folded into the values for the Michaelis constant (KM) and kcat (Fig. 2C) (
      • Johnson K.A.
      Transient-state kinetic analysis of enzyme reaction pathways.
      ). This means that it is not possible to detect the two-step kinetic cycle from simply measuring the steady-state rate at different substrate concentrations. When k3 is much larger than k2, the kinetic terms simplify to the standard forms where KM is (k2 + k1)/k1 and kcat is k2. On the other hand, when k2 is larger than k3, the kcat reduces to k3 and the apparent KM is decreased by dividing by the burst ratio (k2/k3). This has the curious effect of improving the apparent affinity of enzymes with burst kinetics for their substrates.
      The remarkable stability of the PRL phosphocysteine intermediate leads to almost complete conversion to the phosphoenzyme form when substrate is present (Fig. 2D). At saturation, the ratio of the two forms is equal to the burst ratio. Detection of the phosphoenzyme intermediate is most easily accomplished by SDS-PAGE gel electrophoresis with the Phostag reagent that decreases migration of phosphorylated proteins (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ). The instability of phosphocysteine allows the presence of phosphocysteine to be confirmed by heating prior to electrophoresis. Phosphocysteine can also be detected by its phosphorus NMR signal, which has been used to measure its half-life (Fig. 2E) (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ).
      A common misconception about PRLs is that the low steady-state activity is due the absence of a true, bona fide substrate. As the Vmax depends only on the rate of hydrolysis (k3), it is by nature independent of the substrate. At saturation, all substrates have the same turnover number. This may explain why attempts to identify PRL substrates have failed to reach a clear consensus. While these issues do not apply to the burst phase, it is difficult to observe and frequently ignored in assays of PRL activity. As a result, PRLs appear to have low activity and ill-defined substrate specificity.
      PRL phosphatase activity is surprisingly easy to detect if the level of PRL cysteine phosphorylation is measured instead of substrate dephosphorylation. Phostag gel analysis of PRL-3 allowed detection of phosphatase activity even with nonphysiological substrates (
      • Zhang H.
      • Kozlov G.
      • Li X.
      • Wu H.
      • Gulerez I.
      • Gehring K.
      PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3.
      ). Low levels of phosphoenzyme intermediate formed when PRL-3 was incubated with a variety of low-molecular-weight diphosphate and triphosphate-containing compounds. This likely explains the observation that the PRLs are partially phosphorylated when purified from E. coli, even though the bacteria are unlikely to possess PRL-specific substrates (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Zhang H.
      • Kozlov G.
      • Li X.
      • Wu H.
      • Gulerez I.
      • Gehring K.
      PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3.
      ).

