The Tuberous Sclerosis 2 Gene Product, Tuberin, Functions as a Rab5 GTPase Activating Protein (GAP) in Modulating Endocytosis

The tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene that plays a causative role in the autosomal dominant syndrome of tuberous sclerosis. The latter is characterized by the development of hamartomas and occasional malignancies. Expression of the wild-type gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This “suppressor− activity is encoded by functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2, a cytosolic factor, rabaptin-5, was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possessed activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity was specific for Rab5 and showed no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possessed minimal Rab5GAP activity and were associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduced the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis. The tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene that plays a causative role in the autosomal dominant syndrome of tuberous sclerosis. The latter is characterized by the development of hamartomas and occasional malignancies. Expression of the wild-type gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This “suppressor− activity is encoded by functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2, a cytosolic factor, rabaptin-5, was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possessed activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity was specific for Rab5 and showed no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possessed minimal Rab5GAP activity and were associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduced the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis. INTRODUCTIONTumor suppressor genes consist of a diverse group of genetic elements that encode proteins whose normal functions are to suppress cell proliferation and tumor formation. Their inactivation often plays a critical role in neoplastic transformation and is responsible for the initiation of the majority of hereditary cancers in humans. The TSC2 gene is a new member of the tumor suppressor gene family that is involved in the autosomal dominant syndrome of tuberous sclerosis (TSC) 1The abbreviations used are: TSCtuberous sclerosis complexCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferaseGAPGTPase activating proteinHRPhorseradish peroxidaseDTTdithiothreitol. (1Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1498) Google Scholar). The latter is a multi-organ disease of benign tumors (i.e. hamartomas) and malformations affecting tissues of mesodermal and ectodermal derivation (2Gomez M.R. Tuberous Sclerosis. Raven Press, New York1988Google Scholar). Occasionally, additional tumorigenic events can lead to malignant transformation affecting mainly the kidneys (3Bernstein J. Robbins T.O. Ann. N. Y. Acad. Sci. 1991; 615: 36-49Crossref PubMed Scopus (79) Google Scholar).The study of the Eker rat model of hereditary cancer has provided additional evidence for the tumor suppressor role of TSC2 (4Yeung R.S. Xiao G.-H. Jin F. Lee W-C. Testa J.R. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (270) Google Scholar, 5Kobayashi T. Hirayama Y. Kobayashi E. Kubo Y. Hino O. Nat. Genet. 1995; 9: 70-74Crossref PubMed Scopus (289) Google Scholar). These animals carry a germline mutation of TSC2, and tumors arising from the kidneys and uterus showed frequent loss of heterozygosity at this locus resulting in loss of protein expression (6Xiao G.-H. Jin F. Yeung R.S. Oncogene. 1995; 11: 81-87PubMed Google Scholar, 7Yeung R.S. Xiao G.H. Everitt J. Jin F. Walker C. Mol. Carcinogen. 1995; 14: 28-36Crossref PubMed Scopus (64) Google Scholar). Introduction of a wild-type TSC2 gene or its 3′ region into the Eker tumor cell lines inhibited cell proliferation and tumorigenicity, thus providing direct experimental evidence for tuberin tumor suppressor function (8Jin F. Wienecke R. Xiao G.H. Maize J. DeClue J.E. Yeung R.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9154-9159Crossref PubMed Scopus (119) Google Scholar).The TSC2 gene, identified through positional cloning, encodes an open reading frame of 1870 amino acids with a region of sequence homology with the catalytic domain of Rap1GAP near the C terminus (1Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1498) Google Scholar). Multiple splice variants that are conserved between rodents and human have been identified and are differentially expressed in adult tissues (9Xiao G.