Interaction of Farnesylated PRL-2, a Protein-tyrosine Phosphatase, with the β-Subunit of Geranylgeranyltransferase II
2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês
10.1074/jbc.m010400200
ISSN1083-351X
AutoresXiaoning Si, Qi Zeng, Chee H. Ng, Wanjin Hong, Catherine J. Pallen,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoProtein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the β-subunit of Rab geranylgeranyltransferase II (βGGT II). The specific interaction of βGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with βGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for βGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for βGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the α-subunit (α) of GGT II, βGGT II, and PRL-2 resulted in α/βGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous α/βGGT II activity in HeLa cells. Together, these results indicate that the binding of αGGT II and PRL-2 to βGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity. Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the β-subunit of Rab geranylgeranyltransferase II (βGGT II). The specific interaction of βGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with βGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for βGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for βGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the α-subunit (α) of GGT II, βGGT II, and PRL-2 resulted in α/βGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous α/βGGT II activity in HeLa cells. Together, these results indicate that the binding of αGGT II and PRL-2 to βGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity. farnesyltransferase farnesyltransferase inhibitor geranylgeranyltransferase protein-tyrosine phosphatase protein of regenerating liver-protein-tyrosine phosphatase high performance liquid chromatography polyacrylamide gel electrophoresis Protein prenylation is a post-translational modification with an important role in targeting proteins to membranes and in protein-protein interactions (reviewed in Refs. 1Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1733) Google Scholar and 2Seabra M.C. Cell. Signal. 1998; 10: 167-172Crossref PubMed Scopus (223) Google Scholar). Prominent among a variety of prenylated proteins are numerous small GTP-binding proteins, including Ras, RhoB, and the Rab proteins, and prenylation is essential for their cellular functions in signal transduction and vesicle trafficking (2Seabra M.C. Cell. Signal. 1998; 10: 167-172Crossref PubMed Scopus (223) Google Scholar). Isoprenoid modification can be catalyzed by one of three different transferases, depending on the isoprenoid and the prenylation motif of the target protein. Farnesyltransferase (FT)1 and geranylgeranyltransferase I (GGT I) are α/β heterodimers which share a common α-subunit and utilize the C15 farnesyl or the C20 geranylgeranyl, respectively (3Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (702) Google Scholar, 4Seabra M.C. Reiss Y. Casey P.J. Brown M.S. Goldstein J.L. Cell. 1991; 65: 429-434Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 5Moomaw J.F. Casey P.J. J. Biol. Chem. 1992; 267: 17438-17443Abstract Full Text PDF PubMed Google Scholar). Both recognize the prenylation motif CAAX, where C is cysteine, A is aliphatic, and X is preferred as Met, Ser of Gln by FT, and as Leu for GGT I. Ras and Rho proteins can be farnesylated by FT or geranylgeranylated by GGT I (6Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (779) Google Scholar, 7Adamson P. Marshall C.J. Hall A. Tilbrook P.A. J. Biol. Chem. 1992; 267: 20033-20038Abstract Full Text PDF PubMed Google Scholar, 8Rowell C.A. Kowalczyk J.J. Lewis M.D. Garcia A.M. J. Biol. Chem. 1997; 272: 14093-14097Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 9Whyte D.B. Kirschmeier P. Hockenberry T.N. Nunez-Oliva I. James L. Catino J.J. Bishop W.R. Pai J.-K. J. Biol. Chem. 1997; 272: 14459-14464Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). The Rab proteins are the only known substrates of GGT II, a distinct α/β dimer that prenylatesXXCC, XCXC, or CCXXC-terminal sequences when the Rab proteins are bound to a carrier called REP (Rab escort protein) (10Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Abstract Full Text PDF PubMed Google Scholar, 11Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Abstract Full Text PDF PubMed Scopus (285) Google Scholar). Ras oncogenes are associated with many human cancers, and as Ras transforming function requires farnesylation (12Jackson J.H. Cochrane C.G. Bourne J.R. Solski P.A. Buss J.E. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3042-3046Crossref PubMed Scopus (296) Google Scholar, 13Kato K. Cox A.D. Hisaka M.M. Graham S.M. Buss J.E. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6403-6407Crossref PubMed Scopus (555) Google Scholar), FT inhibitors (FTI) have been developed and tested as anti-cancer therapeutics. While Ras-mediated oncogenesis is in many cases compromised by such inhibitors (14James G.L. Goldstein J.L. Brown M.S. Rawson T.E. Somers T.C. McDowell R.S. Crowley C.W. Lucas B.K. Levinson A.D. Marsters Jr., J.C. Science. 1993; 260: 1937-1942Crossref PubMed Scopus (607) Google Scholar, 15Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompliano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (619) Google Scholar, 16Kohl N.E. Wilson F.R. Mosser S.D. Giuliani E. deSolms S.J. Conner M.W. Anthony N.J. Holtz W.J. Gomez R.P. Lee T.J. Smith R.L. Graham S.L. Hartman G.D. Gibbs J.B. Oliff A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9141-9145Crossref PubMed Scopus (322) Google Scholar, 17Kohl N.E. Omer C.A. Conner M.W. Anthony N.J. Davide J.P. deSolms S.J. Giuliani E.A. Gomez R.P. Graham S.L. Hamilton K. Handt L.K. Hartman G.D. Koblan K.S. Kral A.M. Miller P.J. Mosser S.M. O'Neill T.J. Rands E. Schaber M.D. Gibbs J.B. Oliff A. Nat. Med. 1995; 1: 792-797Crossref PubMed Scopus (513) Google Scholar), in other cases it appears that other non-Ras farnesylated proteins are the effective targets of the inhibitor (18Gibbs J.B. Graham S.L. Hartman G.D. Koblan K.S. Kohl N.E. Omer C.A. Oliff A. Curr. Opin. Chem. Biol. 1997; 1: 197-203Crossref PubMed Scopus (75) Google Scholar). One candidate is RhoB, which is farnesylated and geranylgeranylated in cells, and a gain in the level of geranylgeranylated RhoB is observed upon FTI treatment (19Lebowitz P.F. Prendergast G.C. Oncogene. 1998; 17: 1439-1445Crossref PubMed Scopus (202) Google Scholar, 20Lebowitz P. Casey P.J. Prendergast G.C. Thissen J. J. Biol. Chem. 1997; 272: 15591-15594Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). This prenylation switch has been proposed to mediate a gain of the tumor growth inhibitory function of RhoB (21Du W. Lebowitz P.F. Prendergast G.C. Mol. Cell. Biol. 1999; 19: 1831-1840Crossref PubMed Scopus (231) Google Scholar), however, recent studies with human cancer cells suggest that both prenylated forms of RhoB are potent anti-transforming molecules and that RhoB thus may not be the unidentified FTI target in cancer cells (22Chen Z. Sun J. Pradines A. Favre G. Adnane J. Sebti S.M. J. Biol. Chem. 2000; 275: 17974-17978Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The nature and role of other farnesylated proteins that could account for the anti-tumorigenic effects of FTI need to be investigated. The PRL subgroup (PRL-1, -2, -3) of protein-tyrosine phosphatases (PTPs) are closely related intracellular enzymes (23Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (245) Google Scholar, 24Montagna M. Serova O. Sylla B.S. Feunteun J. Lenoir G.M. Hum. Genet. 1995; 96: 532-538Crossref PubMed Scopus (28) Google Scholar, 25Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S.K. Crowell P.L. Crowell D.N. Canc. Lett. 1996; 110: 49-55Crossref PubMed Scopus (191) Google Scholar, 26Zhao Z. Lee C.