Abstract
The human BCR gene encodes a protein with serine/threonine kinase activity and regulatory domains for the small G-proteins RAC and CDC42. Previous work in our laboratory has established that BCR is a substrate for c-FES, a non-receptor tyrosine kinase linked to myeloid growth and differentiation. Tyrosine phosphorylation led to the association of BCR with the RAS guanine nucleotide exchange complex GRB2-SOS in vivo via the GRB2 SH2 domain, linking BCR to RAS signaling (Maru, Y., Peters, K. L., Afar, D. E. H., Shibuya, M., Witte, O. N., and Smithgall, T. E. (1995) Mol. Cell. Biol. 15, 835-842). In the present study, we demonstrate that BCR Tyr-246 and at least one of the closely spaced tyrosine residues, Tyr-279, Tyr-283, and Tyr-289 (3Y cluster), are phosphorylated by FES both in vitro and in 32Pi-labeled cells. Mutagenesis of BCR Tyr-177 to Phe completely abolished FES-induced BCR binding to the GRB2 SH2 domain, identifying Tyr-177 as an additional phosphorylation site for FES. Co-expression of BCR and FES in human 293T cells stimulated the tyrosine autophosphorylation of FES. By contrast, tyrosine phosphorylation of BCR by FES suppressed BCR serine/threonine kinase activity toward the 14-3-3 protein and BCR substrate, BAP-1. These data show that tyrosine phosphorylation by FES affects the interaction of BCR with multiple signaling partners and suggest a general role for BCR in non-receptor protein-tyrosine kinase regulation and signal transduction.
Highlights
The human BCR gene encodes a 160-kDa protein (BCR) with multiple biochemical functions
Phosphorylation of the BCR 162– 413 Region by FES in Vitro—Recent work in our laboratory established that BCR is a target for the v-FPS and c-FES tyrosine kinases [21]
All of the BCR double mutants exhibited diminished binding to the ABL and p85 SH2 domains while the triple mutant (Y177F/Y246F/⌬3Y) did not bind to either of these SH2 domains. These results demonstrate that FES-mediated phosphorylation of multiple BCR tyrosine residues in the 162– 413 region is required for maximal ABL and p85 SH2 domain binding
Summary
Mutagenesis of the BCR 162– 413 Region and Expression of GSTBCR2 Fusion Proteins in Escherichia coli—DNA encoding BCR N-terminal amino acids 162– 413 was amplified by polymerase chain reaction and cloned into pGEX-2T (Pharmacia Biotech Inc.). Phosphorylation, Tryptic Phosphopeptide Mapping, and Phosphoamino Acid Analysis of GST-BCR Fusion Proteins in Vitro—Recombinant FES was expressed as a C-terminal FLAG fusion protein using a baculovirus/Sf9 cell system and purified using the anti-FLAG M2 affinity gel [21]. BCR sequences encoding Tyr mutations in the 162– 413 region were cut from the pGEX-2T constructs described above with NcoI and BglII and swapped with the corresponding wild-type BCR fragment in the pSP72/ BCR construct. BCR proteins were immunoprecipitated from the cell lysates with an anti-BCR monoclonal antibody (Santa Cruz Biotechnology) and protein G-Sepharose, separated by SDS-PAGE, and visualized by storage phosphor technology. Phosphorylation of BAP-1 in Vitro—BCR was expressed either alone or with FES in human 293T cells and immunoprecipitated from cell lysates using the BCR monoclonal antibody. A pGEX vector for expression of GST-BAP-1 was generously provided by Dr Yoshiro Maru
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