Structural Insights into Molecular Function of the Metastasis-associated Phosphatase PRL-3

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys49 that forms an intramolecular disulfide bond with the catalytic Cys104 even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity. Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys49 that forms an intramolecular disulfide bond with the catalytic Cys104 even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity. PRL (for phosphatase of regenerating liver) phosphatases constitute a novel class of small tyrosine phosphatases involved in the modulation of cell growth. Initial studies identified PRL-1 as an intermediate-early gene expressed in the early response of regenerating liver tissue to mitogens (1Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (248) Google Scholar). Overexpression of this protein was shown to lead to cellular transformation (1Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (248) Google Scholar, 2Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S.K. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (191) Google Scholar, 3Zeng Q. Dong J.M. Guo K. Li J. Tan H.X. Koh V. Pallen C.J. Manser E. Hong W. Cancer Res. 2003; 63: 2716-2722PubMed Google Scholar). The biological role of PRL-1 is tissue-dependent. Its overexpression is associated with cell proliferation in the liver (1Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (248) Google Scholar) but with differentiation of epithelial cells in the digestive system (4Diamond R.H. Peters C. Jung S.P. Greenbaum L.E. Haber B.A. Silberg D.G. Traber P.G. Taub R. Am. J. Physiol. 1996; 271: G121-G129Crossref PubMed Google Scholar). The closely related phosphatases PRL-2 and PRL-3 are also involved in growth regulation, proliferation, and cell invasion (3Zeng Q. Dong J.M. Guo K. Li J. Tan H.X. Koh V. Pallen C.J. Manser E. Hong W. Cancer Res. 2003; 63: 2716-2722PubMed Google Scholar, 5Matter W.F. Estridge T. Zhang C. Belagaje R. Stancato L. Dixon J. Johnson B. Bloem L. Pickard T. Donaghue M. Acton S. Jeyaseelan R. Kadambi V. Vlahos C.J. Biochem. Biophys. Res. Commun. 2001; 283: 1061-1068Crossref PubMed Scopus (116) Google Scholar, 6Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (158) Google Scholar). All three proteins are prenylated at their C terminus, which critically affects their cellular localization and function (6Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (158) Google Scholar, 7Zeng Q. Si X. Horstmann H. Xu Y. Hong W. Pallen C.J. J. Biol. Chem. 2000; 275: 21444-21452Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8Wang J. Kirby C.E. Herbst R. J. Biol. Chem. 2002; 277: 46659-46668Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). As shown for the human PRL-2, the role of PRLs is associated with the regulation of progression through mitosis, and their cellular localization is likely controlled by the cell cycle (8Wang J. Kirby C.E. Herbst R. J. Biol. Chem. 2002; 277: 46659-46668Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). PRL phosphatases are widely distributed in eukaryotes. In humans, PRL-1 and PRL-2 are ubiquitously expressed in various tissues (6Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (158) Google Scholar), whereas PRL-3 is normally expressed in cardiac and skeletal muscles (5Matter W.F. Estridge T. Zhang C. Belagaje R. Stancato L. Dixon J. Johnson B. Bloem L. Pickard T. Donaghue M. Acton S. Jeyaseelan R. Kadambi V. Vlahos C.J. Biochem. Biophys. Res. Commun. 2001; 283: 1061-1068Crossref PubMed Scopus (116) Google Scholar). Comprising typically only 140-180 amino acids, PRLs are among the smallest phosphatases. They consist of a single catalytic domain lacking any auxiliary docking/regulatory domains other than the prenylation site at the C terminus. PRLs contain the protein-tyrosine phosphatase (PTPase) 1The abbreviations used are: PTPase, protein-tyrosine phosphatase; DSP, dual specificity phosphatase; VHR, vaccinia H1-related phosphatase; CDC, cell division cycle phosphatase; KAP, kinase-associated phosphatase; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect correlation spectroscopy; DTT, dithiothreitol; OMFP, 3-O-methylfluorescein phosphate. active consensus motif HCXXGXXR, referred to as the P-loop; however, their primary sequence shows only remote similarity to phosphatases in other regions. Tyrosine-specific phosphatases as well as dual specificity phosphatases (DSP), enzymes capable of dephosphorylating both phosphotyrosine and phosphothreonine/serine residues, share the same general catalytic mechanism. The key structural elements include the positively charged phosphate-binding region of the P-loop and a catalytic cysteine residue that possesses an unusually low pKa of ∼5 such that its side chain exists as a thiolate at a physiological pH. During catalysis, this cysteine acts as a nucleophile to form a thiophosphoryl enzyme intermediate, and a conserved aspartic acid in a neighboring loop participates in both the formation and the hydrolysis of the phosphoenzyme intermediate. The conserved arginine in the P-loop is important for the stabilization of the transition state (for a review, see Ref. 9Zhang Z.Y. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 209-234Crossref PubMed Scopus (380) Google Scholar). In many PTPases, correct positioning of the flexible loop containing the catalytic aspartate is critical for activation and catalytic performance of these enzymes (10Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar). Recent interest in PRL phosphatases relates to their role in cell proliferation, including promotion of cell migration, invasion, and metastasis (2Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S.K. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (191) Google Scholar, 3Zeng Q. Dong J.M. Guo K. Li J. Tan H.X. Koh V. Pallen C.J. Manser E. Hong W. Cancer Res. 2003; 63: 2716-2722PubMed Google Scholar, 4Diamond R.H. Peters C. Jung S.P. Greenbaum L.E. Haber B.A. Silberg D.G. Traber P.G. Taub R. Am. J. Physiol. 1996; 271: G121-G129Crossref PubMed Google Scholar, 7Zeng Q. Si X. Horstmann H. Xu Y. Hong W. Pallen C.J. J. Biol. Chem. 2000; 275: 21444-21452Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 11Bardelli A. Saha S. Sager J.A. Romans K.E. Xin B. Markowitz S.D. Lengauer C. Velculescu V.E. Kinzler K.W. Vogelstein B. Clin. Cancer Res. 2003; 9: 5607-5615PubMed Google Scholar, 12Werner S.R. Lee P.A. DeCamp M.W. Crowell D.N. Randall S.K. Crowell P.L. Cancer Lett. 2003; 202: 201-211Crossref PubMed Scopus (74) Google Scholar). SAGE (serial analysis of gene expression) experiments showed that PRL-3 is massively overexpressed in colon tumors metastasizing to the liver but not in nonmetastatic tumors and in normal colorectal epithelium (13Saha 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. Science. 2001; 294: 1343-1346Crossref PubMed Scopus (587) Google Scholar). Further support for the involvement of PRL-3 in metastasis was provided by the finding of gene amplification in a significant fraction of metastatic lesions from different patients (13Saha 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. Science. 2001; 294: 1343-1346Crossref PubMed Scopus (587) Google Scholar). Because of the massive levels of overexpression, PRL-3 constitutes a useful marker for metastasis and possibly a new therapeutic target. More studies at both the physiological and biochemical level are needed to better understand the function of PRL phosphatases and, in particular, to evaluate their involvement and role in metastasis. Here, we determined the solution structure of PRL-3 and classify it as a member of the family of dual specificity phosphatases. The structure and site-directed mutagenesis experiments identify residues that are important for PRL-3 catalytic activity and reveal unique features that distinguish PRL-3 from other phosphatases. PRL-3 Expression and Purification—The human phosphatase PRL-3 sequence, comprising amino acids 1-169, was subcloned into the pET15b vector (Novagen Inc., Madison, WI) and overexpressed in Escherichia coli BL21(DE3) as a His-tagged fusion protein. The protein was purified as described previously (14Kozlov G. Cheng J. Lievre C. Banville D. Gehring K. Ekiel I. J. Biomol. NMR. 2002; 24: 169-170Crossref PubMed Scopus (10) Google Scholar). The resulting protein contains the from the site of the vector and not include the prenylation site PRL-3 was from cells containing with without The PRL-3 expressed and purified as described PRL-3 by site-directed mutagenesis The of the proteins by and at the protein level by All of the expressed and purified the same as for the with a protein of into DTT, and experiments at K. and side chain signal of PRL-3 