Mutations in the N-terminal regulatory region reduce the catalytic activity of Csk, but do not affect its recognition of Src

In addition to the C-terminal catalytic domain, Csk is a protein tyrosine k inase that has an N-terminal regulatory region that contains SH3 and SH2 do mains. The role this region plays relative to the function of the catalytic domain is not clear. To study its role, we introduced either deletion or s ite-specific mutations within this region and analyzed the effect of such m utations on the catalytic activity of Csk and its ability to phosphorylate/ inactivate Src protein tyrosine kinase, its physiological substrate in the cell. Deletion of the SH3 domain and the SH2 domain resulted in reductions of kinase activity by 70 and 96%, respectively, Mutations within the SH2 do main that abolished its ability to bind phosphotyrosine did not result in a significant loss of kinase activity. Mutation of Ser78 to Asp, located bet ween the SH3 and the SH2 domains, resulted in a reduction of over 90% of th e catalytic activity, The reduction in specific activity is not the result of any apparent physical instability of the mutants. Kinetic analyses indic ate that the mutations did not affect the K-m values for ATP-Mg or the poly peptide substrate. The ability of the mutants to phosphorylate and inactiva te Src is directly correlated to their kinase activity. These results indic ate that the regulatory region is important in optimizing the kinase activi ty of the catalytic domain, but apparently plays no direct or specific role in substrate recognition. (C) 1999 Academic Press.