AUTOPHOSPHORYLATION OF THE FES TYROSINE KINASE - EVIDENCE FOR AN INTERMOLECULAR MECHANISM INVOLVING 2 KINASE DOMAIN TYROSINE RESIDUES

The human c-fes proto oncogene encodes a cytoplasmic tyrosine kinase ( Fes) that is associated with multiple hematopoietic cytokine receptors , Fes tyrosine autophosphorylation sites may regulate kinase activity and recruit downstream signaling proteins with SH2 domains, To localiz e the Fes autophosphorylation sites, full-length Fes and deletion muta nts lacking either the unique N-terminal or SH2 domain were autophosph orylated in vitro and analyzed by CNBr cleavage, Identical phosphopept ides of 10 and 4 kDa were produced with all three proteins, localizing the tyrosine autophosphorylation sites to the C-terminal kinase domai n, Substitution of kinase domain tyrosine residues 713 or 811 with phe nylalanine resulted in a loss of the 10- and 4-kDa phosphopeptides, re spectively, identifying these tyrosines as in vitro autophosphorylatio n sites, CNBr cleavage analysis of Fes isolated from (PO4)-P-32-labele d 293T cells showed that Tyr-713 and Tyr-811 are also autophosphorylat ed in vivo. Mutagenesis of Tyr-713 reduced both autophosphorylation of Tyr-811 and transphosphorylation of Bcr, a recently identified Fes su bstrate, supporting a major regulatory role for Tyr-713, Wild-type Fes transphosphorylated a kinase-inactive Fes mutant on Tyr-713 and Tyr-8 11, suggesting that Fes autophosphorylation occurs via an intermolecul ar mechanism analogous to receptor tyrosine kinases.