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.
Subject(s)
Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Cell Differentiation , Cell Line , Cloning, Molecular , DNA, Complementary/genetics , HL-60 Cells , Humans , Molecular Sequence Data , Proline/chemistry , Protein Binding , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-hck , Rats , Recombinant Fusion Proteins/chemistry , Spleen/metabolism , src Homology DomainsABSTRACT
The hck gene is member of src family of non-receptor type tyrosine kinases. Here we report the nucleotide sequence of the rat hck eDNA of 1.94 kb. The nuclcotide sequence shows an open reading frame coding for a polypeptide of 503 amino acids. A vector expressing a fusion protein of glutathione-S-transferase with 82 amino acids of the N-terminal region of hck (from amino acids 32 to 113) was constructed, Using this bacterially expressed fusion protein antibodies were prepared which recognize the cellular hck gene product. These antibodies identified, by immunoblotting, two polypeptides of 56 and 59 kDa in rat spleen where hck transcripts are present at high level. Immunoprecipitated hck polypeptides were enzymatically active and were autophosphorylated in the presence of ATP and Mg2+. 1mmunoprecipitated hck could phosphorylate exogenous substrates. Treatment of immunoprecipitated hck by a purified protein tyrosine phosphatase decreased its enzymatic acitivity. Our results suggest that the enzymatic activity of hck tyrosine kinase is regulated by phosphorylation and dephosphorylation.