PTEN Associates with the Vault Particles in HeLa Cells

PTEN is a tumor suppressor that primarily dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to down-regulate the phosphoinositide 3-kinase/Akt signaling pathway. Although the cellular functions of PTEN as a tumor suppressor have been well characterized, the mechanism by which PTEN activity is modulated by other signal molecules in vivo remains poorly understood. In searching for potential PTEN modulators through protein-protein interaction, we identified the major vault protein (MVP) as a dominant PTEN-binding protein in a yeast two-hybrid screen. MVP is the major structural component of vault, the largest intracellular ribonucleoprotein particle. Co-immunoprecipitation confirmed the interaction between PTEN and MVP in transfected mammalian cells. More importantly, we found that a significant portion of endogenous PTEN associates with vault particles in human HeLa cells. Deletion mutation analysis demonstrated that MVP binds to the C2 domain of PTEN and that PTEN interacts with MVP through its EF hand-like motif. Furthermore, the in vitro binding experiments revealed that the interaction of PTEN with MVP is Ca2+-dependent. PTEN is a tumor suppressor that primarily dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to down-regulate the phosphoinositide 3-kinase/Akt signaling pathway. Although the cellular functions of PTEN as a tumor suppressor have been well characterized, the mechanism by which PTEN activity is modulated by other signal molecules in vivo remains poorly understood. In searching for potential PTEN modulators through protein-protein interaction, we identified the major vault protein (MVP) as a dominant PTEN-binding protein in a yeast two-hybrid screen. MVP is the major structural component of vault, the largest intracellular ribonucleoprotein particle. Co-immunoprecipitation confirmed the interaction between PTEN and MVP in transfected mammalian cells. More importantly, we found that a significant portion of endogenous PTEN associates with vault particles in human HeLa cells. Deletion mutation analysis demonstrated that MVP binds to the C2 domain of PTEN and that PTEN interacts with MVP through its EF hand-like motif. Furthermore, the in vitro binding experiments revealed that the interaction of PTEN with MVP is Ca2+-dependent. PTEN was originally identified as a tumor suppressor gene based on its high frequency of mutation in a variety of tumors (1Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Google Scholar, 2Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C., Hu, R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Google Scholar, 3Li D.-M. Sun H. Cancer Res. 1997; 57: 2124-2129Google Scholar). Germ-line mutations of PTEN are the cause of Cowden disease, an autosomal-dominant hamartoma syndrome that results in an increased risk for development of tumors in a variety of tissues (4Marsh D.J. Dahia P.L. Zheng Z. Liaw D. Parsons R. Gorlin R.J. Eng C. Nat. Genet. 1997; 16: 333-349Google Scholar, 5Liaw D. Marsh D.J., Li, J. Dahia P.L. Wang S.I. Zheng Z. Bose S. Call K.M. Tsou H.C. Peacocke M. Eng C. Parsons R. Nat. Genet. 1997; 16: 64-67Google Scholar, 6Lynch E.D. Ostermeyer E.A. Lee M.K. Arena J.F., Ji, H. Dann J. Swisshelm K. Suchard D. MacLeod P.M. Kvinnsland S. Gjertsen B.T. Heimdal K. Lubs H. Møller P. King M.-C. Am. J. Hum. Genet. 1997; 61: 1254-1260Google Scholar, 7Nelen M.R. van Staveren C.G. Peeters E.A.J. Ben Hassel M. Gorlin R.J. Hamm H. Lindboe C.F. Fryns J.-P. Sijmons R.H. Woods D.G. Mariman E.C.M. Padberg G.W. Kremer H. Hum. Mol. Genet. 1997; 6: 1383-1387Google Scholar). The genetic evidence that PTEN is an important tumor suppressor is supported by the fact that heterozygous disruption of the PTEN gene in knockout mice results in the spontaneous development of tumors (8Di Cristofano A. Pesce B. Cordon-Cardo C. Pandolfi P.P. Nat. Genet. 1998; 19: 348-355Google Scholar, 9Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Google Scholar, 10Podsypanina K. Ellenson L.H. Nemes A., Gu, J. Tamura M. Yamada K.M. Cordon-Cardo C. Catoretti G. Fisher P.E. Parsons R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1563-1568Google Scholar). Although PTEN as a protein phosphatase is capable of dephosphorylating tyrosine and threonine/serine residues (11Myers M.P. Stolarov J.P. Eng C., Li, J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Google Scholar, 12Tamura M., Gu, J. Matsumoto K. Aota S. Parsons R. Yamada K. Science. 