Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416) - Effect of introducing a high affinity intramolecular SH2 ligand

The Src family tyrosine kinase Hck possesses two phosphorylation sites,Tyr( 527) and Tyr(416), that affect the catalytic activity in opposite ways. Whe n phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this s equence does not conform to the sequence of the high affinity SH2 ligand, p YEEI, We mutated this sequence to YEEI and show that this mutant form of Hc k cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is al so involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to w ild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr (416) is required for Hck activation. We mutated this residue to alanine an d characterized its catalytic activity. The Y416A mutant shows a higher K-m value for peptide and a lower V-max than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and th e intramolecular binding of the SH2 and SH3 domains.