The Tyrosine Phosphatase PRL-1 Localizes to the Endoplasmic Reticulum and the Mitotic Spindle and Is Required for Normal Mitosis

Abstract

PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics. PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics. Protein-tyrosine phosphatases (PTPs) 1The abbreviations used for: PTP, protein-tyrosine phosphatase; GST, glutathioneS-transferase; DOX, doxycycline; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FTI, farnesyltransferase inhibitor; DAPI, 4,6-diamidino-2-phenylindole. 1The abbreviations used for: PTP, protein-tyrosine phosphatase; GST, glutathioneS-transferase; DOX, doxycycline; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FTI, farnesyltransferase inhibitor; DAPI, 4,6-diamidino-2-phenylindole. have been recognized as important modulators in signal transduction pathways that regulate cell proliferation and cell differentiation, as well as other cellular functions of eukaryotic cells (1Dixon J.E. Recent Prog. Horm. Res. 1996; 51: 405-415PubMed Google Scholar, 2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar). PTPs can function as positive or negative regulators of signal transduction pathways, depending on the particular substrate and the effect of phosphorylation on its activity (3Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar, 4Frearson J.A. Alexander D.R. Bioessays. 1997; 19: 417-427Crossref PubMed Scopus (62) Google Scholar). Like kinases, PTPs have been implicated in various malignancies, including cancer.The PTP superfamily can be divided into two classes based on sequence similarity and substrate specificity: “classic” PTPs, which dephosphorylate phosphotyrosine residues, and dual specificity phosphatases, which can also use phosphoserine and phosphothreonine residues as substrates. In addition to the conserved catalytic domain, PTPs frequently contain additional domains or sequence motifs important for subcellular localization, substrate recognition, or regulation of their activities (2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar, 5Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (317) Google Scholar).Over the past few years, a new subfamily of PTPs, PRL (also termed PTPCAAX), has been identified (6Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (242) Google Scholar, 7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar, 8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar). Although PRL phosphatases contain the PTP signature motif (I/V(HCXAGXXR)S/T)G at the active site, their sequences are distinct from other PTPs. Overall, their catalytic domain is most similar to that of dual specificity phosphatases. Their closest relatives are CDC14, a mitotic regulator, and the tumor suppressor PTEN (8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar, 9Hooft van Huijsduijnen R. Gene (Amst.). 1998; 225: 1-8Crossref PubMed Scopus (67) Google Scholar). To date, three family members (PRL-1, PRL-2, PRL-3) have been identified in mammals. PRL phosphatases are relatively small enzymes with an apparent molecular mass of 22 kDa and contain a C-terminal CAAX motif as the signal for protein farnesylation. All three family members can serve as substrates for farnesyltransferasesin vitro, and PRL-2 has been shown to be prenylated in vivo (7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar, 10Zeng 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 (180) Google Scholar).The three enzymes share a high degree of amino acid sequence similarity with each other. For example, PRL-1 shares 87 and 75% sequence identity with PRL-2 and PRL-3, respectively (8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar). The amino acid sequence similarity of the three family members is also reflected on the cDNA level within the coding region. However, their 5′ non-coding sequences are quite divergent, suggesting possible differences in the regulation of gene expression (8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar).PRL-1 was first cloned as an immediate-early gene in rat liver following hepatectomy. More recently, it has been shown that the expression of human PRL-1 is regulated by Egr-1, a growth factor-activated transcription factor (11Peng Y. Genin A. Spinner N.B. Diamond R.H. Taub R. J. Biol. Chem. 1998; 273: 17286-17295Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Peng Y. Du K. Ramirez S. Diamond R.H. Taub R. J. Biol. Chem. 1999; 274: 4513-4520Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Interestingly, overexpression of PRL-1 and PRL-2 in epithelial cells results in a transformed phenotype and the transfected cells are able to form tumors in nude mice (7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar). Further, stable cell lines overexpressing PRL-1 have enhanced saturation density and a shorter doubling time relative to control cell lines (6Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (242) Google Scholar, 7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar). More recently PRL-2 was shown to be overexpressed in prostate cancer (13Wang Q. Holmes D.I. Powell S.M. Lu Q.L. Waxman J. Cancer Lett. 2002; 175: 63-69Crossref PubMed Scopus (53) Google Scholar). Further, Saha et al.