1998; 280: 1614-1617Google Scholar), the primary substrates of PTEN are 3′-phosphoinositides, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 (13Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Google Scholar). Genetic and biochemical studies have demonstrated that the tumor suppressor functions of PTEN are linked primarily with the lipid phosphatase activity and its association with the well defined phosphoinositide 3-kinase pathway (reviewed in Refs. 14Maehama T. Taylor G.S. Dixon J.E. Annu. Rev. Biochem. 2001; 70: 247-729Google Scholar, 15Simpson L. Parsons R. Exp. Cell Res. 2001; 264: 29-41Google Scholar, 16Waite K.A. Eng C. Am. J. Hum. Genet. 2002; 70: 829-844Google Scholar, 17Leslie N.R. Downes C.P. Cell. Signal. 2002; 14: 285-295Google Scholar, 18Downes C.P. Bennett D. McConnachie G. Leslie N.R. Pass I. MacPhee C. Patel L. Gray A. Biochem. Soc. Trans. 2001; 29: 846-851Google Scholar, 19Yamada K.M. Araki M. J. Cell Sci. 2001; 114: 2375-2382Google Scholar, 20Mills G.B., Lu, Y. Fang X. Wang H. Eder A. Mao M. Swaby R. Cheng K.W. Stokoe D. Siminovitch K. Jaffe R. Gray J. Semin. Oncol. 2001; 28: 125-141Google Scholar). Substantial progress has been made in the characterization of PTEN as a tumor suppressor as well as in the regulation of many cellular processes including growth, adhesion, migration, invasion, and apoptosis. Nevertheless, the mechanism by which PTEN activity is modulated in various cellular signaling complexes remains elusive. It is assumed that the activity and the cellular function of PTEN may be regulated through in vivoprotein-protein interactions. PTEN contains a number of putative regulatory modules, including the N-terminal phosphoinositide binding motif, a C2 domain, a PDZ-binding site, and two proline-, glutamic acid-, serine-, and threonine-rich segments (21Georgescu M.M. Kirsch K.H. Akagi T. Shishido T. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10182-10187Google Scholar). The C2 domain of PTEN has been implicated in mediating membrane association (22Lee J.O. Yang H. Georgescu M.M., Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Google Scholar). The C-terminal tail of PTEN interacts with several PDZ domain-containing proteins such as hDLG, hMAST205, MAGI-2, and MAGI-3 (23Wu X. Hepner K. Castelino-Prabhu S., Do, D. Kaye M.B. Yuan X.J. Wood J. Ross C. Sawyers C.L. Whang Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4233-4238Google Scholar, 24Wu Y. Dowbenko D. Spencer S. Laura R. Lee J., Gu, Q. Lasky L.A. J. Biol. Chem. 2000; 275: 21477-21485Google Scholar, 25Adey N.B. Huang L. Ormonde P.A. Baumgard M.L. Pero R. Byreddy D.V. Tavtigian S.V. Bartel P.L. Cancer Res. 2000; 60: 35-37Google Scholar). The interaction of PTEN with these proteins may be important for its biological function, as it has been reported that MAGI-2 and MAGI-3 can enhance the activity of PTEN (23Wu X. Hepner K. Castelino-Prabhu S., Do, D. Kaye M.B. Yuan X.J. Wood J. Ross C. Sawyers C.L. Whang Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4233-4238Google Scholar, 24Wu Y. Dowbenko D. Spencer S. Laura R. Lee J., Gu, Q. Lasky L.A. J. Biol. Chem. 2000; 275: 21477-21485Google Scholar). In contrast, several groups found that the PDZ-binding site of PTEN is not required for tumor suppression or other biological activities (21Georgescu M.M. Kirsch K.H. Akagi T. Shishido T. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10182-10187Google Scholar, 25Adey N.B. Huang L. Ormonde P.A. Baumgard M.L. Pero R. Byreddy D.V. Tavtigian S.V. Bartel P.L. Cancer Res. 2000; 60: 35-37Google Scholar, 26Leslie N.R. Gray A. Pass I. Orchiston E.A. Downes C.P. Biochem. J. 2000; 346: 827-833Google Scholar, 27Morimoto A.M. Berson A.E. Fujii G.H. Teng D.H.F. Tavtigian S.V. Bookstein R. Steck P.A. Bolen J.B. Oncogene. 1999; 18: 1261-1266Google Scholar, 28Tolkacheva T. Chan A.M. Oncogene. 