-H. Jin F. Yeung R.S. Cell Growth 6: 1185-1191PubMed Google Scholar, 10Xu L. Sterner C. Maheshwar M.M. Wilson P.J. Nellist M. Short P.M. Haines J.L. Sampson J.R. Ramesh V. Genomics. 1995; 27: 475-480Crossref PubMed Scopus (60) Google Scholar). The ∼190-kDa protein product, tuberin, is widely expressed and separates with the membrane/particulate (100,000 × g pellet) fraction (1Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1498) Google Scholar, 4Yeung R.S. Xiao G.-H. Jin F. Lee W-C. Testa J.R. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (270) Google Scholar, 11Wienecke R. Konig A. DeClue J.E. J. Biol. Chem. 1995; 270: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Immunofluoresence analysis has sublocalized the protein to the perinuclear region where it co-localized with Rap1 (12Wienecke R. Maize J.C. Shoarinejad F. Vass W.C. Reed J. Bonifacino J.S. Resau J.H. de Gunzburg J. Yeung R.S. DeClue J.E. Oncogene. 1996; 13: 913-923PubMed Google Scholar). Biochemical analysis has demonstrated in vitro GAP activity toward Rap1a, but the degree of stimulation of the intrinsic GTPase by tuberin is weak (11Wienecke R. Konig A. DeClue J.E. J. Biol. Chem. 1995; 270: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). While the physiologic significance of this activity in vivo remains undefined, it has been postulated that tuberin defective in its GAP activity could lead to the constitutive activation of Rap1a or other monomeric GTPase proteins which may result in deregulated mitogenic signaling in the target cells. Such a model would be analogous to the role of mutant neurofibromin in modulating Ras GTPase activity in schwannomas (13DeClue J.E. Papageorge A.G. Fletcher J.A. Diehl S.R. Ratner N. Vass W.C. Lowy D.R. Cell. 1992; 69: 265-273Abstract Full Text PDF PubMed Scopus (513) Google Scholar).The Ras superfamily of small GTP-binding proteins are central to a wide variety of cellular processes and are regulated by different classes of proteins that determine the “on-off− state of the GTPases. GAPs stimulate the intrinsic rate of GTPases and serve as negative regulators of these binary switches. Unique GAPs exist for specific families of GTPases and perhaps for each member of the family. Substrate specificity exhibited by GAP proteins appears quite stringent. The p120GAP specifically stimulates GTPase activities of Ha-Ras, N-Ras, Ki-Ras and R-Ras, but not those of Rho, Rac, or Rab (14Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar). Rap1a, a closely related member of Ras, can bind to p120GAP, but its GTPase activity is unaffected (15Bollag G. McCormick F. Annu. Rev. Cell Biol. 1991; 7: 601-632Crossref PubMed Scopus (288) Google Scholar). Proteins that activate GTPase of Rap1 include Rap1GAP, Spa1, and tuberin, but they share no similarity with p120GAP. To understand the mechanism of tuberin function, studies were undertaken to identify proteins that interact with the C-terminal fragment containing the GAP homology domain. Surprisingly, we identified a tuberin-binding molecule that associates with the small GTPase Rab5 and demonstrated specific GAP activity of tuberin toward Rab5. These findings have implications for tuberin function in the endocytic pathway.RESULTS AND DISCUSSIONWe focused on the tuberin C-terminal region encompassing the GAP domain as a probe to identify potential binding molecules. An ∼1-kilobase BamHI fragment of the 3′ end of the rat TSC2 gene (nucleotides 4351-5352) was cloned into pJK202 as bait vector in a yeast two-hybrid system (16Golemis E. Gyuris J. Brent R. Current Protocols in Molecular Biology. John Wiley 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar). Of the four partial rabaptin-5 clones identified in our screen, the smallest overlapping region corresponded to nucleotides 455-717 (amino acids 90-176) near the 5′ end of the gene. In contrast, Stenmark et al. (19Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar) reported interaction of GTP-Rab5 with the C terminus of rabaptin-5 amino acids 551-862 (19Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar). Thus there exist at least two protein-protein-binding domains in rabaptin-5. To define the structural requirement of tuberin for rabaptin-5 binding, deletion subclones of the LexA-TSC2 constructs were tested for β-galactosidase activity and leucine auxotrophy. The smallest region of overlap was mapped to a 59-amino acid fragment near the C terminus of tuberin amino acids 1668-1726 (Fig. 1). The TSC2-GAP homology domain lies adjacent but distinct from this region. Of significance, there exist at least two examples of missense mutations within this rabaptin-5-binding domain in two affected TSC individuals (20Wilson P.J. Ramesh V. Kristiansen A. Bove C. Jozwiak S. Kwiatkowski D.J. Short M.P. Haines J.L. Hum. Mol. Genet. 