      Interactions with CNNM proteins

      A major step forward in understanding the function of PRLs was the discovery that they tightly bind CNNM (CBS-pair domain divalent metal cation transport mediators) proteins (
      • Funato Y.
      • Yamazaki D.
      • Mizukami S.
      • Du L.
      • Kikuchi K.
      • Miki H.
      Membrane protein CNNM4-dependent Mg2+ efflux suppresses tumor progression.
      ,
      • Hardy S.
      • Uetani N.
      • Wong N.
      • Kostantin E.
      • Labbe D.P.
      • Begin L.R.
      • Mes-Masson A.
      • Miranda-Saavedra D.
      • Tremblay M.L.
      The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis.
      ) (Fig. 3A). CNNM proteins are membrane proteins that mediate magnesium transport and are associated with the diseases of magnesium metabolism: familial dominant hypomagnesemia (
      • Stuiver M.
      • Lainez S.
      • Will C.
      • Terryn S.
      • Gunzel D.
      • Debaix H.
      • Sommer K.
      • Kopplin K.
      • Thumfart J.
      • Kampik N.B.
      • Querfeld U.
      • Willnow T.E.
      • Nemec V.
      • Wagner C.A.
      • Hoenderop J.G.
      • et al.
      CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.
      ) and Jalili syndrome (
      • Parry D.A.
      • Mighell A.J.
      • El-Sayed W.
      • Shore R.C.
      • Jalili I.K.
      • Dollfus H.
      • Bloch-Zupan A.
      • Carlos R.
      • Carr I.M.
      • Downey L.M.
      • Blain K.M.
      • Mansfield D.C.
      • Shahrabi M.
      • Heidari M.
      • Aref P.
      • et al.
      Mutations in CNNM4 cause Jalili syndrome, consisting of autosomal-recessive cone-rod dystrophy and amelogenesis imperfecta.
      ). CNNMs have also been implicated in growth control and energy metabolism through regulation of magnesium levels (
      • Funato Y.
      • Yamazaki D.
      • Mizukami S.
      • Du L.
      • Kikuchi K.
      • Miki H.
      Membrane protein CNNM4-dependent Mg2+ efflux suppresses tumor progression.
      ,
      • Hardy S.
      • Uetani N.
      • Wong N.
      • Kostantin E.
      • Labbe D.P.
      • Begin L.R.
      • Mes-Masson A.
      • Miranda-Saavedra D.
      • Tremblay M.L.
      The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis.
      ,
      • Yamazaki D.
      • Funato Y.
      • Miura J.
      • Sato S.
      • Toyosawa S.
      • Furutani K.
      • Kurachi Y.
      • Omori Y.
      • Furukawa T.
      • Tsuda T.
      • Kuwabata S.
      • Mizukami S.
      • Kikuchi K.
      • Miki H.
      Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: A mouse model.
      ). Studies of CNNM magnesium transport showed that PRL binding inhibits transport (
      • Funato Y.
      • Yamazaki D.
      • Mizukami S.
      • Du L.
      • Kikuchi K.
      • Miki H.
      Membrane protein CNNM4-dependent Mg2+ efflux suppresses tumor progression.
      ,
      • Hirata Y.
      • Funato Y.
      • Takano Y.
      • Miki H.
      Mg2+-dependent interactions of ATP with the cystathionine-beta-synthase (CBS) domains of a magnesium transporter.
      ) suggesting that PRL oncogenicity is a consequence of the growth-promoting effects of elevated cytoplasmic magnesium levels (
      • Rubin H.
      The logic of the Membrane, Magnesium, Mitosis (MMM) model for the regulation of animal cell proliferation.
      ,
      • Trapani V.
      • Wolf F.I.
      Dysregulation of Mg(2+) homeostasis contributes to acquisition of cancer hallmarks.
      ). A role for PRLs in magnesium homeostasis was confirmed by observations that PRL levels are regulated by magnesium availability, diet, and circadian rhythms (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Hardy S.
      • Uetani N.
      • Wong N.
      • Kostantin E.
      • Labbe D.P.
      • Begin L.R.
      • Mes-Masson A.
      • Miranda-Saavedra D.
      • Tremblay M.L.
      The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis.
      ,
      • Uetani N.
      • Hardy S.
      • Gravel S.P.
      • Kiessling S.
      • Pietrobon A.
      • Wong N.N.
      • Chenard V.
      • Cermakian N.
      • St-Pierre J.
      • Tremblay M.L.
      PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms.
      ,
      • Gungabeesoon J.
      • Tremblay M.L.
      • Uetani N.
      Localizing PRL-2 expression and determining the effects of dietary Mg(2+) on expression levels.
      ,
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ,
      • Hardy S.
      • Kostantin E.
      • Wang S.J.
      • Hristova T.
      • Galicia-Vazquez G.
      • Baranov P.V.
      • Pelletier J.
      • Tremblay M.L.
      Magnesium-sensitive upstream ORF controls PRL phosphatase expression to mediate energy metabolism.
      ,
      • Yoshida A.
      • Funato Y.
      • Miki H.
      Phosphatase of regenerating liver maintains cellular magnesium homeostasis.
      ).
      Figure thumbnail gr3
      Figure 3CNNM proteins bind PRLs via the catalytic site. A, model of PRL bound to a Mg2+ transporter CNNM protein. The model is based on structures of bacterial and human CNNM proteins and their complexes with PRLs (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Zhang H.
      • Kozlov G.
      • Li X.
      • Wu H.
      • Gulerez I.
      • Gehring K.
      PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3.
      ,
      • Gimenez-Mascarell P.
      • Gonzalez-Recio I.
      • Fernandez-Rodriguez C.
      • Oyenarte I.
      • Muller D.
      • Martinez-Chantar M.L.
      • Martinez-Cruz L.A.
      Current structural knowledge on the CNNM family of magnesium transport mediators.
      ,
      • Gimenez-Mascarell P.
      • Oyenarte I.
      • Hardy S.
      • Breiderhoff T.
      • Stuiver M.
      • Kostantin E.
      • Diercks T.
      • Pey A.L.
      • Ereno-Orbea J.
      • Martinez-Chantar M.L.
      • Khalaf-Nazzal R.
      • Claverie-Martin F.
      • Muller D.
      • Tremblay M.L.
      • Martinez-Cruz L.A.
      Structural basis of the oncogenic interaction of phosphatase PRL-1 with the magnesium transporter CNNM2.
      ,
      • Chen Y.S.
      • Kozlov G.
      • Fakih R.
      • Yang M.
      • Zhang Z.
      • Kovrigin E.L.
      • Gehring K.
      Mg(2+)-ATP sensing in CNNM, a putative magnesium transporter.
      ,
      • Huang Y.
      • Jin F.
      • Funato Y.
      • Xu Z.
      • Zhu W.
      • Wang J.
      • Sun M.
      • Zhao Y.
      • Yu Y.
      • Miki H.
      • Hattori M.
      Structural basis for the Mg(2+) recognition and regulation of the CorC Mg(2+) transporter.
      ,
      • Chen Y.S.
      • Kozlov G.
      • Fakih R.
      • Funato Y.
      • Miki H.
      • Gehring K.
      The cyclic nucleotide–binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity.
      ,
      • Corral-Rodriguez M.A.
      • Stuiver M.
      • Abascal-Palacios G.
      • Diercks T.
      • Oyenarte I.
      • Ereno-Orbea J.
      • de Opakua A.I.
      • Blanco F.J.
      • Encinar J.A.
      • Spiwok V.
      • Terashima H.
      • Accardi A.
      • Muller D.
      • Martinez-Cruz L.A.
      Nucleotide binding triggers a conformational change of the CBS module of the magnesium transporter CNNM2 from a twisted towards a flat structure.
      ,
      • Chen Y.S.
      • Kozlov G.
      • Moeller B.E.
      • Rohaim A.
      • Fakih R.
      • Roux B.
      • Burke J.E.
      • Gehring K.
      Crystal structure of an archaeal CorB magnesium transporter.
      ). B, the PRL catalytic site controls the affinity of CNNM binding. A negative charge from either the catalytic cysteine or a substituted aspartic acid is required for high affinity (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). Oxidation or phosphorylation of the cysteine decrease or prevent binding (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ). C, CNNM binding, but not phosphatase activity, is required for PRL-3 promotion of lung metastases by B16 melanoma cells (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). The PRL-3 C104D and R138E mutants are reciprocally deficient in either catalytic activity or CNNM binding while the C104E mutant is doubly deficient. D, phosphorylation of PRL-1 and -2 in vivo. The extent of phosphorylation changes in different culture media and tissues (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ).
      There are four CNNM proteins in humans, CNNM1, CNNM2, CNNM3, and CNNM4, composed of an uncharacterized N-terminal extracellular domain, a transmembrane DUF21 domain, a CBS-pair (cystathionine-β-synthase) domain, and cyclic nucleotide-binding homology (CNBH) C-terminal domain (
      • Funato Y.
      • Miki H.
      Molecular function and biological importance of CNNM family Mg2+ transporters.
      ,
      • Gimenez-Mascarell P.
      • Gonzalez-Recio I.
      • Fernandez-Rodriguez C.
      • Oyenarte I.
      • Muller D.
      • Martinez-Chantar M.L.
      • Martinez-Cruz L.A.
      Current structural knowledge on the CNNM family of magnesium transport mediators.
      ). The proteins are dimers and mediate magnesium transport through the conserved transmembrane domain. The CBS-pair domains regulate transport activity via binding of magnesium, nucleotides, and PRLs. Structurally, CBS-pair domains are composed of two CBS motifs with a mixed αβ-fold and an ATP-binding site at the dimerization interface.