2000; 19: 680-689Google Scholar). Therefore, the complete spectrum of PTEN-interacting proteins and the effects of the interactions on PTEN function are not yet defined. To date, the vault complex with a molecular mass of 13 MDa is the largest intracellular ribonucleoprotein particle to be described. Vaults were first observed in preparations of clathrin-coated vesicles (29Kedersha N.L. Rome L.H. J. Cell Biol. 1986; 103: 699-709Google Scholar) and were so-named because of their arched morphology, reminiscent of the vaulted ceilings of a cathedral (29Kedersha N.L. Rome L.H. J. Cell Biol. 1986; 103: 699-709Google Scholar). Vaults are conserved in phylogenetic groups as diverse as mammals, avians, amphibians, sea urchins, and slime molds. The mammalian vaults comprise three proteins, the major vault protein (MVP) 1The abbreviations used are: MVP, major vault protein; VPARP, vault poly(ADP-ribose) polymerase; HA, hemagglutinin; GST, glutathione S-transferase; PLC, phospholipase C. 1The abbreviations used are: MVP, major vault protein; VPARP, vault poly(ADP-ribose) polymerase; HA, hemagglutinin; GST, glutathione S-transferase; PLC, phospholipase C. (30Kedersha N.L. Heuser J.E. Chugani D.C. Rome L.H. J. Cell Biol. 1991; 12: 225-235Google Scholar), the vault poly(ADP-ribose) polymerase (VPARP) (31Kickhoefer V.A. Siva A.C. Kedersha N.L. Inman E.M. Ruland C. Streuli M. Rome L.H. J. Cell Biol. 1999; 146: 917-928Google Scholar), and the telomerase-associated protein 1 (32Kickhoefer V.A. Stephen A.G. Harrington L. Robinson M.O. Rome L.H. J. Biol. Chem. 1999; 274: 32712-32717Google Scholar), as well as one or more small untranslated RNA molecules (33Kickhoefer V.A. Searles R.P. Kedersha N.L. Garber M.E. Johnson D.L. Rome L.H. J. Biol. Chem. 1993; 268: 7868-7873Google Scholar). MVP constitutes >70% of the total mass and is the major vault structural component. The precise cellular function(s) of the vaults are not yet completely understood. However, several studies have implicated that the vaults are involved in nucleocytoplasmic transport. The vaults apparently reside in the cytoplasm, but ∼5% of the vaults are occasionally localized to the cytoplasmic face of the nuclear membrane at or near nuclear pore complexes (34Chugani D.C. Rome L.H. Kedersha N.L. J. Cell Sci. 1993; 106: 23-29Google Scholar). Moreover, the 31-Å resolution structure of vault determined by cryoelectron microscopy reveals a hollow interior that is big enough to enclose a complex as large as intact ribosome (35Kong L.B. Siva A.C. Rome L.H. Stewart P.L. Structure. 1999; 7: 371-379Google Scholar). The hollow structure may indicate an important role for vault in the transport/sequestration of cellular molecules. In addition, MVP has been identified as the lung resistance-related protein (36Scheffer G.L. Wijngaard P.L.J. Flens M.J. Izquierdo M.A. Slovak M.L. Pinedo H.M. Meijer C.J.L.M. Clevers H.C. Scheper R.J. Nat. Med. 1995; 1: 578-582Google Scholar). Many multidrug-resistant cancer cells frequently overexpress MVP/lung resistance-related protein and intact vault particles (reviewed in Ref. 37Scheffer G.L. Schroeijers A.B. Izquierdo M.A. Wiemer E.A. Scheper R.J. Curr. Opin. Oncol. 2000; 12: 550-556Google Scholar). How vault functions in drug resistance is unknown. It has been proposed that vault may function as a transporter or sequester, but proteins or protein complexes that can be transported or sequestered by this particle have yet to be identified. We have employed the protein-protein interaction approach to screen for signaling molecules potentially modulating the activity of PTEN and found that PTEN associates with both MVP and intact vault particles. Rabbit anti-PTEN polyclonal antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, NY) and Cell Signaling Technology Inc. (Beverly, MA). Anti-lung resistance-related protein (MVP) monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY). Anti-Myc (clone 9E10) monoclonal antibody and protein A-Sepharose CL-4B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-hemagglutinin (anti-HA) monoclonal antibody (clone 3F10) used for immunoprecipitation was purchased from Roche Molecular Biochemicals, and anti-HA antibody (clone 12CA5) used for was from the of membrane was from and were from Laboratories CA). was purchased from were from Roche HeLa and obtained from were in in a at PTEN was by from a polymerase and to was by was of the binding domain of in the K. Oncogene. 