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 kinase that has an N-terminal regulatory region that contains SH3 and SH2 domains. 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 site-specific mutations within this region and analyzed the effect of such mutations 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 domain that abolished its ability to bind phosphotyrosine did not result in a significant loss of kinase activity. Mutation of Ser78 to Asp, located between the SH3 and the SH2 domains, resulted in a reduction of over 90% of the catalytic activity. The reduction in specific activity is not the result of any apparent physical instability of the mutants. Kinetic analyses indicate that the mutations did not affect the Km values for ATP-Mg or the polypeptide substrate. The ability of the mutants to phosphorylate and inactivate Src is directly correlated to their kinase activity. These results indicate that the regulatory region is important in optimizing the kinase activity of the catalytic domain, but apparently plays no direct or specific role in substrate recognition.
Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030, USA.
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D000081247:CSK Tyrosine-Protein Kinase; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008957:Models, Genetic; D016296:Mutagenesis; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D013696:Temperature; D018909:src Homology Domains; D019061:src-Family Kinases
D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases