Evidence for a novel Cdc42GAP domain at the carboxyl terminus of BNIP-2.
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 Cdc42GAP homology (BCH) domain. Using BNIP-2 glutathione S-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into alpha-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 ((288)EYV(290) 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.
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Signal Transduction Laboratory, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Republic of Singapore.
Seow
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Guy
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D020703:ras GTPase-Activating Proteins
C499184:BNIP2 protein, human; D002352:Carrier Proteins; C101203:IQ motif containing GTPase activating protein 1; D008840:Microfilament Proteins; D020703:ras GTPase-Activating Proteins