Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: Insights from NMR mapping studies and site-directed mutagenesis

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the S rc homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphory lates the C-terminal tail of Src-family members, resulting in downregulatio n of the Src family kinase activity. The Src family kinases share 37% homol ogy with Csk but, unlike Src-family kinases, the catalytic domain of Csk al one is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolec ular interaction were studied by nuclear magnetic resonance (NMR) and site- directed mutagenesis techniques. Chemical shift perturbation of backbone nu clei (H' and N-15) has been used to map the Csk catalytic domain binding si te on the Csk-SH3. The experimentally determined interaction surface includ es three structural elements: th N-terminal tail, a small part of the RT-lo op, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed t hat mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinas e activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a m ajor role in the activation of the Csk catalytic domain. (C) 2001 Academic Press.