Tyrosine phosphorylation of the Bcl-2-associated protein BNIP-2 by fibroblast growth factor receptor-1 prevents its binding to Cdc42GAP and Cdc42

Fibroblast growth factor (FGF) receptor tyrosine kinases are involved in th e regulation of cell growth, development, and differentiation in a variety of tissues. To isolate potential signaling molecules in the FGF signaling p athway, we have initiated a yeast two-hybrid screening using the cytosolic domain of FGF receptor-1 (Plg). Here we report the identification of BNIP-2 , a previously cloned Bcl-2- and adenovirus E1B-associated protein, as a pu tative substrate of the receptor. When cotransfected in 293T cells, BNIP-2 was tyrosine-phosphorylated via Flg, but their interaction was transient an d could only be seen by "capture" experiments with catalytically inert kina se mutants. When responsive cells were challenged with basic FGF, endogenou s tyrosine-phosphorylated BNIP-2 could be precipitated with a BNIP-2 antibo dy. In addition, the recombinant BNIP-2 expressed in bacteria could be phos phorylated by active Flg in vitro. BNIP-2 shares a region of homology with the noncatalytic domain of Cdc42GAP, a GTPase-activating protein for the sm all GTP-binding molecule, Cdc42, We show here that BNIP-2 and Cdc42GAP coul d directly bind to each other and they also compete for the binding to the same target, Cdc42. Unexpectedly, BNIP-2, either produced as a bacterial re combinant protein or expressed in 293T cells, could stimulate the intrinsic GTPase activity of Cdc42, In all cases, tyrosine phosphorylation of BNIP-2 severely impaired its association with Cdc42GAP and its induced GTPase-act ivating protein-like activity toward Cdc42, These findings should allow us to further characterize the integration of signaling between receptor tyros ine kinases, GTP-binding molecules, and apoptotic pathways.