INACTIVATION OF C-YES TYROSINE KINASE BY ELEVATION OF INTRACELLULAR CALCIUM LEVELS

We have previously shown that the c-Src tyrosine kinase is activated f our- to fivefold when cultured keratinocytes differentiate following t he elevation of intracellular calcium levels. In contrast to c-Src, an other Src family tyrosine kinase, c-Yes, was rapidly inactivated in th ese same cells, despite its marked similarity in structure and enzymat ic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes we re not altered, but the phosphotyrosine content of both proteins was g reatly reduced. As has been demonstrated for c-Src, in vitro dephospho rylation of c-Yes by incubation with protein tyrosine phosphatases als o resulted in its activation, not inactivation. In vitro reconstitutio n experiments showed that c-Yes can be inactivated by preincubation wi th a Ca2+-supplemented cell extract and that this inhibition was rever sed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether )-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complex es were not seen in c-Src immunoprecipitates. One of these two protein s has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca2+-dependent inactivation of c-Yes was observed in kidney tubula r cells and fibroblasts, suggesting that the Ca2+ -dependent regulatio n of c-Yes tyrosine kinase is not unique to keratinocytes. We postulat e that c-Yes is inactivated through a Ca2+-dependent association with cellular proteins, which seems to override its activation resulting fr om tyrosine dephosphorylation.