      PRL-CNNM complexes

      Nine structures of complexes of PRL and CNNM CBS-pair domains have been determined (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Zhang H.
      • Kozlov G.
      • Li X.
      • Wu H.
      • Gulerez I.
      • Gehring K.
      PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3.
      ,
      • Gimenez-Mascarell P.
      • Oyenarte I.
      • Hardy S.
      • Breiderhoff T.
      • Stuiver M.
      • Kostantin E.
      • Diercks T.
      • Pey A.L.
      • Ereno-Orbea J.
      • Martinez-Chantar M.L.
      • Khalaf-Nazzal R.
      • Claverie-Martin F.
      • Muller D.
      • Tremblay M.L.
      • Martinez-Cruz L.A.
      Structural basis of the oncogenic interaction of phosphatase PRL-1 with the magnesium transporter CNNM2.
      ) (Fig. 3A). The first structure was PRL-2 bound to the CBS-pair domain from CNNM3 and showed that binding is mediated by an extended CNNM loop that inserts into the PRL catalytic site (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ). Subsequent structures with other PRLs and CBS-pair domains showed that the recognition mechanism is shared across all members of both families (Table 1). The most important binding interaction is between a CNNM aspartic acid and the PRL arginine in the P-loop. The negative charge of the aspartic acid carboxyl group mimics the phosphate group of a substrate and is positioned above the catalytic cysteine by hydrogen bonds with the positively charged arginine. Mutation of either the aspartic acid or arginine residue completely prevents binding.
      Isothermal titration calorimetry measurements have shown that there is no specificity in the interactions between the different PRL and CNNM isoforms in vitro. All three PRLs bind CBS-pair domains, and the interaction loop is conserved across the four members of the CNNM family. The binding affinities are roughly 10 nM and very sensitive to modifications of the catalytic site (Fig. 3B) (
      • Zhang H.
      • Kozlov G.
      • Li X.
      • Wu H.
      • Gulerez I.
      • Gehring K.
      PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3.
      ,
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). Substitution of the catalytic cysteine by either alanine or serine decreases the affinity as does disulfide bond formation (
      • Zhang H.
      • Kozlov G.
      • Li X.
      • Wu H.
      • Gulerez I.
      • Gehring K.
      PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3.
      ,
      • Gimenez-Mascarell P.
      • Oyenarte I.
      • Hardy S.
      • Breiderhoff T.
      • Stuiver M.
      • Kostantin E.
      • Diercks T.
      • Pey A.L.
      • Ereno-Orbea J.
      • Martinez-Chantar M.L.
      • Khalaf-Nazzal R.
      • Claverie-Martin F.
      • Muller D.
      • Tremblay M.L.
      • Martinez-Cruz L.A.
      Structural basis of the oncogenic interaction of phosphatase PRL-1 with the magnesium transporter CNNM2.
      ) (Fig. 3B). Despite this, all the crystal structures of complexes of PRL and CNNM CBS-pair domains except one contain either mutations of the catalytic cysteine or the cysteine disulfide. This occurs because, even with a 100-fold loss of affinity, the protein concentrations used to crystallize the complexes are higher than the Kd of binding.

      Pseudophosphatase activity

      Confirmation that CNNM binding rather than phosphatase activity is responsible for some of the biological functions of PRLs required the identification of mutations that selectively inactivate phosphatase or pseudophosphatase activity. The majority of mutations in the catalytic site disrupt both functions. The PRL-3 C104S mutation has been widely used as a negative control. Finding a mutant that blocks CNNM binding was straightforward as there are protein–protein contacts outside the catalytic site that are required for binding. Finding the reciprocal mutation was more difficult, but eventually it was observed that substitution of the catalytic cysteine by aspartic acid retained binding while inactivating catalytic activity (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). Not typically considered a conservative substitution, the aspartic acid preserves the negative charge of the thiolate in the active site (Fig. 3B). Remarkably, the cysteine-to-aspartic acid mutation increases the thermal stability of PRLs by 8 to 10 °C due to relaxation of the conformational strain required to shift the cysteine pKa in the wild-type enzyme (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ).
      The PRL-3 C104D mutation provided the first unambiguous evidence that PRL pseudophosphatase activity is responsible for a biological function (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). The mutant inhibited CNNM4-associated magnesium transport in cells and promoted tumor formation in a murine model of cancer metastasis (Fig. 3C). In contrast, the R138E mutation, which disrupts binding but not catalysis (with small-molecule substrates), was inactive in both assays. While the R138E mutation could disrupt PRL-3 interactions with protein substrates, the C104D mutation unequivocally shows that at least two biological functions of PRLs do not require catalytic activity. The importance of phosphatase activity versus CNNM binding for other functions should become clearer as researchers test these mutants in different cellular and animal models.

      Regulation of PRLs

      PRLs are regulated at the gene, mRNA, and protein level. A variety of transcriptional factors bind and regulate the transcription of PRL genes in response to different stimuli and growth signals, see (
      • Hardy S.
      • Kostantin E.
      • Hatzihristidis T.
      • Zolotarov Y.
      • Uetani N.
      • Tremblay M.L.
      Physiological and oncogenic roles of the PRL phosphatases.
      ). PRL-1 was first identified due to the increase in mRNA and protein levels in the liver following partial hepatectomy (
      • Diamond R.H.
      • Cressman D.E.
      • Laz T.M.
      • Abrams C.S.
      • Taub R.
      PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth.
      ). Similarly, PRL-3 levels are dramatically increased in metastatic colon cancer (
      • Saha S.
      • Bardelli A.
      • Buckhaults P.
      • Velculescu V.E.
      • Rago C.
      • St Croix B.
      • Romans K.E.
      • Choti M.A.
      • Lengauer C.
      • Kinzler K.W.
      • Vogelstein B.
      A phosphatase associated with metastasis of colorectal cancer.
      ). Of particular relevance, considering that PRLs bind magnesium transporters, is the increase in PRL protein levels in response to magnesium depletion in cultured cells (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Hardy S.
      • Uetani N.
      • Wong N.
      • Kostantin E.
      • Labbe D.P.
      • Begin L.R.
      • Mes-Masson A.
      • Miranda-Saavedra D.
      • Tremblay M.L.
      The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis.
      ,
      • Gungabeesoon J.
      • Tremblay M.L.
      • Uetani N.
      Localizing PRL-2 expression and determining the effects of dietary Mg(2+) on expression levels.
      ,
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ,
      • Hardy S.
      • Kostantin E.
      • Wang S.J.
      • Hristova T.
      • Galicia-Vazquez G.
      • Baranov P.V.
      • Pelletier J.
      • Tremblay M.L.
      Magnesium-sensitive upstream ORF controls PRL phosphatase expression to mediate energy metabolism.
      ,
      • Yoshida A.
      • Funato Y.
      • Miki H.
      Phosphatase of regenerating liver maintains cellular magnesium homeostasis.
      ). The change in protein levels is also accompanied by changes in the levels of cysteine phosphorylation (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ) (Fig. 3D).
      Reactive oxygen species (ROS) have been widely studied as a mechanism of regulation of CBPs, including PRLs (
      • Tonks N.K.
      Protein tyrosine phosphatases: From genes, to function, to disease.
      ,
      • Dustin C.M.
      • Heppner D.E.
      • Lin M.J.
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      Redox regulation of tyrosine kinase signalling: More than meets the eye.
      ,
      • Miki H.
      • Funato Y.
      Regulation of intracellular signalling through cysteine oxidation by reactive oxygen species.
      ). The catalytic cysteine is highly susceptible to oxidation, which can lead to temporary or permanent inactivation of catalytic activity. There are five possible oxidized forms of the sulfur ranging from addition of 1, 2, or 3 oxygen atoms to sulfenamide and disulfide bond formation (
      • Funato Y.
      • Miki H.
      Reversible oxidation of PRL family protein-tyrosine phosphatases.
      ,
      • Salmeen A.
      • Barford D.
      Functions and mechanisms of redox regulation of cysteine-based phosphatases.
      ). In addition, cysteine modifications by nitric oxide (S-nitrosylation) and sulfhydryls (persulfidation) have been reported for some phosphatases (
      • Heneberg P.
      Reactive nitrogen species and hydrogen sulfide as regulators of protein tyrosine phosphatase activity.
      ,
      • Dóka É.
      • Ida T.
      • Dagnell M.
      • Abiko Y.
      • Luong N.C.
      • Balog N.
      • Takata T.
      • Espinosa B.
      • Nishimura A.
      • Cheng Q.
      • Funato Y.
      • Miki H.
      • Fukuto J.M.
      • Prigge J.R.
      • Schmidt E.E.
      • et al.
      Control of protein function through oxidation and reduction of persulfidated states.
      ).
      Relatively little is known about the oxidation of PRLs in cells. They are easily oxidized and form disulfides even under mildly reducing conditions in vitro (
      • Kozlov G.
      • Cheng J.
      • Ziomek E.
      • Banville D.
      • Gehring K.
      • Ekiel I.
      Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.
      ,
      • Sun J.P.
      • Wang W.Q.
      • Yang H.
      • Liu S.
      • Liang F.
      • Fedorov A.A.
      • Almo S.C.
      • Zhang Z.Y.
      Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion.
      ,
      • Skinner A.L.
      • Vartia A.A.
      • Williams T.D.
      • Laurence J.S.
      Enzyme activity of phosphatase of regenerating liver is controlled by the redox environment and its C-terminal residues.
      ). High concentrations of reducing agents are required to maintain the catalytic cysteine residue fully reduced. In cells, they are partially oxidized in addition to their extensive phosphorylation (
      • Skinner A.L.
      • Vartia A.A.
      • Williams T.D.
      • Laurence J.S.
      Enzyme activity of phosphatase of regenerating liver is controlled by the redox environment and its C-terminal residues.
      ,
      • Ishii T.
      • Funato Y.
      • Miki H.
      Thioredoxin-related protein 32 (TRP32) specifically reduces oxidized phosphatase of regenerating liver (PRL).
      ). PRLs are particularly sensitive to inhibition by redox-active compounds that promote their oxidation or inhibit their reduction (
      • Ishii T.
      • Funato Y.
      • Miki H.
      Thioredoxin-related protein 32 (TRP32) specifically reduces oxidized phosphatase of regenerating liver (PRL).
      ,
      • Zhang Z.
      • Kozlov G.
      • Chen Y.S.
      • Gehring K.
      Mechanism of thienopyridone and iminothienopyridinedione inhibition of protein phosphatases.
      ,
      • Lazo J.S.
      • Blanco I.K.
      • Tasker N.R.
      • Rastelli E.J.
      • Burnett J.C.
      • Garrott S.R.
      • Hart D.J.
      • McCloud R.L.
      • Hsu K.L.
      • Wipf P.
      • Sharlow E.R.
      Next-generation cell-active inhibitors of the undrugged oncogenic PTP4A3 phosphatase.
      ).
      The biological consequences of PRL oxidation are not fully understood. Disulfide bond formation blocks both CNNM binding and catalysis, but it may also protect PRLs from overoxidation to the sulfinyl (SO2H) or sulfonyl (SO3H) forms (
      • Funato Y.
      • Miki H.
      Reversible oxidation of PRL family protein-tyrosine phosphatases.
      ,
      • Salmeen A.
      • Barford D.
      Functions and mechanisms of redox regulation of cysteine-based phosphatases.
      ). Disulfide bond formation is reversible unlike the more oxidized states. Cysteine phosphorylation could be similarly protective. Much more work is needed to characterize the modifications of PRLs and CBPs in vivo.

