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• W2090200732 abstract "We recently identified BNIP-2, a previously cloned Bcl-2- and E1B-associated protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite the lack of homology to previously described catalytic domains of GTPase-activating proteins (GAPs). BNIP-2 contains many arginine residues at the carboxyl terminus, which includes the region of homology to the noncatalytic domain of Cdc42GAP, termed BNIP-2 and Cdc42GAPhomology (BCH) domain. Using BNIP-2 glutathioneS-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into α-helical bundles similar to the topology of the catalytic GAP domain of Cdc42GAP. Alignment of exposed arginine residues in this domain helped to identify Arg-235 and Arg-238 as good candidates for catalysis. Arg-238 matched well to the arginine “finger” required for enhanced GTP hydrolysis in homodimerized Cdc42. Site-directed mutagenesis confirmed that an R235K or R238K mutation severely impaired the BNIP-2 GAP activity without affecting its binding to Cdc42. From deletion studies, a region adjacent to the arginine patch (288EYV290 on BNIP-2) and the Switch I and Rho family-specific “Insert” region on Cdc42 are involved in the binding. The results indicate that the BCH domain of BNIP-2 represents a novel GAP domain that employs an arginine patch motif similar to that of the Cdc42-homodimer. We recently identified BNIP-2, a previously cloned Bcl-2- and E1B-associated protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite the lack of homology to previously described catalytic domains of GTPase-activating proteins (GAPs). BNIP-2 contains many arginine residues at the carboxyl terminus, which includes the region of homology to the noncatalytic domain of Cdc42GAP, termed BNIP-2 and Cdc42GAPhomology (BCH) domain. Using BNIP-2 glutathioneS-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into α-helical bundles similar to the topology of the catalytic GAP domain of Cdc42GAP. Alignment of exposed arginine residues in this domain helped to identify Arg-235 and Arg-238 as good candidates for catalysis. Arg-238 matched well to the arginine “finger” required for enhanced GTP hydrolysis in homodimerized Cdc42. Site-directed mutagenesis confirmed that an R235K or R238K mutation severely impaired the BNIP-2 GAP activity without affecting its binding to Cdc42. From deletion studies, a region adjacent to the arginine patch (288EYV290 on BNIP-2) and the Switch I and Rho family-specific “Insert” region on Cdc42 are involved in the binding. The results indicate that the BCH domain of BNIP-2 represents a novel GAP domain that employs an arginine patch motif similar to that of the Cdc42-homodimer. GTPase-activating protein BNIP-2 and Cdc42GAP homology breakpoint cluster region homology glutathioneS-transferase guanosine 5′-3-O-(thio)triphosphate hemagglutinin polyacrylamide gel electrophoresis Ras superfamily GTPase proteins act as molecular switches for signal transduction pathways to control cell growth, differentiation, and motility. The Ras superfamily consists of the Ras, Rho, Rab, and Arf families, which are classified according to their sequence similarities and functions (1.Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 2.Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (341) Google Scholar). These proteins cycle between two guanine-nucleotide bound states, the GTP-bound form, which is active, and the inactive GDP-bound form. Activation occurs as a result of a change in the conformation of discrete “switch regions” in these proteins that allow them to interact with their appropriate effector proteins. The GTP/GDP-regulated proteins possess a low intrinsic activity for hydrolyzing GTP to GDP, but for efficient physiological catalysis, they associate with other proteins, which can enhance their GTPase activity further. These proteins are termed GTPase-activating proteins (GAPs),1 and they have been recognized by conserved amino acid sequence motifs that are characteristic of each family (3.Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar).The Rho subfamily of GTPases, which includes RhoA, RhoB, RhoC, RhoE, RhoG, Rac1, Rac2, Cdc42, and TC10, is involved in various aspects of cytoskeletal organization, cell polarity, and motility (4.Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 5.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar, 6.Hall A. Br. J. Cancer. 1999; 80 Suppl 1: 25-27PubMed Google Scholar). For example, RhoA is involved in the regulation of stress fibers and focal adhesion formation (7.Chardin P. Boquet P. Modaule P. Popoff M.R. Rubin E.J. Gill D.M. EMBO J. 1989; 8: 1087-1092Crossref PubMed Scopus (416) Google Scholar, 8.Paterson H.F. Self A.J. Garrett M.D. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1009Crossref PubMed Scopus (567) Google Scholar), Rac1 is involved in the formation of lamellipodia and membrane ruffling (9.Waterman-Storer C.M. Worthylake R.A. Liu B.P. Burridge K. Salmon E.D. Nat. Cell Biol. 1999; 1: 45-50Crossref PubMed Scopus (395) Google Scholar, 10.Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar), and Cdc42 is necessary for actin microspikes/filopodia to form (10.Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar, 11.Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3698) Google Scholar). Furthermore, all three can activate the Jun amino-terminal kinase, affect the transcription of certain target genes, and regulate the progression of the cell cycle (12.Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1443) Google Scholar, 13.Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (284) Google Scholar). Structural and biochemical studies show that all GAPs, although bearing no close overall sequence homology to each other, exert their effect either by contributing catalytic residuesin-trans, by lowering the activation energy for GTP hydrolysis, or by stabilizing the conformation of the inherent GTPases. These mechanisms are employed by 120-kDa RasGAP and the 50-kDa RhoGAP/Cdc42GAP through a highly conserved arginine “finger” catalytic motif and by similar binding topology (3.Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 14.Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (122) Google Scholar, 15.Rittinger K. Walker P.A. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 16.Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 17.Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1176) Google Scholar, 18.Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (292) Google Scholar, 19.Barrett T. Xiao B. Dodson E.J. Dodson G. Ludbrook S.B. Nurmahomed K. Gamblin S.J. Musacchio A. Smerdon S.J. Eccleston J.F. Nature. 1997; 385: 458-461Crossref PubMed Scopus (98) Google Scholar, 20.Bax B. Nature. 1998; 392: 447-448Crossref PubMed Scopus (15) Google Scholar, 21.Rittinger K. Taylor W.R. Smerdon S.J. Gamblin S.J. Nature. 1998; 392: 448-449Crossref PubMed Scopus (26) Google Scholar, 22.Calmels T.P. Callebaut I. Leger I. Durand P. Bril A. Mornon J.P. Souchet M. FEBS Lett. 1998; 426: 205-211Crossref PubMed Scopus (14) Google Scholar). Recently, however, the crystal structure of rna1p, the Schizosaccharomyces pombe ortholog of the mammalian GAP of Ran, revealed a completely different folding pattern that nevertheless contributed to both the catalysis and the stabilization effect on Ran (23.Hillig R.C. Renault L. Vetter I.R. Drell T., IV Wittinghofer A. Becker J. Mol. Cell. 1999; 3: 781-791Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar).GAPs have been identified that act preferentially on members of the Rho subfamily. It is interesting to note that two different catalytic domains have been described for GAPs acting on Cdc42. The 50-kDa Cdc42GAP was shown in various crystallographic studies to possess the conventional arginine finger that interacted closely with the Switch I domain from Cdc42 to affect catalysis. The second type of catalytic domain, albeit of unproven physiological relevance, was identified when Cdc42 forms a homodimer. In this circumstance, one molecule can act as a GAP toward the other partner, with catalysis being mediated by a conserved arginine patch in the carboxyl terminus (24.Zhang B. Zheng Y. J. Biol. Chem. 1998; 273: 25728-25733Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25.Zhang B. Zhang Y. Collins C.C. Johnson D.I. Zheng Y. J. Biol. Chem. 1999; 274: 2609-2612Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).We have recently shown BNIP-2, a previously cloned Bcl-2 interacting protein, to be a putative substrate of the FGF receptor tyrosine kinase and to bind both Cdc42GAP and Cdc42 when not tyrosine-phosphorylated. Interestingly, BNIP-2 was also shown to possess a “GAP-like” activity toward Cdc42 (26.Low B.C. Lim Y.P. Lim J. Wong E.S.M. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This protein contains no sequence homology to the canonical catalytic domain of a GAP, but it shares a highly conserved sequence with a region in the amino-terminal, noncatalytic half of Cdc42GAP (26.Low B.C. Lim Y.P. Lim J. Wong E.S.M. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 27.Boyd J.M. Malstrom S. Subramanian T. Venkatesh L.K. Schaeper U. Elangovan B. D'Sa-Eipper C. Chinnadurai G. Cell. 1994; 79: 341-351Abstract Full Text PDF PubMed Scopus (392) Google Scholar). We wished to determine the sequences in BNIP-2 that were responsible for the binding to Cdc42 and for the catalysis. To do this, we used a combination of site-directed mutagenesis and molecular modeling based on both the known biochemistry and structural topology of the catalytic Cdc42GAP domains. It enabled a hypothetical model to be constructed that formed the basis for the mutational studies. Using this approach, we have found that theBNIP-2 and Cdc42GAP homology (BCH) domain contains the GAP activity in BNIP-2, but not in Cdc42GAP. Within this novel domain, there are two critical arginine residues that are important for conferring the GAP activity. This arginine patch shares reasonable similarity to those residues that mediate the homodimerization-induced GAP activity seen with Cdc42 homodimers (24.Zhang B. Zheng Y. J. Biol. Chem. 1998; 273: 25728-25733Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar,25.Zhang B. Zhang Y. Collins C.C. Johnson D.I. Zheng Y. J. Biol. Chem. 1999; 274: 2609-2612Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Other discrete regions likely to be important for the BNIP-2 and Cdc42 interaction were also identified.DISCUSSIONWe have identified several arginines as key residues within the BNIP-2 BCH domain that are responsible for the GAP activity of the protein. This region containing the arginines bears no similarity to the arginine motifs employed by the “cradle-fold” structural topology in the RasGAP or Cdc42GAP/RhoGAP catalytic structures (3.Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar,14.Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (122) Google Scholar, 15.Rittinger K. Walker P.A. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 16.Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 17.Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1176) Google Scholar, 18.Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (292) Google Scholar, 19.Barrett T. Xiao B. Dodson E.J. Dodson G. Ludbrook S.B. Nurmahomed K. Gamblin S.J. Musacchio A. Smerdon S.J. Eccleston J.F. Nature. 1997; 385: 458-461Crossref PubMed Scopus (98) Google Scholar, 20.Bax B. Nature. 1998; 392: 447-448Crossref PubMed Scopus (15) Google Scholar, 21.Rittinger K. Taylor W.R. Smerdon S.J. Gamblin S.J. Nature. 1998; 392: 448-449Crossref PubMed Scopus (26) Google Scholar, 22.Calmels T.P. Callebaut I. Leger I. Durand P. Bril A. Mornon J.P. Souchet M. FEBS Lett. 1998; 426: 205-211Crossref PubMed Scopus (14) Google Scholar) or to the leucine repeat folds of RanGAP binding to Ran (23.Hillig R.C. Renault L. Vetter I.R. Drell T., IV Wittinghofer A. Becker J. Mol. Cell. 1999; 3: 781-791Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). It does, however, show a striking similarity to the polybasic carboxyl terminus of Cdc42. Although not required for the formation of dimers, this polybasic patch in Cdc42 was identified to be responsible for its GAP-like activity when homodimers are formed (24.Zhang B. Zheng Y. J. Biol. Chem. 1998; 273: 25728-25733Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25.Zhang B. Zhang Y. Collins C.C. Johnson D.I. Zheng Y. J. Biol. Chem. 1999; 274: 2609-2612Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Of particular interest is that in this local alignment, the Arg-238 in BNIP-2 matches the critical arginine residue that is indispensable for the homodimer-enhanced GAP activity. In those members of the Rho subfamily that fail to display GAP activity when homodimerized, i.e.RhoA and the yeast form of Cdc42 (25.Zhang B. Zhang Y. Collins C.C. Johnson D.I. Zheng Y. J. Biol. Chem. 1999; 274: 2609-2612Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), this arginine is absent, as it is from the BCH domain of Cdc42GAP, which is also catalytically inactive (Fig. 2 D). Although it lacks GAP activity, the BCH domain of Cdc42GAP can still bind to Cdc42 (data not shown).Despite the varying degrees of the loss of GAP activity displayed by the R238K, R235K, and R236K mutants, BNIP-2 still retains its ability to bind Cdc42, indicating that these residues are not involved in the binding to Cdc42. The conserved arginine fingers in the GAP domains of Cdc42GAP (34.Graham D.L. Eccleston J.F. Lowe P.N. Biochemistry. 1999; 38: 985-991Crossref PubMed Scopus (69) Google Scholar) or RasGAP (18.Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (292) Google Scholar, 35.Geyer M. Schweins T. Herrmann C. Prisner T. Wittinghofe A. Kalbitzer H.R. Biochemistry. 1996; 35: 10308-10320Crossref PubMed Scopus (189) Google Scholar) have all been shown to be important for catalysis and not for binding to their respective partners. In both Cdc42GAP and RasGAPs, catalytic enhancement is likely to be the result of stabilization of the conformation most complementary to the transition state and from ground state destabilization (3.Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 14.Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (122) Google Scholar, 15.Rittinger K. Walker P.A. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 16.Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 17.Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1176) Google Scholar, 18.Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (292) Google Scholar, 19.Barrett T. Xiao B. Dodson E.J. Dodson G. Ludbrook S.B. Nurmahomed K. Gamblin S.J. Musacchio A. Smerdon S.J. Eccleston J.F. Nature. 1997; 385: 458-461Crossref PubMed Scopus (98) Google Scholar, 20.Bax B. Nature. 1998; 392: 447-448Crossref PubMed Scopus (15) Google Scholar, 21.Rittinger K. Taylor W.R. Smerdon S.J. Gamblin S.J. Nature. 1998; 392: 448-449Crossref PubMed Scopus (26) Google Scholar, 22.Calmels T.P. Callebaut I. Leger I. Durand P. Bril A. Mornon J.P. Souchet M. FEBS Lett. 1998; 426: 205-211Crossref PubMed Scopus (14) Google Scholar,34.Graham D.L. Eccleston J.F. Lowe P.N. Biochemistry. 1999; 38: 985-991Crossref PubMed Scopus (69) Google Scholar, 35.Geyer M. Schweins T. Herrmann C. Prisner T. Wittinghofe A. Kalbitzer H.R. Biochemistry. 1996; 35: 10308-10320Crossref PubMed Scopus (189) Google Scholar, 36.Ahmed S. Lee J. Wen L.P. Zhao Z. Ho J. Best A. Kozma R. Lim L. J. Biol. Chem. 1994; 269: 17642-17648Abstract Full Text PDF PubMed Google Scholar, 37.Morcos P. Thapar N. Tusneem N. Stacey D. Tamanoi F. Mol. Cell. Biol. 1996; 16: 2496-2503Crossref PubMed Scopus (28) Google Scholar). It is most likely that the reactive “arginine patch” in BNIP-2 would confer one or more of the residues in-trans for the catalysis. In this regard, we observed that Arg-235 and Arg-238 are potent residues. The current work utilizes semiquantitative measurements to demonstrate the involvement of such arginine fingers in the BCH domain of BNIP-2. The actual relative contribution from each arginine residue or combination of arginines 235, 236, and 238 awaits a more thorough kinetic determination.In some of the most efficient phosphoryl-transferring enzymes, such as adenylate kinase and uridylate kinase (38.Abele U. Schulz G.E. Protein Sci. 1995; 4: 1262-1271Crossref PubMed Scopus (129) Google Scholar, 39.Scheffzek K. Kliche W. Wiesmuller L. Reinstein J. Biochemistry. 1996; 35: 9716-9727Crossref PubMed Scopus (79) Google Scholar), several arginine residues are located in the active sites and they catalyze the reaction by stabilizing developing negative charges in the transition state. Although the involvement of multiple neighboring polybasic residues had not been tested in the similar arginine patches in Cdc42 and other Rho family members, it is tempting to speculate that these tandem arginine residues in BNIP-2 would provide a positive-charge interface to stabilize the negative charges that develop during the transition state of GTP hydrolysis in Cdc42. In Cdc42GAP, the Arg-305 within the classical GAP domain has been identified both structurally and biochemically as the key catalytic residue in promoting GTP hydrolysis in Cdc42. It does not account, however, for the full GAP activity. Recently, its adjacent arginine residue, Arg-306, was identified to be necessary to further augment its GAP activity (40.Leonard D.A. Lin R. Cerione R.A. Manor D. J. Biol. Chem. 1998; 273: 16210-16215Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Similarly, the p120-RasGAP and the other RasGAP, neurofibromin NF1, both require at least the input of Arg-789 and Arg-903 (for p120-RasGAP (17.Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1176) Google Scholar, 41.Scheffzek K. Lautwein A. Kabsch W. Ahmadian M.R. Wittinghofer A. Nature. 1996; 384: 591-596Crossref PubMed Scopus (141) Google Scholar)) or Arg-1276 and Arg-1391 (for NF1 (42.Sermon B.A. Lowe P.N. Strom M. Eccleston J.F. J. Biol. Chem. 1998; 273: 9480-9485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar)) for optimal catalysis. Therefore, it appears that although the BCH domain of BNIP-2 shows no clear sequence homology to any of these molecules, it too could utilize the same means to facilitate GTP hydrolysis by Cdc42.Recently, evidence has begun accumulating that demonstrates that various GTPases are the preferred eukaryotic substrates of diverse bacterial toxins and exoenzymes (43.Aktories K. Trends Microbiol. 1997; 5: 282-288Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 44.Fu Y. Galan J.E. Nature. 1999; 401: 293-297Crossref PubMed Scopus (445) Google Scholar, 45.Goehring U.-M. Schmidt G. Pederson K.J. Aktories K. Barbieri J.T. J. Biol. Chem. 1999; 274: 36369-36372Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Several of these bacterial products have been shown to interfere with Rho family GTPase activity by means of chemical modifications on important residues (46.Sekine A. Fujiwara M. Narimuya M. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar, 47.Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar) or increasing the lifetime of the active GTP-bound form of the Rho proteins (48.Horiguchi Y. Inoue N. Masuda M. Kashimoto T. Katahira J. Sugimoto N. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11623-11626Crossref PubMed Scopus (100) Google Scholar). Recently, the amino-terminal domain of Pseudomonas aeruginosa exoenzyme S was shown to activate the GTPase activity of Rho, Rac, and Cdc42 (45.Goehring U.-M. Schmidt G. Pederson K.J. Aktories K. Barbieri J.T. J. Biol. Chem. 1999; 274: 36369-36372Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). It was interesting to note that the catalytic domain on this protein had no strong sequence homology to any canonical Rho-GAPs, thus providing evidence that dissimilar primary sequences are capable of performing a similar catalytic function when folded in the appropriate conformation.Based on modeling, we attempted to identify the candidate regions for interaction between BNIP-2 and Cdc42. We observed that at least three sites on each protein are involved in interprotein binding. We showed that the region 288EYV290 on BNIP-2 together with the Switch I or Insert region of Cdc42 synergistically caused a 50% loss in binding, suggesting that they include at least three of these binding sites. These data differ from those derived from studies on the “classical” GAP domain of Cdc42GAP, in which deletion of the Switch I region on Cdc42 alone was already sufficient to markedly reduce the binding to this domain.More than 10 direct interacting partners of Cdc42 have been identified to date (Ref. 26.Low B.C. Lim Y.P. Lim J. Wong E.S.M. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar and references therein). It remains a major issue as to how the incoming signals determine the specificity of functional coupling between this molecule and its effectors/regulators. Recently, Li et al. (49.Li R. Debreceni B. Jia B. Gao Y. Tigyi G. Zheng Y. J. Biol. Chem. 1999; 274 (27654): 29648Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) utilized a mutational and chimeric approach and mapped unique regions on Cdc42 responsible for binding to three different target proteins. They showed that the Switch I and the immediate neighboring region on Cdc42 contain all the necessary determinants for PAK1 binding, whereas WASP and IQGAP1 did not bind the same sequence. Two distantly located regions on Cdc42, residues 155–184 and residues 83–120, constitute the WASP- and IQGAP1-binding regions, respectively. Furthermore, it was shown that the Rho family-unique Insert region of Cdc42 is dispensable for PAK1 and WASP binding but is required for high affinity binding by IQGAP1. Recently, solution structures for the complex of Cdc42 binding to ACK (50.Mott H.R. Owen D. Nietlispach D. Lowe P.N. Manser E. Lim L. Laue E.D. Nature. 1999; 399: 384-388Crossref PubMed Scopus (150) Google Scholar) and Cdc42 binding to WASP (51.Abdul-Manan N. Aghazadeh B. Liu G.A. Majumdar A. Ouerfelli O. Siminovitch K.A. Rosen M.K. Nature. 1999; 399: 379-383Crossref PubMed Scopus (280) Google Scholar) have highlighted the involvement of unique regions within Cdc42 and the respective effectors in mediating the specificity in the binding. Our results show that the binding of BNIP-2 to Cdc42 was mainly mediated by the Switch I and the Insert region. It will be interesting to test how BNIP-2 affects the binding of other effectors to Cdc42 and to what extent this would affect cellular functions. We have recently shown that BNIP-2, when tyrosine-phosphorylated, failed to bind Cdc42 (26.Low B.C. Lim Y.P. Lim J. Wong E.S.M. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This phosphorylation could conceivably act as an additional switch to regulate the formation/dissociation of the complex.Based on our model, the BCH domain of BNIP-2 was predicted to display folding similar to that of the classical GAP domain of Cdc42GAP. Nevertheless, the refined structure is likely to be different from that of the predicted structure. As noted previously, BNIP-2 binds equally well to each form of Cdc42, whereas Cdc42GAP recognizes only the GDP or GTP-bound form of Cdc42 (26.Low B.C. Lim Y.P. Lim J. Wong E.S.M. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 52.Lancaster C.A. Taylor-Harris P.M. Self A.J. Brill S. van Erp H.E. Hall A. J. Biol. Chem. 1994; 269: 1137-1142Abstract Full Text PDF PubMed Google Scholar). This difference in binding selectivity is further supported by our observations that a deletion in the Switch I region of Cdc42 caused severe loss of binding to Cdc42GAP but not to BNIP-2, suggesting that this region alone is not sufficient to confer tight binding to BNIP-2.The identification of the BCH domain as a binding domain for Cdc42 in BNIP-2 and Cdc42GAP has further implications. Whereas the BCH domain of BNIP-2 acts as a GAP, the corresponding region in Cdc42GAP does not, partly because of its lack of Arg-236 and Arg-238 (see Fig.2 D). This raises an interesting issue as to what role this region may play in the physiological function of Cdc42GAP. Most previous biochemical and functional studies involving the interaction of various GAPs with their binding partners were performed using the minimal GAP domains, and any potential regulation via the non-GAP domains would have been overlooked. We have evidence that suggests that the BCH domain is also involved in homophilic and heterophilic interactions involving BNIP-2 and Cdc42GAP. The discrete regions that mediate these interactions are different from those involved in the binding to Cdc42.2 It therefore seems likely that, because of this binding specificity, this domain will have some regulatory function.In conclusion, our present work has shown that the BCH domain of BNIP-2 define a new catalytic GAP domain that contains an arginine patch similar to that found in certain members of the Rho subfamily when they form homodimers. Although the actual mechanism of GAP catalysis by either BNIP-2 or Cdc42 has yet to be determined by x-ray or NMR structural analyses, current evidence suggests that strategically placed arginine residues may represent GAP domains in different primary sequences and tertiary structures. This notion is supported by the recent identification of the arginine finger of rna1p/RanGAP present in a novel folding structure that is mediated by leucine-rich repeats (23.Hillig R.C. Renault L. Vetter I.R. Drell T., IV Wittinghofer A. Becker J. Mol. Cell. 1999; 3: 781-791Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Ras superfamily GTPase proteins act as molecular switches for signal transduction pathways to control cell growth, differentiation, and motility. The Ras superfamily consists of the Ras, Rho, Rab, and Arf families, which are classified according to their sequence similarities and functions (1.Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 2.Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (341) Google Scholar). These proteins cycle between two guanine-nucleotide bound states, the GTP-bound form, which is active, and the inactive GDP-bound form. Activation occurs as a result of a change in the conformation of discrete “switch regions” in these proteins that allow them to interact with their appropriate effector proteins. The GTP/GDP-regulated proteins possess a low intrinsic activity for hydrolyzing GTP to GDP, but for efficient physiological catalysis, they associate with other proteins, which can enhance their GTPase activity further. These proteins are termed GTPase-activating proteins (GAPs),1 and they have been recognized by conserved amino acid sequence motifs that are characteristic of each family (3.Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). The Rho subfamily of GTPases, which includes RhoA, RhoB, RhoC, RhoE, RhoG, Rac1, Rac2, Cdc42, and TC10, is involved in various aspects of cytoskeletal organization" @default.
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• W2090200732 title "Evidence for a Novel Cdc42GAP Domain at the Carboxyl Terminus of BNIP-2" @default.
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