12: Scholar) to the The human lung as proteins with the domain of in the were obtained from The two-hybrid screen was the to the the and were the yeast on and were for of The was from the and identified by To a for the of MVP in mammalian a in the yeast two-hybrid the residues the of the of MVP in was and the was the at the The an MVP C-terminal PTEN was obtained by the of PTEN by with an site, at the The was transfected cells the the cells were with and in 1 and 1 The were at for at of this was and the was to immunoprecipitation as J. Mol. Cell. Biol. 2002; Scholar). The proteins were on a and to The were with in and with the first antibodies for with the were with the antibody to for 1 and with The were the to the The residues of MVP was by and the The protein was by with at for and as L. M.L. D. J. Biol. Chem. 1998; 273: Scholar). HeLa cells were with and and at for at The was as binding with of or protein were at for with 1 of HeLa in the of various of and as a with 1 of as The were with the of as that in the binding and the proteins were by and HeLa cells were with and with 1 and The were at for at and of were on the of in and for at a were from the of the to The were used for and To PTEN-interacting proteins and its potential we a yeast two-hybrid screen with the of an K. Oncogene. 12: Scholar). The PTEN with a mutation of to which the phosphatase activity but the binding to its substrates T. D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: Scholar), was the K. Oncogene. 12: Scholar). of the yeast results in the of the binding protein and to both tyrosine and interactions. from primary a human lung were for both and revealed that of these the MVP, the major component of vault particles. The MVP identified contains an to the The interaction was confirmed by an MVP with PTEN and such as the domain-containing and The revealed that MVP with PTEN but not with or both were well as by their interaction with in this We determined the interaction between PTEN and MVP is tyrosine To we PTEN the that not and a yeast two-hybrid this in the of is not required for the interaction of MVP with interacts with PTEN but not with in yeast two-hybrid yeast of yeast of yeast not of not in a yeast of yeast of yeast not of not To MVP interacts with PTEN in mammalian we with MVP the residues to the cells. were with an antibody or an anti-HA and the were to in 1 was in the that was with a not The confirmed the association of these two proteins in as was with by the anti-HA antibody 1 MVP is the major component of the vault which is of three proteins and at one small untranslated RNA To PTEN interacts with the vault we vault particles from HeLa cells by on a analysis of the with antibody that the vault particles were in to with a in MVP was in the the that MVP molecules are the vault particles in HeLa cells. analysis of the with anti-PTEN antibody that PTEN was in the of the as well as in the the vault particles were PTEN was not in that the PTEN with the vault was not from the To the of of PTEN in the vault from the we two proteins and in the in was in the vault results that endogenous PTEN associates with intact vault particles in HeLa cells. The association of endogenous PTEN with vault particles to the mechanism of the association between PTEN and We first the MVP binding of PTEN by analysis in the yeast two-hybrid PTEN is of an N-terminal domain, by a C2 domain, two and a PDZ domain binding motif. We made mutations with of C-terminal in the of the PDZ domain binding three and the segments not the However, the of the C2 domain completely the that the C2 domain is for the interaction of PTEN with we determined which of MVP was for the we first putative structural or in reported by van A. M.H. M. G.L. Scheper R.J. P. Wiemer E.A. Biochem. Res. 2002; Scholar), the MVP contains three putative EF in the N-terminal and a in the C-terminal EF is a motif. In residues in and of the structure are involved in residues are conserved in the first two EF but not are conserved in the one the between the and the EF is that between the first and the The MVP we identified in the yeast two-hybrid screen the from to the and contains both the EF and the on this we made that of the N-terminal of including the EF and its C-terminal including the domain, The experiments that PTEN with the N-terminal but not with the C-terminal on these we made of the N-terminal from both the and the in of a N-terminal to of the first EF in complete disruption of the interaction between MVP and that the first EF is for the the C-terminal the interaction for MVP was to a residues that is of the first two EF PTEN interacts with MVP through the C2 domain of PTEN and the first two EF of EF are binding and the binding of the of EF we were in the interaction of MVP with PTEN We experiments a protein residues of MVP in PTEN was by from HeLa in the of but not in its In the PTEN was not by a we proteins with in the to