      PRLs as molecular switches

      The formation of phosphocysteine can be thought of as a mechanism for regulating PRL activity. It is a reversible modification, changes in response to stimuli (magnesium availability) and inhibits both catalytic activity and CNNM binding. Endogenous PRLs are stably phosphorylated in cultured cells and the levels change in response to magnesium in the culture medium (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ) (Fig. 3D). Unphosphorylated PRLs slowly accumulate upon magnesium withdrawal and are rapidly phosphorylated upon readdition of magnesium (
      • Gulerez I.
      • Funato Y.
      • Wu H.
      • Yang M.
      • Kozlov G.
      • Miki H.
      • Gehring K.
      Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.
      ,
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). PRLs are also highly phosphorylated in tissues (
      • Kozlov G.
      • Funato Y.
      • Chen Y.S.
      • Zhang Z.
      • Illes K.
      • Miki H.
      • Gehring K.
      PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.
      ). CNNMs were recently shown to bind ARL15, a member of the ARF-like family of G proteins (
      • Zolotarov Y.
      • Ma C.
      • Gonzalez-Recio I.
      • Hardy S.
      • Franken G.A.C.
      • Uetani N.
      • Latta F.
      • Kostantin E.
      • Boulais J.
      • Thibault M.P.
      • Cote J.F.
      • Diaz-Moreno I.
      • Quintana A.D.
      • Hoenderop J.G.J.
      • Martinez-Cruz L.A.
      • et al.
      ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs.
      ).

      Inhibitors of PRL phosphatase activity

      Due to their oncogenicity, PRLs have long been the target of searches for inhibitors (
      • Frankson R.
      • Yu Z.H.
      • Bai Y.
      • Li Q.
      • Zhang R.Y.
      • Zhang Z.Y.
      Therapeutic targeting of oncogenic tyrosine phosphatases.
      ,
      • Lazo J.S.
      • McQueeney K.E.
      • Burnett J.C.
      • Wipf P.
      • Sharlow E.R.
      Small molecule targeting of PTPs in cancer.
      ). This has not been an easy task due to the shallow catalytic pocket, low activity, and ill-defined substrate specificity. Pentamidine, an antiparasitic drug, was the first proposed inhibitor, but it is not specific or potent (
      • Cuevas I.C.
      • Rohloff P.
      • Sanchez D.O.
      • Docampo R.
      Characterization of farnesylated protein tyrosine phosphatase TcPRL-1 from Trypanosoma cruzi.
      ,
      • Pathak M.K.
      • Dhawan D.
      • Lindner D.J.
      • Borden E.C.
      • Farver C.
      • Yi T.
      Pentamidine is an inhibitor of PRL phosphatases with anticancer activity.
      ). Compounds based on rhodanine, a chemical group frequently involved in nonspecific interactions, have also been developed (
      • Ahn J.H.
      • Kim S.J.
      • Park W.S.
      • Cho S.Y.
      • Ha J.D.
      • Kim S.S.
      • Kang S.K.
      • Jeong D.G.
      • Jung S.K.
      • Lee S.H.
      • Kim H.M.
      • Park S.K.
      • Lee K.H.
      • Lee C.W.
      • Ryu S.E.
      • et al.
      Synthesis and biological evaluation of rhodanine derivatives as PRL-3 inhibitors.
      ,
      • Min G.
      • Lee S.K.
      • Kim H.N.
      • Han Y.M.
      • Lee R.H.
      • Jeong D.G.
      • Han D.C.
      • Kwon B.M.
      Rhodanine-based PRL-3 inhibitors blocked the migration and invasion of metastatic cancer cells.
      ). Redox-active compounds such as hydrogen peroxide are effective inhibitors both in vitro and in vivo (
      • Sun J.P.
      • Wang W.Q.
      • Yang H.
      • Liu S.
      • Liang F.
      • Fedorov A.A.
      • Almo S.C.
      • Zhang Z.Y.
      Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion.
      ,
      • Ishii T.
      • Funato Y.
      • Miki H.
      Thioredoxin-related protein 32 (TRP32) specifically reduces oxidized phosphatase of regenerating liver (PRL).
      ). Thienopyridine and a related compound, JMS-053, have been proposed to be PRL-specific inhibitors although their specificity is debated due to their ability to promote oxidation (
      • Zhang Z.
      • Kozlov G.
      • Chen Y.S.
      • Gehring K.
      Mechanism of thienopyridone and iminothienopyridinedione inhibition of protein phosphatases.
      ,
      • Lazo J.S.
      • Blanco I.K.
      • Tasker N.R.
      • Rastelli E.J.
      • Burnett J.C.
      • Garrott S.R.
      • Hart D.J.
      • McCloud R.L.
      • Hsu K.L.
      • Wipf P.
      • Sharlow E.R.
      Next-generation cell-active inhibitors of the undrugged oncogenic PTP4A3 phosphatase.
      ).
      Compounds that block protein–protein interactions rather than catalytic activity have also been proposed. Small molecules that inhibit PRL trimerization reportedly prevent cell proliferation and migration but not phosphatase activity (
      • Bai Y.
      • Yu Z.H.
      • Liu S.
      • Zhang L.
      • Zhang R.Y.
      • Zeng L.F.
      • Zhang S.
      • Zhang Z.Y.
      Novel anticancer agents based on targeting the trimer interface of the PRL phosphatase.
      ). Cai et al. (
      • Cai F.
      • Huang Y.
      • Wang M.
      • Sun M.
      • Zhao Y.
      • Hattori M.
      A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2.
      ) developed a FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL-2.
      Surprisingly, the most promising approach has been the design of antibodies against PRL-3 for use in immunotherapy to kill PRL-3 expressing tumors (
      • Thura M.
      • Al-Aidaroos A.Q.
      • Gupta A.
      • Chee C.E.
      • Lee S.C.
      • Hui K.M.
      • Li J.
      • Guan Y.K.
      • Yong W.P.
      • So J.
      • Chng W.J.
      • Ng C.H.
      • Zhou J.
      • Wang L.Z.
      • Yuen J.S.P.
      • et al.
      PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein.
      ). This unconventional approach is based on the presence of PRL-3 antigens on the cell surface, which leads to antibody-dependent cytotoxicity or phagocytosis of the tumor cells. The humanized antibody, PRL3-zumab, reduced tumor relapse in an animal model and has been approved for Phase 2 clinical trials against solid tumors in Singapore, United States, and China (
      • Thura M.
      • Ye Z.
      • Al-Aidaroos A.Q.
      • Xiong Q.
      • Ong J.Y.
      • Gupta A.
      • Li J.
      • Guo K.
      • Ang K.H.
      • Zeng Q.
      PRL3 induces polypoid giant cancer cells eliminated by PRL3-zumab to reduce tumor relapse.
      ).

