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-associate d protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite th e lack of homology to previously described catalytic domains of GTPase-acti vating proteins (GAPs). BNIP-2 contains many arginine residues at the carbo xyl terminus, which includes the region of homology to the noncatalytic dom ain of Cdc42GAP, termed BNIP-2 and Cdc42GAP homology (BCH) domain. Using BN IP-2 glutathione S-transferase recombinants, it was found that its BCH boun d Cdc42, and contributed the GAP activity. This domain was predicted to fol d into cw-helical bundles similar to the topology of the catalytic GAP doma in of Cdc42GAP. Alignment of exposed arginine residues in this domain helpe d 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 affecti ng its binding to Cdc42. From deletion studies, a region adjacent to the ar ginine patch ((EYV290)-E-288 on BNIP-2) and the Switch I and Rho family-spe cific "Insert" region on Cdc42 are involved in the binding. The results ind icate that the BCH domain of BNIP-2 represents a novel GAP domain that empl oys an arginine patch motif similar to that of the Cdc42-homodimer.