the binding of anti-PTEN antibody to the proteins or the proteins in the The with a molecular mass of by anti-PTEN antibody in the analysis was not in the of and experiments revealed that is required for binding results that PTEN interacts with MVP in a In this we found that endogenous PTEN associates with the vault the largest intracellular ribonucleoprotein particle to The precise cellular function(s) of the vault complex are not yet completely However, its structure and indicate that vault may be involved in the transport/sequestration of cellular molecules. Although the of vaults are in the cytoplasm, a of vaults to the nuclear membrane at or near the nuclear pore complexes (34Chugani D.C. Rome L.H. Kedersha N.L. J. Cell Sci. 1993; 106: 23-29Google Scholar). The structure of vault a hollow interior that is big enough to enclose a complex as large as an intact ribosome (35Kong L.B. Siva A.C. Rome L.H. Stewart P.L. Structure. 1999; 7: 371-379Google Scholar). of vault its role in molecular It has been reported that the vaults with the in the of and this association in to C. V. A. L. A. G. V. A.M. B. J. Cell Biol. 1998; Scholar). the function of the association between PTEN and vault is not It is that the of PTEN is localized to the PTEN is in the However, in PTEN is localized in the M., Gu, J. Matsumoto K. Aota S. Parsons R. Yamada K. Science. 1998; 280: 1614-1617Google Scholar, A. Marsh D.J. U. R. L. Robinson P. H. Eng C. Am. J. 2000; Scholar, M.B. P. S. R. S. Ross A.H. J. 2000; Scholar, A. P. P. C. S. Eng C. Am. J. 2000; Scholar, D.C. S. N.K. Eng C. J. 2002; 99: Scholar). PTEN not a nuclear and the mechanism by which PTEN is localized to the is unknown. the endogenous PTEN interacts with vault that can be localized in the nuclear it is that vault the cellular of has been that both MVP and the vault particle are frequently in multidrug-resistant cancer cells (reviewed in Ref. 37Scheffer G.L. Schroeijers A.B. Izquierdo M.A. Wiemer E.A. Scheper R.J. Curr. Opin. Oncol. 2000; 12: 550-556Google Scholar). Although it has not been that the is by the of studies have demonstrated that of MVP the and of MVP drug resistance M. T. Y. T. S. A. S. T. S. J. Natl. Cancer 1999; Scholar, S.H. Lee 2000; Scholar, Y. Stephen A.G. J. V. A.H. Rome L.H. J. 2002; 97: Scholar). for drug resistance to the such as protein 1 and protein which by intracellular drug (reviewed in Ref. 37Scheffer G.L. Schroeijers A.B. Izquierdo M.A. Wiemer E.A. Scheper R.J. Curr. Opin. Oncol. 2000; 12: 550-556Google Scholar). How vaults drug resistance in cancer cells is It may by binding or proteins and or to a cellular the drug are not it may by the function of other proteins to drug as an that dephosphorylates the phosphoinositide 3-kinase pathway and of PTEN activity its to membrane that in resistance to drug vaults may PTEN function through their interaction to drug resistance in cancer cells. The experiments that the interaction between these two proteins is through the C2 domain of PTEN and the two EF of The of EF is both and for the vault of of three proteins, and MVP is the major vault component. vault particle contains VPARP, two telomerase-associated protein and MVP molecules. van A. M.H. M. G.L. Scheper R.J. P. Wiemer E.A. Biochem. Res. 2002; Scholar) the function of structural domain of these three proteins for the of the vault particles. It was found that MVP through its C-terminal and interacts with through its N-terminal MVP not with telomerase-associated protein the of to MVP molecules in the vault particle is the of MVP not be completely by a of the EF may be it for vault to with other the C2 and the EF are as binding the C2 domain of PTEN not to We found that vitro interaction between PTEN and MVP that may the association of PTEN with the vault particles in interaction through C2 domain and EF is not a However, proteins both the C2 and the EF such as the phospholipase proteins 1999; Scholar). The structure revealed that the of the EF of an interaction with its C2 domain 1999; Scholar). The C2 domain of PTEN has structural to that of with an of for (21Georgescu M.M. Kirsch K.H. Akagi T. Shishido T. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10182-10187Google Scholar). It be to the EF of MVP with the C2 domain of PTEN with a mechanism to that of the interaction of the EF and the C2 domain of We J. A. for the and we and for on the

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