-C. Monckton D.G. Yazdani A. Coolbaugh M.I. Li X. Bailey J. Shen Y. Caskey C.T. Genomics. 1996; 35: 172-186Crossref PubMed Scopus (27) Google Scholar, 27Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (155) Google Scholar) with the highest sequence homology to two dual specificity PTPs, Cdc14p and PTEN. The PRLs are unique among PTP superfamily members in possessing a C-terminal prenylation motif and being prenylated in vivo. Prenylated PRLs are found in the early endosome and at the plasma membrane (28Zeng 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 (181) Google Scholar). Inhibition of prenylation, by treatment of cells with a farnesyltransferase inhibitor, results in PRL translocation to the nucleus (28Zeng 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 (181) Google Scholar). The prenylation motifs of the PRLs (CCIQ, CCVQ, CCVM) conform to either the CAAX motif preferentially recognized by FT or to the CCXX motif recognized by GGT II, although their relocalization in response to FT inhibition indicates that they are likely farnesylated proteins in vivo. The prenylation-dependent subcellular localization of the PRLs suggests that regulated prenylation is a mechanism which controls the access of these PTPs to early endosomal or nuclear substrates. Cellular substrates of the PRL phosphatases have not yet been identified, although PRL-1 interacts with a basic leucine zipper protein, ATF-7, and can dephosphorylate it in vitro (29Peters C.S. Liang X. Li S. Kannan S. Peng Y. Taub R. Diamond R.H. J. Biol. Chem. 2001; 276: 13718-13726Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Although the specific substrates of the PRLs are unknown, PRL expression is associated with two distinct cell processes. PRL-1 was first identified as an immediate early gene expressed in regenerating liver and in serum-treated fibroblasts, and overexpression of PRL-1 in NIH 3T3 cells results in cell transformation, suggesting that the gene product plays a role in proliferation (23Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (245) Google Scholar, 30Mohn K.L. Laz T.M. Hsu J.-C. Melby A.E. Bravo R. Taub R. Mol. Cell. Biol. 1991; 11: 381-390Crossref PubMed Scopus (187) Google Scholar). Another role for PRL-1 has been proposed in the development and maintenance of differentiating epithelial tissues, as it is expressed in several developing tissues in fetal rat, and in the adult rat is found in terminally differentiated cells such as renal tubular epithelium, bronchiolar epithelium of the lung, and in villus but not crypt enterocytes of the intestine (31Diamond R.H. Peters C. Jung S.P. Greenbaum L.E. Haber B.A. Silberg D.G. Traber P.G. Taub R. Am. J. Physiol. Gastrointest. Liver Physiol. 1996; 271: G121-G129Crossref PubMed Google Scholar,32Kong W. Swain G.P. Li S. Diamond R.H. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G613-G621Crossref PubMed Google Scholar). Similarly, PRL-3 is specifically expressed in the differentiated epithelial cells of the villus but not in the proliferating crypt cells of the mouse small intestine (28Zeng 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 (181) Google Scholar). Yeast-two hybrid screening was carried out to identify PRL-2 interacting proteins, the nature of which could give insight into the cellular role of this PTP. The β-subunit of a prenyltransferase, GGT II, was found to specifically interact with PRL-2. This was intriguing, as this enzyme is only known to prenylate Rab proteins. The association of βGGT II with PRL-2 in mammalian cells was confirmed, and found to depend on the prenylation status of PRL-2, even though PRL-2 was farnesylated in cells. Association also required a unique region of PRL-2 that is not present in PRL-1 or -3. We present evidence that the binding of PRL-2 and αGGT II to βGGT II is mutually exclusive, and propose that through displacement of αGGT II, PRL-2 may function as a regulator of Rab GGT II activity. The pAS2-1 vector (CLONTECH) was used to generate the yeast expression PRL-PTP plasmids. In general, the cDNAs encoding the full-length PRL-1, -2, and -3 were amplified by polymerase chain reaction, and each subcloned in-frame into BamHI andPstI cut pAS2-1 vector. The pXJ40-myc vector (a gift from V. Yu) was used to generate the PRL-PTP expression plasmids for use in transient transfections. A BamHI-XhoI fragment encoding each full-length PRL-PTP or a mutant PRL-2 lacking the four C-terminal amino acids (PRL-2(cd)) was excised from pGEX-KG-PRL-1, -2, -3 or pGEX-KG-PRL-2(cd) (28Zeng 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 (181) Google Scholar), respectively, and subcloned in-frame into pXJ40-myc. The plasmid pXJ40-myc-PRL-2(CSVQ) was generated by in-frame insertion into BamHI/XhoI-cut pXJ40-myc of a polymerase chain reaction fragment from amplification of PRL-2 using an appropriate forward primer incorporating a BamHI site, and a reverse primer with a nucleotide substitution giving the desired C165S mutation and an engineered XhoI site. To generate the PRL-1/-2 chimera expression plasmids, a PRL-1 or PRL-2 cDNA fragment was excised from pXJ40-myc-PRL-2 or pXJ40-myc-PRL-1 atEcoRII and/or AflII sites, and was replaced by the corresponding fragment from the other PRL-PTP. The βGGT II cDNA (lacking the nucleotides encoding the four N-terminal amino acids) was amplified from the pGADGH-GGT II-B plasmid and subcloned into an NcoI/XhoI-cut pGEX-KG vector. The βGGT II cDNA was then excised using NcoI and SacI flanking sites, and subcloned into anNcoI/SacI-cut pBKS-flag vector (a gift from S. Lin). The DNA encoding flag-βGGT II was released by digestion withEcoRI and SacI, and subcloned in-frame into pXJ40 to create pXJ40-flag-βGGT II. The rat αGGT II cDNA was a gift from M. C. Seabra and was subcloned in-frame into aBamHI/KpnI cut pXJ41-HA vector. All of the plasmids were sequenced prior to use. The interaction screen was performed essentially as recommended in theCLONTECH user manual. A HeLa cDNA library (CLONTECH) was transformed into the yeast strain Y190, which had been pretransformed with pAS2-1-PRL-2. The transformants were plated on His, Leu, and Trp− medium containing 25 mm 3-amino-1,2,4-triazole (Sigma). Plasmids were isolated from positive colonies that fulfilled all criteria, and retested. One plasmid that was consistently positive for interaction with PRL-2 was sequenced and compared against the Entrez data base using a Blast search, and was identified as the cDNA encoding the β-subunit of human GGT II. HeLa cells were maintained in Dulbecco's modified Eagle's medium and transiently transfected using LipofectAMINE reagent (Life Technologies, Inc.). The empty expression vector pXJ40-myc was used to normalize the amount of DNA in each transfection. After 24 h of culture, the cells were harvested in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 2 mm phenylmethylsulfonyl fluoride). The cytosol fraction was prepared by passing the cells 6–8 times through a 26-gauge needle, followed by clarification of the lysate by centrifugation at 12,000 rpm for 30 min at 4 °C. The supernatant (cytosol) was used for immunoprecipitation and Western blotting. For farnesyltransferase inhibition, 10 µm FTI-277 (a gift from S. M. Sebti) (33Lerner E.C. Qian Y. Blaskovich M.A. Fossum R.D. Vogt A. Sun J. Cox A.D. Der C.J. Hamilton A.D. Sebti S.M. J. Biol. Chem. 1995; 270: 26802-26806Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar) in Dulbecco's modified Eagle's medium, 10% fetal calf serum was added to the cells 5 h after transfection. Anti-Myc (9E10, Santa Cruz) and anti-FLAG (M2, Sigma) antibodies were used for immunoprecipitation and Western blotting. Typically the lysates were incubated with the specific antibody overnight at 4 °C. Immunofluorescence was carried out using fluorescein isothiocyanate-conjugated Myc