determined as described previously (14Kozlov G. Cheng J. Lievre C. Banville D. Gehring K. Ekiel I. J. Biomol. NMR. 2002; 24: 169-170Crossref PubMed Scopus (10) Google Scholar). for the structure from and at and a The was for these from an H. S. A. J. Biomol. NMR. 1994; PubMed Scopus Google Scholar). and M.A. J. Biomol. NMR. Scholar) and with C. M. P. K. J. Biomol. NMR. 1995; PubMed Scopus Google Scholar). from and experiments and from The and from and G. A. J. Biomol. NMR. PubMed Scopus Google Scholar) and with resulting from an The the M. H. J. Mol. Biol. PubMed Scopus Google Scholar) in the P. J. M. T. Biol. 1998; PubMed Scopus Google Scholar). The of was for the protein and used to the first of to the as and NOESY, NOESY, and used in the to and The of used to the of The of the was J.A. M.W. R. J.M. J. Biomol. NMR. 1996; PubMed Scopus Google Scholar). The for the structure are shown in I. The with the and the with the for for structure bond from from of the from the for residues in most in in in in a new with 3-O-methylfluorescein as a at for of the Xu X. Burke S.P. Dixon J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). All of the at in containing DTT, The from to for the protein and the and determined from the as and of the was to was by the of the enzyme to the and for a The to is the is the and is the for the of the The by the of the with the The from the for the is to the enzyme The kinetic to the is the is the is the is the phosphoenzyme intermediate, and is the The was used to and the of the and to and was to of the was to of the to is and represents the catalytic of the phosphatase Xu X. Burke S.P. Dixon J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Molecular of phosphatases PRL-1 and PRL-2 by homology modeling the M.A. A. R. A. Annu. Rev. Biophys. Biomol. 2000; PubMed Scopus Google Scholar), the determined structure of PRL-3 as a St. was used for structure and was by an P. T. P.A. J. Am. Chem. 1995; Scopus Google Scholar) with the a and an The and C with and and of determined the structure of the human phosphatase PRL-3, the first of the PRL protein The previously (14Kozlov G. Cheng J. Lievre C. Banville D. Gehring K. Ekiel I. J. Biomol. NMR. 2002; 24: 169-170Crossref PubMed Scopus (10) Google Scholar) used to from and and The a of was to and These by determined from an in the PRL-3 region used to the PRL-3 The of to the The structural are shown in I. PRL-3 is of a and is with to the and The and are side of the and the and form a the side of the of the structure elements and is for DSP, PRL-3 as a member of this class of phosphatases. reveals the closest similarity to the dual specificity phosphatases VHR phosphatase and and with of and The PRL-3 and phosphatase is for the in the conserved elements of sequence with other dual specificity phosphatases and tyrosine phosphatases shows sequence which is for this class of enzymes low sequence similarity to significant structural differences are in the the active for both catalytic activity and specificity. shows an of this region in The catalytic residues Cys104 and are at of the catalytic The of conserved amino acids Cys104 and PRL-3 from other dual specificity phosphatases. The amino acids in this region shown to to specificity for phosphatases H. A. T. Y. Dixon P. Full Text Full Text PDF PubMed Scopus Google Scholar, B. W. H. M.A. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The other of the PRL-3 catalytic site is the of which in (10Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar, H. Johnson Barford D. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The terminus, which is in protein-tyrosine phosphatases and to is and in The region which for phosphatase H. Johnson Barford D. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), is and The loop is also and than in the VHR, and phosphatases (10Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar, H. Johnson Barford D. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J.M. Dixon M.A. Science. 