INTERACTION OF SH3 DOMAIN OF HCK TYROSINE KINASE WITH CELLULAR PROTEINS CONTAINING PROLINE-RICH REGIONS - EVIDENCE FOR MODULATION BY UNIQUEDOMAIN

The Hck tyrosine kinase, a member of Src family, is predominantly expr essed in myeloid cells. In this report we have analyzed interaction of cellular proteins with Src homology 3 (SH3) domain of Hck. For this p urpose we used various GST-Hck fusion proteins comprising a part of un ique region, complete unique region and/or complete SH3 domain of Hck, and glutathione S-transferase (GST). When these fusion proteins (or G ST), immobilized on glutathione-agarose beads were incubated with [S-3 5] methionine labelled cell extracts, multiple proteins which interact specifically with SH3 domain of Hck were detected by SDS-PAGE followe d by autoradiography. The Hck interacting proteins could also be detec ted by a tandem blot binding assay in which the blot was incubated wit h purified fusion protein (or GST) and then the interacting proteins w ere identified by using antibody against GST. When a part of or comple te unique domain was present along with SH3 domain, the interaction of some specific proteins was reduced several fold. These results mise t he possibility of unique domain altering the properties of SH3 domain, thus modulating or restricting the interaction of SH3 domain with spe cific cellular proteins.;This modulatory effect of unique domain was l ocalized to 28 amino acids upstream of SH3 domain. SH3 interacting pro teins were associated with serine/threonine and tyrosine kinase activi ties towards exogenous substrates. Most of the SH3 binding proteins we re soluble in Triton X-100. Differentiation of promyelocytic leukemia cell line HL-60 into macrophage like cells resulted in appearance of n ovel SH3 binding proteins. Hck was detected in the eluate of WGA-Sepha rose column, suggesting that it interacts with WGA binding glycoprotei n (s) A rat spleen cDNA library was screened for the SH3 binding prote ins by protein interaction cloning. Sequence analysis of the clones sh owed the presence of proline rich regions containing PPXP motifs.