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. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also 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 wild-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 and 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 the intramolecular binding of the SH2 and SH3 domains.
Porter
M
M
Department of Physiology, School of Medicine, State University of New York, Stony Brook, New York 11794-8661, USA.
Schindler
T
T
Kuriyan
J
J
Miller
W T
WT
D000595:Amino Acid Sequence; D002384:Catalysis; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; 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; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D008024:Ligands; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051572:Proto-Oncogene Proteins c-hck