Tyrosine phosphorylation enhances the SH2 domain-binding activity of Bcr and inhibits Bcr interaction with 14-3-3 proteins.
The cellular Bcr protein consists of an N-terminal serine/threonine kinase domain, a central guanine nucleotide exchange factor homology region and a C-terminal GTPase-activating protein domain. Previous work in our laboratory established that Bcr is a major transformation-related substrate for the v-Fps tyrosine kinase, and tyrosine phosphorylation of Bcr induces Bcr-Grb-2/SOS association in vivo through the Src homology 2 (SH2) domain of Grb-2. In the present study, we mapped the region of Bcr tyrosine phosphorylation by c-Fes, the human homologue of v-Fps, to Bcr N-terminal amino acids 162-413 by using a baculovirus/Sf-9 cell co-expression system. Tyrosine phosphorylation of Bcr by Fes greatly enhanced the binding of Bcr to the SH2 domains of multiple signalling molecules in vitro, including Grb-2, Ras GTPase activating protein, phospholipase C-gamma, the 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, and the Abl tyrosine kinase. In contrast with SH2 binding, tyrosine phosphorylation of Bcr reduced its ability to associate with the 14-3-3 protein Bap-1 (Bcr-associated protein-1), a Bcr substrate and member of a family of phosphoserine-binding adaptor proteins. These experiments provide in vitro evidence that tyrosine phosphorylation may modulate the interaction of Bcr with multiple growth-regulatory signalling pathways.
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68198, USA.
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Peters
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Smithgall
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D051380:GRB2 Adaptor Protein; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051562:Proto-Oncogene Proteins c-bcr; D051578:Proto-Oncogene Proteins c-fes; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase; D018909:src Homology Domains
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011506:Proteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D014446:Tyrosine 3-Monooxygenase; D011505:Protein-Tyrosine Kinases; C496645:FES protein, human; D051578:Proto-Oncogene Proteins c-fes; D016392:Proto-Oncogene Proteins pp60(c-src); C496613:BCR protein, human; D017346:Protein Serine-Threonine Kinases; D051562:Proto-Oncogene Proteins c-bcr