(14Saha 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 (578) Google Scholar) demonstrated that the expression of PRL-3 is associated with liver metastasis of colon cancer. Together these data suggest that PRL-1 and PRL-2 may function in the regulation of cell proliferation and that all three members of this subfamily of PTPs may be involved in tumorigenesis. To date, however, little is known about their possible physiological function and how they might be involved in signal transduction processes. In addition, the mechanism of their regulation and the basis of their transforming activity are not understood.The subcellular localization of a particular protein can sometimes give clues to its physiological function. PRL-1 was originally described as a nuclear protein in transfected cells (6Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (242) Google Scholar). More recently, Zenget al. (10Zeng 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 (180) Google Scholar) reported that PRL phosphatases are localized to the plasma membrane and early endosomes and that this localization pattern is dependent on their farnesylation. This study used an overexpression system, which could lead to non-physiological subcellular localization.By using affinity-purified antibodies against PRL-1, we report that the subcellular localization of endogenous PRL-1 is cell cycle-dependent. In non-mitotic cells PRL-1 localizes to the endoplasmic reticulum (ER), whereas in mitotic cells it is associated with the centrosomes and the spindle apparatus. Further, cell cycle analysis of cells expressing a catalytic domain mutant of PRL-1 indicates that the PRL-1 phosphatase activity is required for progression of cells through mitosis. In addition, expression of a farnesylation site mutant of PRL-1 (PRL-1-C170S), which is enriched in the nucleus throughout the cell cycle, results in mitotic defects without compromising spindle checkpoint function. Like wild type PRL-1, PRL-1-C170S localizes to the spindle apparatus in mitotic cells, demonstrating that farnesylation is required for ER but not for microtubule association. Our results demonstrate for the first time a role for PRL-1 in cell cycle regulation and suggest that PRL-1 activity may be required for proper spindle function or spindle dynamics. Protein-tyrosine phosphatases (PTPs) 1The abbreviations used for: PTP, protein-tyrosine phosphatase; GST, glutathioneS-transferase; DOX, doxycycline; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FTI, farnesyltransferase inhibitor; DAPI, 4,6-diamidino-2-phenylindole. 1The abbreviations used for: PTP, protein-tyrosine phosphatase; GST, glutathioneS-transferase; DOX, doxycycline; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; FTI, farnesyltransferase inhibitor; DAPI, 4,6-diamidino-2-phenylindole. have been recognized as important modulators in signal transduction pathways that regulate cell proliferation and cell differentiation, as well as other cellular functions of eukaryotic cells (1Dixon J.E. Recent Prog. Horm. Res. 1996; 51: 405-415PubMed Google Scholar, 2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar). PTPs can function as positive or negative regulators of signal transduction pathways, depending on the particular substrate and the effect of phosphorylation on its activity (3Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar, 4Frearson J.A. Alexander D.R. Bioessays. 1997; 19: 417-427Crossref PubMed Scopus (62) Google Scholar). Like kinases, PTPs have been implicated in various malignancies, including cancer. The PTP superfamily can be divided into two classes based on sequence similarity and substrate specificity: “classic” PTPs, which dephosphorylate phosphotyrosine residues, and dual specificity phosphatases, which can also use phosphoserine and phosphothreonine residues as substrates. In addition to the conserved catalytic domain, PTPs frequently contain additional domains or sequence motifs important for subcellular localization, substrate recognition, or regulation of their activities (2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar, 5Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (317) Google Scholar). Over the past few years, a new subfamily of PTPs, PRL (also termed PTPCAAX), has been identified (6Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (242) Google Scholar, 7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar, 8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar). Although PRL phosphatases contain the PTP signature motif (I/V(HCXAGXXR)S/T)G at the active site, their sequences are distinct from other PTPs. Overall, their catalytic domain is most similar to that of dual specificity phosphatases. Their closest relatives are CDC14, a mitotic regulator, and the tumor suppressor PTEN (8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar, 9Hooft van Huijsduijnen R. Gene (Amst.). 