1996; 5: 249-256Crossref PubMed Scopus (74) Google Scholar).To further investigate in vivo tuberin-rabaptin-5 interaction in eukaryotic cells, binding assays were conducted following transient transfection in COS-7 cells and with endogenous proteins in human-derived cell lines. A polyclonal anti-rabaptin-5 antiserum, anti-B9, directed against the TSC2-binding domain of rabaptin-5 (residues 455-717) was used to immunoprecipitate lysate from COS-7 cells transfected with full-length rat TSC2 and/or partial rabaptin-5 cDNAs (data not shown). The resultant protein complexes were resolved on SDS-PAGE and analyzed on immunoblot developed with anti-tuberin antibody, anti-L3. The ∼190-kDa tuberin band co-purified with rabaptin-5 in cells transfected with both TSC2 and rabaptin-5 expression vectors and not when either one is omitted. The ability of endogenous tuberin to stably bind to rabaptin-5 was assessed in HeLa and CCL-136 (human sarcoma cell line, ATCC, Rockville, MD) cells. Lysates were immunoprecipitated with anti-L3, and the products were analyzed for the presence of rabaptin-5 by immunoblot detection. The anti-L3 purified tuberin complexed to the 115-kDa rabaptin-5 that was not evident in the preimmune sera immunoprecipitate (data not shown). These results established that tuberin physically associates with rabaptin-5 in vivoAs a membrane-bound GTPase activating protein, tuberin may promote the hydrolysis of Rab5-GTP to its inactive GDP form via its interaction with rabaptin-5. To test this hypothesis, in vitro GAP activity of tuberin was examined using purified Rab5 as the substrate. Native tuberin immunoprecipitated from HeLa cells with anti-L3 showed substantial levels of GAP activity when incubated with γ-32PGTP-Rab5 (Fig. 2A). While the intrinsic GTPase activity of Rab5 in vitro was high as was previously shown (21Stenmark H. Parton R.G. Steele-Mortimer O. Lutcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (767) Google Scholar, V. Ullrich O. M. R. Zerial M. Nature. 1996; PubMed Scopus Google the rate of GTP hydrolysis was further by the anti-L3 immunoprecipitate and not preimmune (Fig. Tuberin GAP activity toward Rab5 was specific no activation of GTPase activity was for Ras and other of the Rab family Rab3a and (Fig. GAP activity was also using purified recombinant protein of the C-terminal region of tuberin, in the of rabaptin-5 (data not shown). This that the latter is not for GTPase activation and may function as an protein to Rab5-GTP to evidence reported that in the of rabaptin-5, the rate of hydrolysis by Rab5 in vitro was reduced V. Ullrich O. M. R. Zerial M. Nature. 1996; PubMed Scopus Google Scholar). This would suggest that the binding of of rabaptin-5 to both Rab5 and tuberin the interaction between the GTPase and its specific GAP activity for Rab5. activation of Rab5 intrinsic GTPase activity by native tuberin was by assay and expressed as the fraction of GTP to Rab5 as a function of immunoprecipitate of preimmune native tuberin immunoprecipitated by HeLa cell analysis of the by showed that the Rab5 was specifically to The of GTP a with preimmune and native tuberin were by analysis and for the of In vitro GAP assay using native tuberin to activity toward Rab3a and as in A intrinsic GTPase activities were in the of each results were in two to cell of and were for Rab5 GAP cells were of endogenous tuberin and possessed minimal activity with has been also as a for tuberin GAP activity in but the of activation is weak (11Wienecke R. Konig A. DeClue J.E. J. Biol. Chem. 1995; 270: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). of our the GAP activity of tuberin on Rap1a was to a that of Rab5 (data not that Rab5 may be the target for tuberin GAP function in HeLa cells. To determine the in vivo of the Rab5GAP activity of tuberin, we examined the levels of GAP activity in cells with and endogenous The latter of cells from an Eker cell lysate from possessed minimal GAP activity toward Rab5 with cell lysate (Fig. cellular Rab5GAP activity with tuberin The evidence for a interaction between tuberin, and Rap1 the of tuberin toward two GTPases. This is in of the specificity of GAP Rab5 and Rap1a to different families of the GTPases and share While Rab5 has a function in the endocytic the physiologic role of Rap1a is well both Rab5 and Rap1 have been in also in the and is with the perinuclear of tuberin (12Wienecke R. Maize J.C. Shoarinejad F. Vass W.C. Reed J. Bonifacino J.S. Resau J.H. de Gunzburg J. Yeung R.S. DeClue J.E. Oncogene. 1996; 13: 913-923PubMed Google Scholar, C. Parton R.G. H. Zerial M. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, V. M. C. Parton R.G. Zerial M. J. Cell Sci. 1994; Google Scholar). results suggest a potential role of TSC2 product in the of Rab5 GTPase is a critical and of the and of the endocytic C. Parton R.G. H. Zerial M. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). This that proteins the state of Rab5 and may have a role in endocytosis. To the potential function of tuberin in we examined the of endogenous TSC2 gene expression in fluid-phase uptake in TSC2 mutant cells from the Eker transient to from of in vitro with those of (Fig. These cells, in tuberin protein expressed of Rab5 and rabaptin-5 (Fig. This in with the endogenous GAP activities for Rab5 of these two cell lines is with a negative role of tuberin on Rab5 activity in vivo in transient transfection tuberin in tumor cells reduced the rate of uptake (data not shown). remains to be which tuberin the rate of fluid-phase endocytosis. of in Eker was with that of following a at of were to HeLa and expressed as the from two with both cell lines expressed of Rab5 and rabaptin-5, but expressed cell were with for for for is specific and has been postulated that of may exist for Rab proteins to for the findings have identified tuberin as a protein with substantial activity and demonstrated that tuberin with Rab5 is by an rabaptin-5. These to the model of the endocytic in which a Rab5-GTP rabaptin-5 to the cytosolic of the in the to a where and can A function of tuberin is to stimulate hydrolysis of the and rabaptin-5 into the for The specificity of this interaction is by at least two Rabaptin-5 specific binding for Rab5 and not other related GTPases (19Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google and tuberin GAP activity not with other Rab5. the functional between rabaptin-5, and tuberin one of specificity that may in with the to evidence that Rab5 is for GTP hydrolysis by Rab5 is not in this but the is on cytosolic V. Ullrich O. M. R. Zerial M. Nature. 1996; PubMed Scopus Google Scholar). the is for is not but tuberin is of with other proteins that may serve as additional of the S. This is with that rabaptin-5 may exist in a high complex (19Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar).The mechanism by which a tumor suppressor gene, as TSC2, the development of and on its on protein remains is to suggest that the loss of tuberin Rab5GAP activity would with the and of the of the endocytic could lead to of or other membrane-bound that would cells in to cells A. Wiley PubMed Scopus Google Scholar). evidence also to the critical role of endocytic not in signaling but also in specific signaling C. 1996; PubMed Scopus Google Scholar). remains to be tuberin could lead to of The of Rab5GAP and Rap1GAP activities in tumor is to be INTRODUCTIONTumor suppressor genes consist of a diverse group of genetic elements that encode proteins whose normal functions are to suppress cell proliferation and tumor formation. Their inactivation often plays a critical role in neoplastic transformation and is responsible for the initiation of the majority of hereditary cancers in humans. The TSC2 gene is a new member of the tumor suppressor gene family that is involved in the autosomal dominant syndrome of tuberous sclerosis (TSC) 1The abbreviations used are: TSCtuberous sclerosis complexCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferaseGAPGTPase activating proteinHRPhorseradish peroxidaseDTTdithiothreitol. (1Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1498) Google Scholar). The latter is a multi-organ disease of benign tumors (i.e. hamartomas) and malformations affecting tissues of mesodermal and ectodermal derivation (2Gomez M.R. Tuberous Sclerosis. Raven Press, New York1988Google Scholar). Occasionally, additional tumorigenic events can lead to malignant transformation affecting mainly the kidneys (3Bernstein J. Robbins T.O. Ann. N. Y. Acad. Sci. 1991; 615: 36-49Crossref PubMed Scopus (79) Google Scholar).The study of the Eker rat model of hereditary cancer has provided additional evidence for the tumor suppressor role of TSC2 (4Yeung R.S. Xiao G.-H. Jin F. Lee W-C. Testa J.R. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (270) Google Scholar, 5Kobayashi T. Hirayama Y. Kobayashi E. Kubo Y. Hino O. Nat. Genet. 1995; 9: 70-74Crossref PubMed Scopus (289) Google Scholar). These animals carry a germline mutation of TSC2, and tumors arising from the kidneys and uterus showed frequent loss of heterozygosity at this locus resulting in loss of protein expression (6Xiao G.-H. Jin F. Yeung R.S. Oncogene. 1995; 11: 81-87PubMed Google Scholar, 7Yeung R.S. Xiao G.H. Everitt J. Jin F. Walker C. Mol. Carcinogen. 1995; 14: 28-36Crossref PubMed Scopus (64) Google Scholar). Introduction of a wild-type TSC2 gene or its 3′ region into the Eker tumor cell lines inhibited cell proliferation and tumorigenicity, thus providing direct experimental evidence for tuberin tumor suppressor function (8Jin F. Wienecke R. Xiao G.H. Maize J. DeClue J.E. Yeung R.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9154-9159Crossref PubMed Scopus (119) Google Scholar).The TSC2 gene, identified through positional cloning, encodes an open reading frame of 1870 amino acids with a region of sequence homology with the catalytic domain of Rap1GAP near the C terminus (1Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1498) Google Scholar). Multiple splice variants that are conserved between rodents and human have been identified and are differentially expressed in adult tissues (9Xiao G.-H. Jin F. Yeung R.S. Cell Growth 6: 1185-1191PubMed Google Scholar, 10Xu L. Sterner C. Maheshwar M.M. Wilson P.J. Nellist M. Short P.M. Haines J.L. Sampson J.R. Ramesh V. Genomics. 1995; 27: 475-480Crossref PubMed Scopus (60) Google Scholar). The ∼190-kDa protein product, tuberin, is widely expressed and separates with the membrane/particulate (100,000 × g pellet) fraction (1Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1498) Google Scholar, 4Yeung R.S. Xiao G.-H. Jin F. Lee W-C. Testa J.R. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (270) Google Scholar, 11Wienecke R. Konig A. DeClue J.E. J. Biol. Chem. 1995; 270: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Immunofluoresence analysis has sublocalized the protein to the perinuclear region where it co-localized with Rap1 (12Wienecke R. Maize J.C. Shoarinejad F. Vass W.C. Reed J. Bonifacino J.S. Resau J.H. de Gunzburg J. Yeung R.S. DeClue J.E. Oncogene. 1996; 13: 913-923PubMed Google Scholar). Biochemical analysis has demonstrated in vitro GAP activity toward Rap1a, but the degree of stimulation of the intrinsic GTPase by tuberin is weak (11Wienecke R. Konig A. DeClue J.E. J. Biol. Chem. 1995; 270: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). While the physiologic significance of this activity in vivo remains undefined, it has been postulated that tuberin defective in its GAP activity could lead to the constitutive activation of Rap1a or other monomeric GTPase proteins which may result in deregulated mitogenic signaling in the target cells. Such a model would be analogous to the role of mutant neurofibromin in modulating Ras GTPase activity in schwannomas (13DeClue J.E. Papageorge A.G. Fletcher J.A. Diehl S.R. Ratner N. Vass W.C. Lowy D.R. Cell. 1992; 69: 265-273Abstract Full Text PDF PubMed Scopus (513) Google Scholar).The Ras superfamily of small GTP-binding proteins are central to a wide variety of cellular processes and are regulated by different classes of proteins that determine the “on-off− state of the GTPases. GAPs stimulate the intrinsic rate of GTPases and serve as negative regulators of these binary switches. Unique GAPs exist for specific families of GTPases and perhaps for each member of the family. Substrate specificity exhibited by GAP proteins appears quite stringent. The p120GAP specifically stimulates GTPase activities of Ha-Ras, N-Ras, Ki-Ras and R-Ras, but not those of Rho, Rac, or Rab (14Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar). Rap1a, a closely related member of Ras, can bind to p120GAP, but its GTPase activity is unaffected (15Bollag G. McCormick F. Annu. Rev. Cell Biol. 1991; 7: 601-632Crossref PubMed Scopus (288) Google Scholar). Proteins that activate GTPase of Rap1 include Rap1GAP, Spa1, and tuberin, but they share no similarity with p120GAP. To understand the mechanism of tuberin function, studies were undertaken to identify proteins that interact with the C-terminal fragment containing the GAP homology domain. Surprisingly, we identified a tuberin-binding molecule that associates with the small GTPase Rab5 and demonstrated specific GAP activity of tuberin toward Rab5. These findings have implications for tuberin function in the endocytic