Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase

c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogeni c counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphor ylated and its catalytic activity is very low. Here we report tyrosine phos phorylation of endogenous c-Abl and a concomitant increase in catalytic act ivity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of act ive Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-m echanism and cause activation of otherwise inactive c-Abl, suggesting a pos itive feedback loop on c-Abl activity. In the recent structure of the Abl c atalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the acti vation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibi tion. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzym e in a phosphorylation-dependent manner.