The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.
The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.
Gonfloni
S
S
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Williams
J C
JC
Hattula
K
K
Weijland
A
A
Wierenga
R K
RK
Superti-Furga
G
G
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D051572:Proto-Oncogene Proteins c-hck; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C496632:Hck protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases