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 (for BNIP-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 217
RRKMP221 as the major BCH interaction site within BNIP-2. In comparison, deletion of either the argininepatch 235 RRLRK 239 (necessary for GAP activity) or region 288 EYV 290 (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 identified BNIP-2, a previously cloned Bcl-2 and adenovirus E1B-interacting protein (1), 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 (2).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 (3-12). 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 (13-15). 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 (15-24).Despite lacking obvious sequence homology to the canonical catalytic domain of GAP proteins, BNIP-2 was shown to possess a GAP...