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 the regulation of cell growth, development, and differentiation in a variety of tissues. To isolate potential signaling molecules in the FGF signaling pathway, we have initiated a yeast two-hybrid screening using the cytosolic domain of FGF receptor-1 (Flg). Here we report the identification of BNIP-2, a previously cloned Bcl-2- and adenovirus E1B-associated protein, as a putative substrate of the receptor. When cotransfected in 293T cells, BNIP-2 was tyrosine-phosphorylated via Flg, but their interaction was transient and could only be seen by "capture" experiments with catalytically inert kinase mutants. When responsive cells were challenged with basic FGF, endogenous tyrosine-phosphorylated BNIP-2 could be precipitated with a BNIP-2 antibody. In addition, the recombinant BNIP-2 expressed in bacteria could be phosphorylated by active Flg in vitro. BNIP-2 shares a region of homology with the noncatalytic domain of Cdc42GAP, a GTPase-activating protein for the small GTP-binding molecule, Cdc42. We show here that BNIP-2 and Cdc42GAP could 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 recombinant 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-activating protein-like activity toward Cdc42. These findings should allow us to further characterize the integration of signaling between receptor tyrosine kinases, GTP-binding molecules, and apoptotic pathways.
Low
B C
BC
Signal Transduction Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Republic of Singapore.
Lim
Y P
YP
Lim
J
J
Wong
E S
ES
Guy
G R
GR
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D000091344:Filaggrin Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
C119020:BNIP1 protein, human; C499184:BNIP2 protein, human; D002352:Carrier Proteins; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017468:Receptors, Fibroblast Growth Factor; D019000:Phosphotyrosine; C496348:FGFR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein