Interaction of SH3 domain of Hck tyrosine kinase with cellular proteins containing proline-rich regions: evidence for modulation by unique domain

Abstract

The Hck tyrosine kinase, a member of Src family, is predominantly expressed in myeloid cells. In this report we have analyzed interaction of cellular proteins with Src homology 3 (SH3) domain of Hck. For this purpose we used various GST-Hck fusion proteins comprising a part of unique region, complete unique region and/or complete SH3 domain of Hck, and glutathione S-transferase (GST). When these fusion proteins (or GST), immobilized on glutathione-agarose beads were incubated with [35S] methionine labelled cell extracts, multiple proteins which interact specifically with SH3 domain of Hck were detected by SDS-PAGE followed by autoradiography. The Hck interacting proteins could also be detected by a tandem blot binding assay in which the blot was incubated with purified fusion protein (or GST) and then the interacting proteins were identified by using antibody against GST. When a part of or complete unique domain was present along with SH3 domain, the interaction of some specific proteins was reduced several fold. These results raise the possibility of unique domain altering the properties of SH3 domain, thus modulating or restricting the interaction of SH3 domain with specific cellular proteins. This modulatory effect of unique domain was localized to 28 amino acids upstream of SH3 domain. SH3 interacting proteins were associated with serine/threonine and tyrosine kinase activities towards exogenous substrates. Most of the SH3 binding proteins were soluble in Triton X-100. Differentiation of promyelocytic leukemia cell line HL-60 into macrophage like cells resulted in appearance of novel SH3 binding proteins. Hck was detected in the eluate of WGA-Sepharose column, suggesting that it interacts with WGA binding glycoprotein (s). A rat spleen cDNA library was screened for the SH3 binding proteins by protein interaction cloning. Sequence analysis of the clones showed the presence of proline rich regions containing PPXP motifs.