antibody as described (28Zeng 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 (181) Google Scholar). HeLa cells were transiently transfected with pXJ40-myc vector (mock) or pXJ40-myc-PRL-2 (PRL-2) for 18 h prior to harvest. Cell lysate was prepared by scraping the cells into lysis buffer containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 10 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride and then passing the cells 6–8 times through a 26-gauge needle. The lysate was clarified by centrifugation at 12,000 rpm for 30 min at 4 °C, and the supernatant was used as the source of GGT II. The in vitro enzymatic reaction was initiated by adding 80 µg of cell lysate to buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 10 mm dithiothreitol) containing 2 µm[3H]GGPP (22 Ci/mmol, PerkinElmer Life Sciences) and with or without 2 µm Rab3a protein (Calbiochem). The 50-µl reaction was incubated at 30 °C and stopped after 4 h by adding SDS sample buffer, and then resolved by SDS-PAGE. The gel was dried and exposed to hyperfilm at −80 °C for 2 weeks and the labeled Rab3a protein was quantified by densitometry. 1-[3H]Mevalonolactone (40 Ci/mmol, [3H]MVA) was purchased from PerkinElmer Life Sciences. HeLa cells were maintained and transfected with pXJ40-myc-PRL-2, and treated with or without FTI-277, as described above. For the final 18 h, cells were treated with [3H]MVA at 100 µCi/ml and 30 µm lovastatin (Calbiochem). Cell lysate was prepared by scraping the cells into 50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm MgCl2, 1% Triton X-100, 2 mm phenylmethylsulfonyl fluoride and then passing the cells 6–8 times through a 26-gauge needle. The lysate was clarified by centrifugation at 12,000 rpm for 30 min at 4 °C, and the supernatant was used to isolate myc-PRL-2 by immunoprecipitation with anti-Myc antibody (9E10, Santa Cruz). The immunoprecipitate was washed twice with acetone and CHCl3/MeOH, 1:2 (v/v) at −20 °C, dried by vacuum centrifugation, and processed for isoprenoid analysis as described by Casey et al. (6Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (779) Google Scholar). The final dried sample was dissolved in 60 µl of 50% CH3CN, 25 mm H3PO4 (solvent A), and a portion was injected onto a C18 reverse-phase HPLC column. The column was eluted with a 4-ml linear gradient of solvent A to 100% CH3CN, 25 mm H3PO4(solvent B), followed by 1 ml of solvent B at a flow rate of 100 µl/min. Fractions of 100 µl were collected and their radioactivity determined by scintillation counting. Trans,trans-farnesol (FOH, Sigma) and all-trans-geranylgeraniol (GGOH, American Radiolabeled Chemicals, Inc.) were used as elution standards. To identify PRL-2 interacting proteins, we performed a yeast two-hybrid screen with full-length PRL-2 fused to the GAL4 DNA-binding domain as bait. The expression of the fusion protein in Y190 yeast cells was verified by immunoblotting (data not shown). A human HeLa cell cDNA library fused to the GAL4 activating domain under the control of the constitutive alcohol dehydrogenase 1 promoter was transfected into Y190 cells that expressed DNA-binding domain-PRL-2. Ten million colonies were selected on medium lacking Trp, Leu, and His and supplemented with 25 mm 3-amino-1,2,4-triazole. Among others, one colony was identified that showed growth on the selective medium and β-galactosidase activity, indicative of an interaction between PRL-2 and a library protein in these cells. The plasmid was isolated and sequenced. The insert was in-frame with the cDNA that coded for the human GGT II β-subunit. PRL-2 is a member of a group of three closely related proteins which also includes PRL-1 and PRL-3 (23Diamond R.H. Cressman D.E. Laz T.M. Abrams C.S. Taub R. Mol. Cell. Biol. 1994; 14: 3752-3762Crossref PubMed Scopus (245) Google Scholar, 24Montagna M. Serova O. Sylla B.S. Feunteun J. Lenoir G.M. Hum. Genet. 1995; 96: 532-538Crossref PubMed Scopus (28) Google Scholar, 25Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S.K. Crowell P.L. Crowell D.N. Canc. Lett. 1996; 110: 49-55Crossref PubMed Scopus (191) Google Scholar, 26Zhao Z. Lee C.-C. Monckton D.G. Yazdani A. Coolbaugh M.I. Li X. Bailey J. Shen Y. Caskey C.T. Genomics. 1996; 35: 172-186Crossref PubMed Scopus (27) Google Scholar, 27Zeng Q. Hong W. Tan Y.H. Biochem. Biophys. Res. Commun. 1998; 244: 421-427Crossref PubMed Scopus (155) Google Scholar). In order to test the specificity of the interaction of PRL-2 with the β-subunit of GGT II, we used PRL-1 and PRL-3 as bait and co-transfected the yeast with a plasmid expressing GAL4(DNA-binding domain)-βGGT II. The expression of the fusion proteins in Y190 yeast cells was verified by immunoblotting (data not shown). Despite the high homology between different PRL-PTP family members, only PRL-2 interacted with the GGT II β subunit in the yeast two-hybrid system (Fig.1). As GGT II is a prenyltransferase, we also tested the dependence of the interaction on the presence of the C-terminal prenylation sequence, the CAAX box, of PRL-2. A prenylation-deficient mutant of PRL-2 lacking the CAAX box, PRL-2(cd), did not interact with βGGT II (Fig. 1). To validate the yeast two-hybrid results, we examined the in vivo interaction of PRL-2, -1, or -3 and βGGT II in mammalian cells. HeLa cells were transiently co-transfected with Myc-tagged PRLs and FLAG-tagged βGGT II. Since βGGT II is a soluble protein mainly localized in the cytosol (10Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Abstract Full Text PDF PubMed Google Scholar), non-detergent-extracted soluble fractions from the HeLa cells were used to carry out anti-Myc and anti-FLAG immunoprecipitations. Virtually all the expressed βGGT II was indeed present in this soluble cytosolic fraction (Fig.2A, upper panel, comparelanes 1–4 with lanes 5–8). More PRL-2 was present in the cytosol than in the pellet (Fig. 2 A, lower panel, compare lanes 3 and 7), while about one-third of the PRL-1 partitioned into the cytosol (Fig. 2 A, lower panel, compare lanes 2 and 6). However, comparatively less of the total PRL-3 was found in the soluble extracts (Fig. 2 A, lower panel, compare lanes 4and 8). PRL-2 and βGGT II co-immunoprecipitated with one another, as evidenced by the presence of FLAG-βGGT II in Myc-PRL-2 immunoprecipitates (Fig. 2 B, lane 6), and by the presence of Myc-PRL-2 in anti-FLAG immunoprecipitates (Fig. 2 C, lane 6). No βGGT II was detected in myc-PRL-1 immunoprecipitates and Myc-PRL-1 was not present in anti-FLAG immunoprecipitates (Fig. 2,B and C, lane 5). Likewise, no association was detected between PRL-3 and βGGT II (Fig. 2, B and C, lane 7). These results confirm the in vivo interaction of PRL-2 and βGGT II and demonstrate that this involves specific features or properties of PRL-2. PRL-2 is localized in the early endosome compartment in a prenylation-dependent manner (28Zeng 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 (181) Google Scholar). In transfected CHO cells and NIH 3T3 cells, PRL-2 showed strong perinuclear staining and weak plasma membrane staining visualized by immunofluorescence. A prenylation-deficient PRL-2 mutant was redirected to the nucleus and a farnesyltransferase inhibitor, FTI-277, caused the same nuclear relocalization (28Zeng 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 (181) Google Scholar). We found essentially the same results in transiently transfected HeLa cells, but with enhanced diffuse cytoplasmic staining of expressed wild-type PRL-2 following treatment with FTI-277, and of expressed PRL-2(cd) (Fig.3). The readily apparent cytoplasmic staining may reflect a higher level of PRL-2 expression in transiently transfected HeLa cells compared to that in the stably transfected CHO and NIH 3T3 cells. Together these results indicate that prenylation plays an important role in the subcellular localization of PRL-2. To determine whether the linked properties of PRL-2 prenylation status and subcellular localization affect its interaction with GGT II, co-immunoprecipitation experiments were carried out from lysates of HeLa cells expressing FLAG-tagged βGGT II and Myc-tagged PRL-2 or PRL-2(cd) mutant. As observed in previous experiments, the prenylated and early endosome-localized wild-type PRL-2 and βGGT II co-immunoprecipitated with one another (Fig.4A, lanes 4 and 7). However, the non-prenylated PRL-2(cd) mutant failed to interact with βGGT II (Fig. 4 A, lanes 5 and 8). The lack of interaction cannot merely be due to physically separate pools of cytoplasmic βGGT II and nuclear PRL-2(cd), as some PRL-2(cd) is present in the cell cytoplasm (Fig. 3) and likely represents the soluble PRL-2(cd) protein that was extracted together with soluble βGGT II (Fig. 4 A, lane 2). Likewise, in the yeast two-hybrid system we observed that the prenylation deficient mutant PRL-2(cd) did not interact with βGGT II, despite these expressed proteins being directed to the yeast nucleus (Fig. 1). Nevertheless, wild-type PRL-2 did interact with βGGT II in the yeast nucleus (Fig.1). These results suggested that co-localization of PRL-2 and βGGT II is not sufficient for interaction, and that the early endosomal localization of PRL-2 per se is not required for the physical association of PRL-2 and βGGT II in vivo. However, prenylation or the C-terminal prenylation sequence of PRL-2 is necessary for interaction. To distinguish between these latter possibilities, we investigated the association between βGGT II and prenylated PRL-2 or unprenylated PRL-2 possessing the CAAX box. HeLa cells were transiently transfected with PRL-2 and βGGT II in the presence or absence of the farnesyltransferase inhibitor FTI-277 for 16 h prior to harvest. Whole cell lysates were prepared and co-immunoprecipitations performed. Once again, wild-type PRL-2 from FTI-277 untreated cells was observed to interact with the GGT II β-subunit as visualized by immunoblotting (Fig. 4 B, lanes 4 and 7). Upon treatment with FTI-277, the expression level of PRL-2 was virtually unchanged (Fig.4 B, lanes 1 and 2) but the association with the GGT II β-subunit was lost (Fig. 4 B, lanes 5 and8). This was consistent with the study of PRL-2(cd) described above, and indicated that not only the CAAX box, but modification of PRL-2 by prenylation was required for interaction. As prenylated PRL-2 is in the early endosome and not in the cytoplasm or in the nucleus, this is the subcellular pool of PRL-2 that can interact with βGGT II. It is well documented that GGT II is a prenyltransferase that recognizes the prenylation motifs of XXCC, CXC, or CCXX, and whose exclusive substrates are Rab GTPases (1Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1733) Google Scholar). The C-terminal prenylation motif of PRL-2, CCVQ, fits the CCXX sequence recognized by geranylgeranyltransferase II or the CAAX motif recognized by farnesyltransferase or GGT I. Indeed, PRL-2 can be geranylgeranylated or farnesylated in vitro (25Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S.K. Crowell P.L. Crowell D.N. Canc. Lett. 1996; 110: 49-55Crossref PubMed Scopus (191) Google Scholar, 28Zeng 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 (181) Google Scholar). To examine the possibility that prenylation of PRL-2 by GGT II was involved in the interaction of these proteins, we examined the lipid modification of PRL-2 in vivo. HeLa cells transiently expressing Myc-tagged PRL-2 were labeled with the isoprenoid precursor [3H]mevalonolactone, and isoprenoid analysis of the immunoprecipitated PRL-2 carried out. SDS-PAGE and autoradiography of the PRL-2 immunoprecipitate revealed that PRL-2 was labeled, and that it contained the majority of the label in the immunoprecipitate (Fig.5A, lane 2). A duplicate Myc-PRL-2
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