SH2-kinase linker mutations release Hck tyrosine kinase and transforming activities in Rat-2 fibroblasts.
Biochemical and structural studies of Src and related kinases demonstrate that two intramolecular interactions suppress kinase activity. These interactions involve binding of the SH2 domain to a phosphotyrosine residue in the C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by amino acids linking the SH2 and kinase domains. Recent studies have shown that high affinity interaction of the SH3 domain of Hck with the human immunodeficiency virus type I Nef protein activates Hck tyrosine kinase and biological activities, suggesting a mechanism that involves disruption of the SH3-linker interaction. To test the role of this interaction in the regulation of Hck kinase activity in living cells, we substituted alanines for prolines 225 and 228 in the linker region and observed that the resulting mutant (Hck-2PA) demonstrated strong transforming activity in a Rat-2 fibroblast focus-forming assay. Hck-2PA also exhibited elevated tyrosine kinase activity in terms of autophosphorylation, endogenous substrate phosphorylation, and in an in vitro kinase assay. The transforming and kinase activities of Hck-2PA were remarkably similar to those observed with a Hck mutant activated by Phe substitution of the conserved tail Tyr residue and with wild-type Hck following co-expression with human immunodeficiency virus Nef. Introduction of the 2PA and tail mutations into a single Hck expression construct did not increase kinase or transforming activity relative to the individual mutations. These data provide new evidence that SH3-linker interaction may represent the dominant mechanism controlling Hck tyrosine kinase activity in vivo.
Briggs
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Eppley Institute for Research in Cancer and the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Smithgall
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D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D005347:Fibroblasts / Q000201:enzymology; D015693:Gene Products, nef / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D051381:Rats; D013379:Substrate Specificity; D018909:src Homology Domains
D015693:Gene Products, nef; D011518:Proto-Oncogene Proteins; D011392:Proline; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; C496633:Hck protein, rat; D051572:Proto-Oncogene Proteins c-hck; D000409:Alanine