1998; 225: 1-8Crossref PubMed Scopus (67) Google Scholar). To date, three family members (PRL-1, PRL-2, PRL-3) have been identified in mammals. PRL phosphatases are relatively small enzymes with an apparent molecular mass of 22 kDa and contain a C-terminal CAAX motif as the signal for protein farnesylation. All three family members can serve as substrates for farnesyltransferasesin vitro, and PRL-2 has been shown to be prenylated in vivo (7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar, 10Zeng 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 (180) Google Scholar). The three enzymes share a high degree of amino acid sequence similarity with each other. For example, PRL-1 shares 87 and 75% sequence identity with PRL-2 and PRL-3, respectively (8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar). The amino acid sequence similarity of the three family members is also reflected on the cDNA level within the coding region. However, their 5′ non-coding sequences are quite divergent, suggesting possible differences in the regulation of gene expression (8Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (154) Google Scholar). PRL-1 was first cloned as an immediate-early gene in rat liver following hepatectomy. More recently, it has been shown that the expression of human PRL-1 is regulated by Egr-1, a growth factor-activated transcription factor (11Peng Y. Genin A. Spinner N.B. Diamond R.H. Taub R. J. Biol. Chem. 1998; 273: 17286-17295Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Peng Y. Du K. Ramirez S. Diamond R.H. Taub R. J. Biol. Chem. 1999; 274: 4513-4520Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Interestingly, overexpression of PRL-1 and PRL-2 in epithelial cells results in a transformed phenotype and the transfected cells are able to form tumors in nude mice (7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar). Further, stable cell lines overexpressing PRL-1 have enhanced saturation density and a shorter doubling time relative to control cell lines (6Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (242) Google Scholar, 7Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (187) Google Scholar). More recently PRL-2 was shown to be overexpressed in prostate cancer (13Wang Q. Holmes D.I. Powell S.M. Lu Q.L. Waxman J. Cancer Lett. 2002; 175: 63-69Crossref PubMed Scopus (53) Google Scholar). Further, Saha et al.(14Saha 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 (578) Google Scholar) demonstrated that the expression of PRL-3 is associated with liver metastasis of colon cancer. Together these data suggest that PRL-1 and PRL-2 may function in the regulation of cell proliferation and that all three members of this subfamily of PTPs may be involved in tumorigenesis. To date, however, little is known about their possible physiological function and how they might be involved in signal transduction processes. In addition, the mechanism of their regulation and the basis of their transforming activity are not understood. The subcellular localization of a particular protein can sometimes give clues to its physiological function. PRL-1 was originally described as a nuclear protein in transfected cells (6Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (242) Google Scholar). More recently, Zenget al. (10Zeng 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 (180) Google Scholar) reported that PRL phosphatases are localized to the plasma membrane and early endosomes and that this localization pattern is dependent on their farnesylation. This study used an overexpression system, which could lead to non-physiological subcellular localization. By using affinity-purified antibodies against PRL-1, we report that the subcellular localization of endogenous PRL-1 is cell cycle-dependent. In non-mitotic cells PRL-1 localizes to the endoplasmic reticulum (ER), whereas in mitotic cells it is associated with the centrosomes and the spindle apparatus. Further, cell cycle analysis of cells expressing a catalytic domain mutant of PRL-1 indicates that the PRL-1 phosphatase activity is required for progression of cells through mitosis. In addition, expression of a farnesylation site mutant of PRL-1 (PRL-1-C170S), which is enriched in the nucleus throughout the cell cycle, results in mitotic defects without compromising spindle checkpoint function. Like wild type PRL-1, PRL-1-C170S localizes to the spindle apparatus in mitotic cells, demonstrating that farnesylation is required for ER but not for microtubule association. Our results demonstrate for the first time a role for PRL-1 in cell cycle regulation and suggest that PRL-1 activity may be required for proper spindle function or spindle dynamics. We are grateful to Dr. James Cupp for help with the confocal microscopy. We thank Emma Lees, David Parry, Bruno Amati, Paul Kirschmeier, Robert Bishop, Dominique Blanchard, Nicholas Cacalano, Leela Basu, and Sherrie Pettiford for helpful discussion and critical reading of the manuscript. We are especially thankful to Rene de Waal Malefyt for help and advice on Taqman analysis. We also thank Maribel Andonian and Gary Burget for graphics support.