Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue.

Intramolecular interactions between the Src homology domains (SH2 and SH3) and the catalytic domains of Src family kinases result in repression of catalytic activity. The crystal structure of the Src family kinase Hck, with its regulatory domains intact, has been solved. It predicts that a conserved residue, Trp260, at the end of the linker between the SH2 and the catalytic domains plays an important role in regulation by the SH3 and SH2 domains. We have mutated this residue and compared the activities of C-terminally phosphorylated wild type Hck and W260A Hck. The W260A mutant has a higher specific activity than wild type Hck. The W260A mutant requires autophosphorylation at Tyr416 for full activity, but it is not activated by ligand binding to the SH3 or SH2 domains. This mutation also changes the accessibility of the SH2 and SH3 domains to their cognate peptide ligands. Our results indicate that Trp260 plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well as in positioning the ligand binding surfaces.

Phosphorylation and activation of cGMP-dependent protein kinase by Src.

Using information obtained from experiments with peptide substrates of v-Src, a motif within the cGMP-binding domain of cGMP-dependent protein kinase (cGK) was identified as a potential phosphorylation site for v-Src. Here we show that the purified Ialpha isozyme of cGK is phosphorylated stoichiometrically and in a time-dependent manner by purified Src in vitro. The kinase activity of cGK is elevated approximately 4-fold (relative to autophosphorylated cGK) or 10-fold (relative to unphosphorylated cGK) upon tyrosine phosphorylation by Src. Phosphorylation of cGK by v-Src produces modest effects on the cGMP-binding properties and dissociation rates of cGK, and reduces the kact for cGMP. We hypothesize that the mechanism of activation may involve coupling of the cGMP binding domain to the catalytic domain.

Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement.

The protein Hck is a member of the Src family of non-receptor tyrosine kinases which is preferentially expressed in haematopoietic cells of the myeloid and B-lymphoid lineages. Src kinases are inhibited by tyrosine-phosphorylation at a carboxy-terminal site. The SH2 domains of these enzymes play an essential role in this regulation by binding to the tyrosine-phosphorylated tail. The crystal structure of the downregulated form of Hck has been determined and reveals that the SH2 domain regulates enzymatic activity indirectly; intramolecular interactions between the SH3 and catalytic domains appear to stabilize an inactive form of the kinase. Here we compare the roles of the SH2 and SH3 domains in modulating the activity of Hck in an investigation of the C-terminally phosphorylated form of the enzyme. We show that addition of the HIV-1 Nef protein, which is a high-affinity ligand for the Hck SH3 domain, to either the downregulated or activated form of Hck causes a large increase in Hck catalytic activity. The intact SH3-binding motif in Nef is crucial for Hck activation. Our results indicate that binding of the Hck SH3 domain by Nef causes a more marked activation of the enzyme than does binding of the SH2 domain, suggesting a new mechanism for regulation of the activity of tyrosine kinases.

Triton X-100-resistant membrane complexes from cultured kidney epithelial cells contain the Src family protein tyrosine kinase p62yes.

We have previously isolated detergent-resistant membrane complexes from lysates of cultured kidney epithelial cells. These structures are enriched in proteins anchored by glycosylphosphatidylinositol, which are localized to the apical plasma membrane of epithelial cells, and have a distinctive lipid composition. We now report that the membranes contain p62yes, a non-receptor tyrosine kinase of the Src family. p62yes is the only major tyrosine-phosphorylated protein in the membrane complexes when they are isolated. Several proteins in the complexes can serve as substrates for phosphorylation by p62yes in vitro. The kinase is enriched at least 10-fold in the insoluble membranes compared to cellular membranes. p60c-src and p60v-src, which are highly homologous to p62yes, are not present in the complexes, even when the membranes are isolated from cells that overexpress the proteins. As p62yes is located near the apical plasma membrane of epithelial cells in several tissues in vivo, interaction of the kinase with other components of a detergent-resistant membrane microdomain may play an important role in transmembrane signal transduction.