1996; PubMed Scopus Google Scholar). The resulting catalytic is and a of specificity for is by an residue in the loop to the catalytic P-loop that as a general acid by a to the serine/threonine J.M. G. Guo Y. Dixon 1995; PubMed Scopus Google Scholar). that in the loop as a general acid for PRL-3 loop also and for its and not loop not to with other of the protein and conformational upon conformational was for and (10Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar, S. Biol. PubMed Scopus Google Scholar). is that PRL-3 the general acid residue to the catalytic cysteine to active activity was with and conserved catalytic residues Cys104 and the motif of and a of in PRL phosphatases. Because the was shown to the catalytic activity of PRL-3 (5Matter W.F. Estridge T. Zhang C. Belagaje R. Stancato L. Dixon J. Johnson B. Bloem L. Pickard T. Donaghue M. Acton S. Jeyaseelan R. Kadambi V. Vlahos C.J. Biochem. Biophys. Res. Commun. 2001; 283: 1061-1068Crossref PubMed Scopus (116) Google Scholar), mutagenesis experiments the other conserved residues of the active site such as and PRL-3 showed low activity with and the used not this a OMFP, was Xu X. Burke S.P. Dixon J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). analysis of the protein showed a with the formation of the phosphoenzyme and of for of the enzyme from the of with other phosphatases Xu X. Burke S.P. Dixon J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. Dixon S. A. 1995; PubMed Scopus Google Scholar). The for PRL-3 activity is the of the The of for the PRL-3 was which is of than that for a such as and VHR Xu X. Burke S.P. Dixon J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). both and are to determined for M.A. A. R. A. Annu. Rev. Biophys. Biomol. 2000; PubMed Scopus Google Scholar), that in the of PRL-3 not but of the phosphoenzyme is for the of kinetic analysis for the PRL-3 and the PRL-3 analysis of the showed that that for PRL-3 is not The analysis of the showed that that The for PRL-3 is not in a new most in the catalytic site of PRL-3 is the presence of an to the catalytic arginine in the tyrosine and dual specificity phosphatases a in this and the of this conserved residue is important for the of the phosphoenzyme and for the thiolate of the catalytic cysteine J.M. Dixon S. A. 1995; PubMed Scopus Google Scholar, Z.Y. B.A. L. Y. 1995; PubMed Scopus Google Scholar). The to both the and the and the used in this it was to only the of the kinetic for the the of the from the in the kinetic of the enzyme from to the of the The also showed at These that the but with a to that of and the in catalytic performance of the the of the than the same for VHR phosphatase Xu X. Burke S.P. Dixon J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). that the presence of in the catalytic site of PRL-3 only the low catalytic and of the aspartic acids and is to as a general acid in the The the to a level it not with its catalytic the other the a to that of PRL-3, that role in catalysis. PRL-3 structure reveals the presence of Cys49 in to the catalytic The disulfide bond formation these for the low catalytic activity. is important a intramolecular disulfide bond in phosphatase was even in the presence of H. Johnson Barford D. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the cysteine residue in the formation of this disulfide bond and the activity In PRL-3, the of the was to that of the that Cys49 is not for the low activity of of the reducing than used for the kinetic disulfide bond formation Cys49 and Cys104 by and shows a of a correlation of PRL-3 under mildly and reducing conditions. are present in the first to a of the and forms of The form as is and only the form is at a of also used to the of phosphatases the and forms different S.R. J. Lee C. W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (191) Google Scholar). of the PRL-3 by showed to the and forms not The a single that Cys49 is involved in disulfide bond These that PRL-3 is capable of an intramolecular disulfide Cys49 and the catalytic Cys104 in a to and S.R. J. Lee C. W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (191) Google Scholar). of of PRL-3 the the of the PRL-3 structure indicates that these from in the first and the and in the the of the to neighboring which the that the are of the dual specificity phosphatase and of PRL-3 expressed and by and and that the the structural of PRL-3, and a of activity. the in of the PRL-3 is to that only the PRL-3 is Molecular of PRL-1 and the differences in biological of modeling was used to for the human PRL-1 and PRL-2 phosphatases their sequence similarity to PRL-3 and amino acid All three phosphatases of amino acids the P-loop and The only amino acid in the of the active