      Perspectives and conclusions

      There are many unanswered questions about the biological function of PRLs but also signs that the gaps in our understanding are closing. One promising development is the realization that CNNM proteins are associated with another family of proteins, the melastatin-related transient receptor potential ion channels, TRPM6 and TRPM7 (
      • Funato Y.
      • Miki H.
      Molecular function and biological importance of CNNM family Mg2+ transporters.
      ,
      • Arjona F.J.
      • de Baaij J.H.
      • Schlingmann K.P.
      • Lameris A.L.
      • van Wijk E.
      • Flik G.
      • Regele S.
      • Korenke G.C.
      • Neophytou B.
      • Rust S.
      • Reintjes N.
      • Konrad M.
      • Bindels R.J.
      • Hoenderop J.G.
      CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia.
      ,
      • Bates-Withers C.
      • Sah R.
      • Clapham D.E.
      TRPM7, the Mg(2+) inhibited channel and kinase.
      ,
      • Funato Y.
      • Yamazaki D.
      • Okuzaki D.
      • Yamamoto N.
      • Miki H.
      Importance of the renal ion channel TRPM6 in the circadian secretion of renin to raise blood pressure.
      ,
      • Kollewe A.
      • Chubanov V.
      • Tseung F.T.
      • Correia L.
      • Schmidt E.
      • Rössig A.
      • Zierler S.
      • Haupt A.
      • Müller C.S.
      • Bildl W.
      • Schulte U.
      • Nicke A.
      • Fakler B.
      • Gudermann T.
      The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics.
      ). Like CNNMs, TRPM6/7 are important for magnesium homeostasis and have been implicated in cancer (
      • Trapani V.
      • Wolf F.I.
      Dysregulation of Mg(2+) homeostasis contributes to acquisition of cancer hallmarks.
      ,
      • Dhennin-Duthille I.
      • Gautier M.
      • Korichneva I.
      • Ouadid-Ahidouch H.
      TRPM7 involvement in cancer: A potential prognostic factor.
      ,
      • Chubanov V.
      • Mittermeier L.
      • Gudermann T.
      Role of kinase-coupled TRP channels in mineral homeostasis.
      ,
      • Meng S.
      • Alanazi R.
      • Ji D.
      • Bandura J.
      • Luo Z.W.
      • Fleig A.
      • Feng Z.P.
      • Sun H.S.
      Role of TRPM7 kinase in cancer.
      ). The convergence of these two well-established research fields promises new insights.
      Finally, while many groups still consider PRLs to function primarily as phosphatases, there is increasing acceptance that PRLs also act as pseudophosphatases and that the two activities are inherently linked. CNNM binding blocks PRL catalytic activity and PRL catalytic activity generates the phosphoenzyme intermediate that is unable to bind CNNMs. Identifying the mechanisms that link these to magnesium levels and cell signaling pathways are key challenges for the future.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgements

      This work was supported by funding from the Canadian Natural Sciences and Engineering Research Council , the Canadian Institutes of Health Research , and the Canada Research Chairs Program .

      Author contributions

      G. K., M. Y., and R. F. writing—original draft; K. G. writing—review and editing.

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