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W2020814966 abstract "We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42. The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (forBNIP-2 and Cdc42GAP homology) domain. Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer. In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive. This raises the possibility that these domains might have other roles in the cell. Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains. Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region 217RRKMP221 as the major BCH interaction site within BNIP-2. In comparison, deletion of either the arginine-patch 235RRLRK239 (necessary for GAP activity) or region 288EYV290 (a Cdc42 binding sequence) had no effect on BCH-BCH interaction. Extensive data base searches showed that the BCH domain is highly conserved across species. The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules. We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42. The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (forBNIP-2 and Cdc42GAP homology) domain. Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer. In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive. This raises the possibility that these domains might have other roles in the cell. Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains. Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region 217RRKMP221 as the major BCH interaction site within BNIP-2. In comparison, deletion of either the arginine-patch 235RRLRK239 (necessary for GAP activity) or region 288EYV290 (a Cdc42 binding sequence) had no effect on BCH-BCH interaction. Extensive data base searches showed that the BCH domain is highly conserved across species. The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules. GTPase-activating protein hemagglutinin glutathioneS-transferase guanosine 5′-O-3-thiotriphosphate polyacrylamide gel electrophoresis breakpoint cluster region homology We recently identified BNIP-2, a previously cloned Bcl-2 and adenovirus E1B-interacting protein (1Boyd 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), as a putative substrate of the fibroblast growth factor receptor tyrosine kinase. When not tyrosine-phosphorylated BNIP-2 can bind to two cellular targets: Cdc42, a small GTPase and its regulator, Cdc42GAP but this binding is abrogated upon its tyrosine phosphorylation (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).Cdc42 is a member of the Rho subfamily of GTPases demonstrated to be involved in various aspects of cytoskeletal organization, regulation of the transcription of certain target genes, and the control of aspects of cell cycle progression (3Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar, 5Hall A. Br. J. Cancer. 1999; 80 Suppl. 1: 25-27PubMed Google Scholar, 6Chardin 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, 7Paterson 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, 8Waterman-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, 9Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar, 10Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3698) Google Scholar, 11Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1443) Google Scholar, 12Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (284) Google Scholar). GTPases cycle between the inactive, GDP-bound form and the active GTP-bound form. The equilibrium between these two states is controlled at least by two major classes of regulators, the guanine nucleotide exchange factors and the GTPase-activating proteins (GAPs)1 (13Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 14Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (341) Google Scholar, 15Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). The guanine nucleotide exchange factors catalyze the exchange of GDP on the inactive GTPase for GTP, which results in enhanced activity of the target protein. The GAPs enhance rates of GTP hydrolysis to GDP mainly by contributing catalytic arginine residues to their substrate target in trans, or by stabilizing the conformation of the inherent GTPases (15Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 16Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (122) Google Scholar, 17Rittinger 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, 18Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 19Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1176) Google Scholar, 20Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (292) Google Scholar, 21Barrett 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, 22Bax B. Nature. 1998; 392: 447-448Crossref PubMed Scopus (15) Google Scholar, 23Rittinger K. Taylor W.R. Smerdon S.J. Gamblin S.J. Nature. 1998; 392: 448-449Crossref PubMed Scopus (26) Google Scholar, 24Calmels 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).Despite lacking obvious sequence homology to the canonical catalytic domain of GAP proteins, BNIP-2 was shown to possess a GAP activity toward Cdc42 (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We recently identified that this unexpected GAP activity is mediated by several key arginine residues within the COOH terminus of BNIP-2 (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) that constitute an apparently catalytic motif similar to the “arginine finger” demonstrated in Cdc42 homodimers (26Zhang B. Zheng Y. J. Biol. Chem. 1998; 273: 25728-25733Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Zhang 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). Interestingly, the COOH-terminal region of BNIP-2 shares a high degree of sequence homology with a region at the NH2-terminal, non-catalytic half of Cdc42GAP, which we termed the BCH (BNIP-2 and Cdc42GAPhomology) domain (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Both BNIP-2 and Cdc42GAP BCH domains can bind Cdc42, but only the BCH domain of BNIP-2 functions as a GAP toward Cdc42 as Cdc42GAP lacks the arginine-finger motif (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).We initially identified Cdc42GAP as a BNIP-2-binding protein by a candidate approach based upon the assumption that both proteins, by having a similar domain, can either bind to a common target or bind to each other (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). There is, however, support for the BCH domain being a potential lipid-targeting sequence. This came from a recent suggestion that the BCH domain shared some homology, albeit low, with Sec14p-like lipid-binding domains (28Aravind L. Neuwald A.F. Ponting C.P. Curr. Biol. 1999; 9: 195-197Abstract Full Text Full Text PDF PubMed Google Scholar). Sec14p is a phospholipid exchange protein in Saccharomyces cerevisiae that mediates the exchange of phosphatidylcholine and phosphatidylinositol between membrane bilayers. Inactive Sec14 mutations inhibit Golgi transport to endosomes and recently it was demonstrated that temperature-sensitive mutants have decreased amounts of phosphatidylinositol 4-phosphate (29Odorizzi G. Babst M. Emr S.D. Trends Biochem. Sci. 2000; 25: 229-235Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). In this context phosphatidylinositol 4-phosphate seems to be a target phospholipid similar to phosphatidylinositol 3-phosphate and both may play a role in intracellular trafficking.We previously employed precipitation experiments and yeast two-hybrid analysis to demonstrate that while BNIP-2 and Cdc42GAP can individually bind to and enhance Cdc42 GTPase activity they could also bind to each other (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Such an interaction between proteins that bind to and activate the same substrate provides a potential controlling mechanism with several layers of complexity. We are primarily interested in establishing which regions of BNIP-2 and Cdc42GAP are responsible for their homophilic and heterophilic interactions. To do this we have used a series of deletion studies and molecular modeling techniques that enabled a hypothetical model to be constructed and formed the basis for more detailed mutational studies. Using these approaches we found that the BCH domains of BNIP-2 and Cdc42GAP are responsible for their homophilic or heterophilic interactions. We further identified a discrete region within the BNIP-2 BCH domain that is responsible for the interactions involving BNIP-2. Having presented evidence to show that the BCH domain was involved in protein-protein interactions we searched various data bases to see what other proteins might contain this domain and to see what other domains they are associated with. The significance of this novel BCH domain-containing family is discussed.DISCUSSIONThe present study examined the interaction between BNIP-2 and Cdc42GAP by using in vitro and in vivo binding experiments and demonstrated that their homologous BCH domains primarily mediate both homophilic and heterophilic interaction between the proteins. Deletion studies aided by computer modeling allowed us to further define a unique region at 217RRKMP221of BNIP-2 as the major determinant in the complex formation. This region is distinct from two other regions of BNIP-2 we had recently identified; the arginine-patch 235RRLRK239 and the region 288EYV290 both of which are important for GAP activity of BNIP-2 and its binding to Cdc42, respectively (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Since all these regions lie within the BCH domain it raises the interesting question as to how the homophilic and heterophilic interaction of BNIP-2 and/or Cdc42GAP (either in their BCH forms or as their full-length entities) would influence their GAP activity toward Cdc42. Our previous studies had shown that the presence of both BNIP-2 and Cdc42GAP led to a decreased GAP activity toward Cdc42 in comparison to when either protein was present alone (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Such experiments suggest that the presence of BNIP-2 could antagonize the GAP activity of Cdc42GAP and vice versa. We have now demonstrated that their binding via BCH domains is at least partly responsible for this inhibitory effect. The BCH-mediated binding of BNIP-2 (specifically via the region-M) negatively regulates the GAP activity of BNIP-2 as well as the Cdc42GAP. We are now trying to establish the site(s) in the Cdc42GAP-BCH domain that is involved in its homophilic and heterophilic interaction, and it remains to be seen whether deletion of such a binding region(s) in Cdc42GAP could lead to an increase in its GAP activity as was seen for BNIP-2.In the case of Cdc42GAP, it was intriguing to see that this molecule harbors two Cdc42-binding domains. In addition to the canonical GAP domain at the carboxyl terminus, we recently identified that the BCH domain of Cdc42GAP can also bind Cdc42 but lacks catalytic activity as it is devoid of the arginine-patch motif found in the BNIP-2 BCH domain. The question arises as to what is the role of the BCH domain of Cdc42GAP? Potentially the Cdc42GAP-BCH domain can act as another binding interface for Cdc42, perhaps by interacting with other regions of the GTPase. Our current findings that Cdc42GAP is also capable of homophilic binding and/or heterophilic interactions with BNIP-2, via the same BCH domain, has added another layer of complexity to the potential regulation of both GAP proteins.The notion that both the BCH and GAP domains in Cdc42GAP and in other members of the RhoGAP subfamily are vital for their possible activation and function is supported by our observations that the spacing between the two domains is well conserved (Fig. 6 B). Nevertheless, the BCH and GAP domains of the various RhoGAPs in tandem are not a universal corollary of RhoGAP catalysis as they appear in only a subset of RhoGAP family proteins. It remains to be seen if there are any unique biochemical or cellular locational peculiarities for this subclass of RhoGAP when compared with those without the proximal BCH domains. Currently there is scant information pertaining to this class of RhoGAP proteins. Similarly, all proteins containing a type-1 BCH distribution, i.e. with their BCH domains at the carboxyl end, have no known functions. Work is currently underway in our laboratory to characterize proteins with each type of BCH domain distribution.To date, all structural studies, and their functional inferences, pertaining to Cdc42GAP and Cdc42 are based on bimolecular complexes between Cdc42 or Rho with the catalytic GAP domain. No studies have been made with either the BCH domain or full-length Cdc42GAP. Structural determinations involving the BCH domain, or better still the whole protein, would give a more complete understanding of the molecular mechanism involved in the regulation of this protein and its interaction with other proteins.The occurrence of at least a dozen distinct proteins with highly conserved BCH domains across so many species suggests that this domain should play a significant role(s) in some biological process(es). With more genomes being sequenced, it is anticipated that the number of proteins harboring similar domains would increase. Although we have shown that the BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain it remains to be seen if all other BCH domains in other proteins are also involved in mediating protein-protein interaction. If they do, interaction among some of these BCH-containing molecules would confer functional diversity for various biological processes. It is worth noting that in the model of the BNIP-2 BCH domain, the region involved in mediating its binding is not conserved in all other “family members” including one of its binding partners, the Cdc42GAP-BCH domain. This non-homology of actual binding region within a conserved structure might provide a mechanism for the regulation of target specificity.Recently, part of the BCH domains of BNIP-2 and Cdc42GAP were deemed, after multiple rounds of iteration in PSI-BLAST analysis, to share a limited homology to the Sec14p-like domain, previously known to mediate the exchange of phosphatidylinositol and phosphatidylcholine inS. cerevisiae (28Aravind L. Neuwald A.F. Ponting C.P. Curr. Biol. 1999; 9: 195-197Abstract Full Text Full Text PDF PubMed Google Scholar). The implications of this observation are manifold. One possibility is that in vivo the BCH domain actually targets to some specific phospholipid moiety and this would direct associated catalytic domains into favorable locations near their substrates. A second possibility is, in addition to mediating protein-protein interaction, BCH domains might bind lipids such that their interaction could be modified and thus regulated, either directly on the protein-binding site or in an allosteric fashion. Regulation of protein-protein interaction by lipids has recently been reported for the intramolecular interaction between the lipid-binding pleckstrin homology and the catalytic Dbl homology domains of Vav or Sos1, two guanine nucleotide exchange factors (39Das B. Shu X. Day G.J. Han J. Krishna U.M. Falck J.R. Broek D. J. Biol. Chem. 2000; 275: 15074-15081Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It was shown that phosphatidylinositol 3-kinase substrate promotes the binding of these two domains and blocks Rac binding to the Dbl homology domain, whereas products of phosphatidylinositol 3-kinase disrupt such interaction and allows Rac binding for activation. A third possibility is that BCH domains are purely protein-protein interaction domains that have diverged sufficiently from Sec14p lipid-binding domains to have evolved a separate function.Our preliminary data on indirect immunofluorescence of BNIP-2 does not show any unique membrane localization of the protein in cells. Neither plasma or organelle membranes appeared to be stained; instead we observed a punctate pattern of distribution more likely linked to a cytoskeletal distribution (data not shown). This observation apparently rules out the notion of at least the BNIP-2 BCH domain being a membrane lipid-targeting device. Current work is aimed at addressing the detailed intracellular localization of BNIP-2.In conclusion, our present work has shown that BCH domains of BNIP-2 and Cdc42GAP define a novel class of protein-protein interaction domain that includes various uncharacterized proteins. It may represent another example of proteins that form dimers as a functional necessity such as: various receptor tyrosine kinases (40Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar), STAT transcription factors (41Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4946) Google Scholar), c-Raf (42Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar, 43Farrar M.A. Alberol I. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar), and various members of the Bcl family (44Diaz J.L. Oltersdorf T. Horne W. McConnell M. Wilson G. Weeks S. Garcia T. Fritz L.C. J. Biol. Chem. 1997; 272: 11350-11355Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 45Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). Our work also highlights the fact that although several structural studies have used the catalytic domain of Cdc42GAP to define a precise interaction with Cdc42 (and Rho) (18Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 46Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (353) Google Scholar) there is still much to be understood about how this protein is targeted and activated and indeed what its actual physiological role is. A better understanding of the structure and functional roles of the BCH domains of BNIP-2, Cdc42GAP, and of other proteins will answer these questions. We recently identified BNIP-2, a previously cloned Bcl-2 and adenovirus E1B-interacting protein (1Boyd 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), as a putative substrate of the fibroblast growth factor receptor tyrosine kinase. When not tyrosine-phosphorylated BNIP-2 can bind to two cellular targets: Cdc42, a small GTPase and its regulator, Cdc42GAP but this binding is abrogated upon its tyrosine phosphorylation (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Cdc42 is a member of the Rho subfamily of GTPases demonstrated to be involved in various aspects of cytoskeletal organization, regulation of the transcription of certain target genes, and the control of aspects of cell cycle progression (3Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar, 5Hall A. Br. J. Cancer. 1999; 80 Suppl. 1: 25-27PubMed Google Scholar, 6Chardin 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, 7Paterson 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, 8Waterman-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, 9Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar, 10Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3698) Google Scholar, 11Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1443) Google Scholar, 12Aspenstrom P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (284) Google Scholar). GTPases cycle between the inactive, GDP-bound form and the active GTP-bound form. The equilibrium between these two states is controlled at least by two major classes of regulators, the guanine nucleotide exchange factors and the GTPase-activating proteins (GAPs)1 (13Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 14Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (341) Google Scholar, 15Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). The guanine nucleotide exchange factors catalyze the exchange of GDP on the inactive GTPase for GTP, which results in enhanced activity of the target protein. The GAPs enhance rates of GTP hydrolysis to GDP mainly by contributing catalytic arginine residues to their substrate target in trans, or by stabilizing the conformation of the inherent GTPases (15Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 16Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (122) Google Scholar, 17Rittinger 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, 18Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 19Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1176) Google Scholar, 20Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (292) Google Scholar, 21Barrett 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, 22Bax B. Nature. 1998; 392: 447-448Crossref PubMed Scopus (15) Google Scholar, 23Rittinger K. Taylor W.R. Smerdon S.J. Gamblin S.J. Nature. 1998; 392: 448-449Crossref PubMed Scopus (26) Google Scholar, 24Calmels 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). Despite lacking obvious sequence homology to the canonical catalytic domain of GAP proteins, BNIP-2 was shown to possess a GAP activity toward Cdc42 (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We recently identified that this unexpected GAP activity is mediated by several key arginine residues within the COOH terminus of BNIP-2 (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) that constitute an apparently catalytic motif similar to the “arginine finger” demonstrated in Cdc42 homodimers (26Zhang B. Zheng Y. J. Biol. Chem. 1998; 273: 25728-25733Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Zhang 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). Interestingly, the COOH-terminal region of BNIP-2 shares a high degree of sequence homology with a region at the NH2-terminal, non-catalytic half of Cdc42GAP, which we termed the BCH (BNIP-2 and Cdc42GAPhomology) domain (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Both BNIP-2 and Cdc42GAP BCH domains can bind Cdc42, but only the BCH domain of BNIP-2 functions as a GAP toward Cdc42 as Cdc42GAP lacks the arginine-finger motif (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We initially identified Cdc42GAP as a BNIP-2-binding protein by a candidate approach based upon the assumption that both proteins, by having a similar domain, can either bind to a common target or bind to each other (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). There is, however, support for the BCH domain being a potential lipid-targeting sequence. This came from a recent suggestion that the BCH domain shared some homology, albeit low, with Sec14p-like lipid-binding domains (28Aravind L. Neuwald A.F. Ponting C.P. Curr. Biol. 1999; 9: 195-197Abstract Full Text Full Text PDF PubMed Google Scholar). Sec14p is a phospholipid exchange protein in Saccharomyces cerevisiae that mediates the exchange of phosphatidylcholine and phosphatidylinositol between membrane bilayers. Inactive Sec14 mutations inhibit Golgi transport to endosomes and recently it was demonstrated that temperature-sensitive mutants have decreased amounts of phosphatidylinositol 4-phosphate (29Odorizzi G. Babst M. Emr S.D. Trends Biochem. Sci. 2000; 25: 229-235Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). In this context phosphatidylinositol 4-phosphate seems to be a target phospholipid similar to phosphatidylinositol 3-phosphate and both may play a role in intracellular trafficking. We previously employed precipitation experiments and yeast two-hybrid analysis to demonstrate that while BNIP-2 and Cdc42GAP can individually bind to and enhance Cdc42 GTPase activity they could also bind to each other (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Such an interaction between proteins that bind to and activate the same substrate provides a potential controlling mechanism with several layers of complexity. We are primarily interested in establishing which regions of BNIP-2 and Cdc42GAP are responsible for their homophilic and heterophilic interactions. To do this we have used a series of deletion studies and molecular modeling techniques that enabled a hypothetical model to be constructed and formed the basis for more detailed mutational studies. Using these approaches we found that the BCH domains of BNIP-2 and Cdc42GAP are responsible for their homophilic or heterophilic interactions. We further identified a discrete region within the BNIP-2 BCH domain that is responsible for the interactions involving BNIP-2. Having presented evidence to show that the BCH domain was involved in protein-protein interactions we searched various data bases to see what other proteins might contain this domain and to see what other domains they are associated with. The significance of this novel BCH domain-containing family is discussed. DISCUSSIONThe present study examined the interaction between BNIP-2 and Cdc42GAP by using in vitro and in vivo binding experiments and demonstrated that their homologous BCH domains primarily mediate both homophilic and heterophilic interaction between the proteins. Deletion studies aided by computer modeling allowed us to further define a unique region at 217RRKMP221of BNIP-2 as the major determinant in the complex formation. This region is distinct from two other regions of BNIP-2 we had recently identified; the arginine-patch 235RRLRK239 and the region 288EYV290 both of which are important for GAP activity of BNIP-2 and its binding to Cdc42, respectively (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Since all these regions lie within the BCH domain it raises the interesting question as to how the homophilic and heterophilic interaction of BNIP-2 and/or Cdc42GAP (either in their BCH forms or as their full-length entities) would influence their GAP activity toward Cdc42. Our previous studies had shown that the presence of both BNIP-2 and Cdc42GAP led to a decreased GAP activity toward Cdc42 in comparison to when either protein was present alone (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Such experiments suggest that the presence of BNIP-2 could antagonize the GAP activity of Cdc42GAP and vice versa. We have now demonstrated that their binding via BCH domains is at least partly responsible for this inhibitory effect. The BCH-mediated binding of BNIP-2 (specifically via the region-M) negatively regulates the GAP activity of BNIP-2 as well as the Cdc42GAP. We are now trying to establish the site(s) in the Cdc42GAP-BCH domain that is involved in its homophilic and heterophilic interaction, and it remains to be seen whether deletion of such a binding region(s) in Cdc42GAP could lead to an increase in its GAP activity as was seen for BNIP-2.In the case of Cdc42GAP, it was intriguing to see that this molecule harbors two Cdc42-binding domains. In addition to the canonical GAP domain at the carboxyl terminus, we recently identified that the BCH domain of Cdc42GAP can also bind Cdc42 but lacks catalytic activity as it is devoid of the arginine-patch motif found in the BNIP-2 BCH domain. The question arises as to what is the role of the BCH domain of Cdc42GAP? Potentially the Cdc42GAP-BCH domain can act as another binding interface for Cdc42, perhaps by interacting with other regions of the GTPase. Our current findings that Cdc42GAP is also capable of homophilic binding and/or heterophilic interactions with BNIP-2, via the same BCH domain, has added another layer of complexity to the potential regulation of both GAP proteins.The notion that both the BCH and GAP domains in Cdc42GAP and in other members of the RhoGAP subfamily are vital for their possible activation and function is supported by our observations that the spacing between the two domains is well conserved (Fig. 6 B). Nevertheless, the BCH and GAP domains of the various RhoGAPs in tandem are not a universal corollary of RhoGAP catalysis as they appear in only a subset of RhoGAP family proteins. It remains to be seen if there are any unique biochemical or cellular locational peculiarities for this subclass of RhoGAP when compared with those without the proximal BCH domains. Currently there is scant information pertaining to this class of RhoGAP proteins. Similarly, all proteins containing a type-1 BCH distribution, i.e. with their BCH domains at the carboxyl end, have no known functions. Work is currently underway in our laboratory to characterize proteins with each type of BCH domain distribution.To date, all structural studies, and their functional inferences, pertaining to Cdc42GAP and Cdc42 are based on bimolecular complexes between Cdc42 or Rho with the catalytic GAP domain. No studies have been made with either the BCH domain or full-length Cdc42GAP. Structural determinations involving the BCH domain, or better still the whole protein, would give a more complete understanding of the molecular mechanism involved in the regulation of this protein and its interaction with other proteins.The occurrence of at least a dozen distinct proteins with highly conserved BCH domains across so many species suggests that this domain should play a significant role(s) in some biological process(es). With more genomes being sequenced, it is anticipated that the number of proteins harboring similar domains would increase. Although we have shown that the BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain it remains to be seen if all other BCH domains in other proteins are also involved in mediating protein-protein interaction. If they do, interaction among some of these BCH-containing molecules would confer functional diversity for various biological processes. It is worth noting that in the model of the BNIP-2 BCH domain, the region involved in mediating its binding is not conserved in all other “family members” including one of its binding partners, the Cdc42GAP-BCH domain. This non-homology of actual binding region within a conserved structure might provide a mechanism for the regulation of target specificity.Recently, part of the BCH domains of BNIP-2 and Cdc42GAP were deemed, after multiple rounds of iteration in PSI-BLAST analysis, to share a limited homology to the Sec14p-like domain, previously known to mediate the exchange of phosphatidylinositol and phosphatidylcholine inS. cerevisiae (28Aravind L. Neuwald A.F. Ponting C.P. Curr. Biol. 1999; 9: 195-197Abstract Full Text Full Text PDF PubMed Google Scholar). The implications of this observation are manifold. One possibility is that in vivo the BCH domain actually targets to some specific phospholipid moiety and this would direct associated catalytic domains into favorable locations near their substrates. A second possibility is, in addition to mediating protein-protein interaction, BCH domains might bind lipids such that their interaction could be modified and thus regulated, either directly on the protein-binding site or in an allosteric fashion. Regulation of protein-protein interaction by lipids has recently been reported for the intramolecular interaction between the lipid-binding pleckstrin homology and the catalytic Dbl homology domains of Vav or Sos1, two guanine nucleotide exchange factors (39Das B. Shu X. Day G.J. Han J. Krishna U.M. Falck J.R. Broek D. J. Biol. Chem. 2000; 275: 15074-15081Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It was shown that phosphatidylinositol 3-kinase substrate promotes the binding of these two domains and blocks Rac binding to the Dbl homology domain, whereas products of phosphatidylinositol 3-kinase disrupt such interaction and allows Rac binding for activation. A third possibility is that BCH domains are purely protein-protein interaction domains that have diverged sufficiently from Sec14p lipid-binding domains to have evolved a separate function.Our preliminary data on indirect immunofluorescence of BNIP-2 does not show any unique membrane localization of the protein in cells. Neither plasma or organelle membranes appeared to be stained; instead we observed a punctate pattern of distribution more likely linked to a cytoskeletal distribution (data not shown). This observation apparently rules out the notion of at least the BNIP-2 BCH domain being a membrane lipid-targeting device. Current work is aimed at addressing the detailed intracellular localization of BNIP-2.In conclusion, our present work has shown that BCH domains of BNIP-2 and Cdc42GAP define a novel class of protein-protein interaction domain that includes various uncharacterized proteins. It may represent another example of proteins that form dimers as a functional necessity such as: various receptor tyrosine kinases (40Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar), STAT transcription factors (41Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4946) Google Scholar), c-Raf (42Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar, 43Farrar M.A. Alberol I. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar), and various members of the Bcl family (44Diaz J.L. Oltersdorf T. Horne W. McConnell M. Wilson G. Weeks S. Garcia T. Fritz L.C. J. Biol. Chem. 1997; 272: 11350-11355Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 45Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). Our work also highlights the fact that although several structural studies have used the catalytic domain of Cdc42GAP to define a precise interaction with Cdc42 (and Rho) (18Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 46Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (353) Google Scholar) there is still much to be understood about how this protein is targeted and activated and indeed what its actual physiological role is. A better understanding of the structure and functional roles of the BCH domains of BNIP-2, Cdc42GAP, and of other proteins will answer these questions. The present study examined the interaction between BNIP-2 and Cdc42GAP by using in vitro and in vivo binding experiments and demonstrated that their homologous BCH domains primarily mediate both homophilic and heterophilic interaction between the proteins. Deletion studies aided by computer modeling allowed us to further define a unique region at 217RRKMP221of BNIP-2 as the major determinant in the complex formation. This region is distinct from two other regions of BNIP-2 we had recently identified; the arginine-patch 235RRLRK239 and the region 288EYV290 both of which are important for GAP activity of BNIP-2 and its binding to Cdc42, respectively (25Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Since all these regions lie within the BCH domain it raises the interesting question as to how the homophilic and heterophilic interaction of BNIP-2 and/or Cdc42GAP (either in their BCH forms or as their full-length entities) would influence their GAP activity toward Cdc42. Our previous studies had shown that the presence of both BNIP-2 and Cdc42GAP led to a decreased GAP activity toward Cdc42 in comparison to when either protein was present alone (2Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Such experiments suggest that the presence of BNIP-2 could antagonize the GAP activity of Cdc42GAP and vice versa. We have now demonstrated that their binding via BCH domains is at least partly responsible for this inhibitory effect. The BCH-mediated binding of BNIP-2 (specifically via the region-M) negatively regulates the GAP activity of BNIP-2 as well as the Cdc42GAP. We are now trying to establish the site(s) in the Cdc42GAP-BCH domain that is involved in its homophilic and heterophilic interaction, and it remains to be seen whether deletion of such a binding region(s) in Cdc42GAP could lead to an increase in its GAP activity as was seen for BNIP-2. In the case of Cdc42GAP, it was intriguing to see that this molecule harbors two Cdc42-binding domains. In addition to the canonical GAP domain at the carboxyl terminus, we recently identified that the BCH domain of Cdc42GAP can also bind Cdc42 but lacks catalytic activity as it is devoid of the arginine-patch motif found in the BNIP-2 BCH domain. The question arises as to what is the role of the BCH domain of Cdc42GAP? Potentially the Cdc42GAP-BCH domain can act as another binding interface for Cdc42, perhaps by interacting with other regions of the GTPase. Our current findings that Cdc42GAP is also capable of homophilic binding and/or heterophilic interactions with BNIP-2, via the same BCH domain, has added another layer of complexity to the potential regulation of both GAP proteins. The notion that both the BCH and GAP domains in Cdc42GAP and in other members of the RhoGAP subfamily are vital for their possible activation and function is supported by our observations that the spacing between the two domains is well conserved (Fig. 6 B). Nevertheless, the BCH and GAP domains of the various RhoGAPs in tandem are not a universal corollary of RhoGAP catalysis as they appear in only a subset of RhoGAP family proteins. It remains to be seen if there are any unique biochemical or cellular locational peculiarities for this subclass of RhoGAP when compared with those without the proximal BCH domains. Currently there is scant information pertaining to this class of RhoGAP proteins. Similarly, all proteins containing a type-1 BCH distribution, i.e. with their BCH domains at the carboxyl end, have no known functions. Work is currently underway in our laboratory to characterize proteins with each type of BCH domain distribution. To date, all structural studies, and their functional inferences, pertaining to Cdc42GAP and Cdc42 are based on bimolecular complexes between Cdc42 or Rho with the catalytic GAP domain. No studies have been made with either the BCH domain or full-length Cdc42GAP. Structural determinations involving the BCH domain, or better still the whole protein, would give a more complete understanding of the molecular mechanism involved in the regulation of this protein and its interaction with other proteins. The occurrence of at least a dozen distinct proteins with highly conserved BCH domains across so many species suggests that this domain should play a significant role(s) in some biological process(es). With more genomes being sequenced, it is anticipated that the number of proteins harboring similar domains would increase. Although we have shown that the BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain it remains to be seen if all other BCH domains in other proteins are also involved in mediating protein-protein interaction. If they do, interaction among some of these BCH-containing molecules would confer functional diversity for various biological processes. It is worth noting that in the model of the BNIP-2 BCH domain, the region involved in mediating its binding is not conserved in all other “family members” including one of its binding partners, the Cdc42GAP-BCH domain. This non-homology of actual binding region within a conserved structure might provide a mechanism for the regulation of target specificity. Recently, part of the BCH domains of BNIP-2 and Cdc42GAP were deemed, after multiple rounds of iteration in PSI-BLAST analysis, to share a limited homology to the Sec14p-like domain, previously known to mediate the exchange of phosphatidylinositol and phosphatidylcholine inS. cerevisiae (28Aravind L. Neuwald A.F. Ponting C.P. Curr. Biol. 1999; 9: 195-197Abstract Full Text Full Text PDF PubMed Google Scholar). The implications of this observation are manifold. One possibility is that in vivo the BCH domain actually targets to some specific phospholipid moiety and this would direct associated catalytic domains into favorable locations near their substrates. A second possibility is, in addition to mediating protein-protein interaction, BCH domains might bind lipids such that their interaction could be modified and thus regulated, either directly on the protein-binding site or in an allosteric fashion. Regulation of protein-protein interaction by lipids has recently been reported for the intramolecular interaction between the lipid-binding pleckstrin homology and the catalytic Dbl homology domains of Vav or Sos1, two guanine nucleotide exchange factors (39Das B. Shu X. Day G.J. Han J. Krishna U.M. Falck J.R. Broek D. J. Biol. Chem. 2000; 275: 15074-15081Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It was shown that phosphatidylinositol 3-kinase substrate promotes the binding of these two domains and blocks Rac binding to the Dbl homology domain, whereas products of phosphatidylinositol 3-kinase disrupt such interaction and allows Rac binding for activation. A third possibility is that BCH domains are purely protein-protein interaction domains that have diverged sufficiently from Sec14p lipid-binding domains to have evolved a separate function. Our preliminary data on indirect immunofluorescence of BNIP-2 does not show any unique membrane localization of the protein in cells. Neither plasma or organelle membranes appeared to be stained; instead we observed a punctate pattern of distribution more likely linked to a cytoskeletal distribution (data not shown). This observation apparently rules out the notion of at least the BNIP-2 BCH domain being a membrane lipid-targeting device. Current work is aimed at addressing the detailed intracellular localization of BNIP-2. In conclusion, our present work has shown that BCH domains of BNIP-2 and Cdc42GAP define a novel class of protein-protein interaction domain that includes various uncharacterized proteins. It may represent another example of proteins that form dimers as a functional necessity such as: various receptor tyrosine kinases (40Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar), STAT transcription factors (41Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4946) Google Scholar), c-Raf (42Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar, 43Farrar M.A. Alberol I. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar), and various members of the Bcl family (44Diaz J.L. Oltersdorf T. Horne W. McConnell M. Wilson G. Weeks S. Garcia T. Fritz L.C. J. Biol. Chem. 1997; 272: 11350-11355Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 45Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). Our work also highlights the fact that although several structural studies have used the catalytic domain of Cdc42GAP to define a precise interaction with Cdc42 (and Rho) (18Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (173) Google Scholar, 46Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (353) Google Scholar) there is still much to be understood about how this protein is targeted and activated and indeed what its actual physiological role is. A better understanding of the structure and functional roles of the BCH domains of BNIP-2, Cdc42GAP, and of other proteins will answer these questions. We thank Dr. Alan Hall for the generous donations of materials." @default.
W2020814966 created "2016-06-24" @default.
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W2020814966 title "The BNIP-2 and Cdc42GAP Homology Domain of BNIP-2 Mediates Its Homophilic Association and Heterophilic Interaction with Cdc42GAP" @default.
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