site that potentially to specificity is in PRL-3, which is in both PRL-1 and amino acids, which are different in and the the active shows the conserved residues in PRL phosphatases from a of eukaryotes. analysis reveals that the most highly conserved are in the of the catalytic site with a small amino acids in other regions. PRLs are a class of small phosphatases the family of They consist of a catalytic domain and a unique prenylation site that is for their cellular localization Q. Si X. Horstmann H. Xu Y. Hong W. Pallen C.J. J. Biol. Chem. 2000; 275: 21444-21452Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8Wang J. Kirby C.E. Herbst R. J. Biol. Chem. 2002; 277: 46659-46668Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The family of a low sequence but PRL phosphatases are closely sequence and human and among of the conserved amino acids a structural role are of the active site The first residues from the hydrophobic and and in elements of the The conserved catalytic which in and include the the general acid and the The sequence the catalytic site a specificity in unique features of the P-loop the catalytic and specificity of PRL phosphatases. the highly conserved amino acids the hydrophobic of the P-loop and a for hydrophobic than of other phosphatases. the sequence of the P-loop is and the residue likely a unique conformational in this critical PRL-3 not the which is for the phosphotyrosine specificity in Because of the unique of the the catalytic the catalytic of PRL-3 is the of phosphatases. The structure of the and showed tyrosine the specificity of from the active site of the catalytic domain H. Johnson Barford D. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). These also various the catalytic The loop the and of PRL-3 is to the loop in the structure and a role in the the unique with the key at its that participates in structural in sequence that is the closest structural shows that PRLs are to VHR, and The most conserved of PRLs is the residue the catalytic other and the identified T. R. T. K. Y. M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), not contain a in this In other in the P-loop the of the conserved in a than an in the catalytic activity Zhang Z.Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In the active site of PRL-3 not contain any that this is for of the low catalytic activity of PRL-3 and that the is provided by the physiological The positioning of the loop containing the general acid is of the activity a phosphatase with a of the general acid a in the was the was by a protein S. Biol. PubMed Scopus Google Scholar). PRL-3 likely a conformational to to Cys104 and catalysis. The of Cys49 in PRL phosphatases a is a interest in the of the catalytic cysteine in phosphatases and its role in the regulation of in response to T. Tonks N.K. Mol. 2002; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). Recent are for the of the catalytic cysteine side chain that its to a acid formation of an intramolecular disulfide bond R.L. M. D. R. H. 2003; PubMed Scopus Google Scholar, A. J.A. Tonks N.K. Barford D. 2003; PubMed Scopus Google Scholar). role for this disulfide is to the catalytic cysteine from a potential regulatory role of the conserved Cys49 in the of under physiological conditions. the other conserved residues in the active likely to the conserved amino acids, and possibly to in and in in is important for phosphoenzyme formation and In PRL-3, is the active site and in in a as for in the VHR phosphatase A. S. S. M. L. A. S.P. T. 2003; PubMed Scopus Google Scholar). The conserved is and and the general acid loop through with at the of this the side of the PRL-3 active a conserved of charged amino acids is These include and a of amino acids at the C of the protein in a conserved motif is an to the prenylation likely participates in with The PRL-3 structure that PRL phosphatases are to dual specificity phosphatases. structural and mutagenesis studies PRL-3 shows low activity. of a serine/threonine residue in the active site is the activity. the general acid loop a conformational change to to the catalytic These are likely in the presence of physiological to reveal catalytic activity to that with other phosphatases. the disulfide bond Cys49 and the catalytic Cys104 potentially a regulatory role and

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