MECHANISM OF DOWN-REGULATION OF C-KIT RECEPTOR - ROLES OF RECEPTOR TYROSINE KINASE, PHOSPHATIDYLINOSITOL 3'-KINASE, AND PROTEIN-KINASE-C

The receptor tyrosine kinase Kit and Hit ligand (KL), encoded at the m urine white spotting (W) and steel (SI) loci, respectively, function i n hematopoiesis, melanogenesis, and gametogenesis. To understand the m echanism of turnover of Bit in mast cells, mutant receptors generated in vitro were heterologously expressed in W-sh/W-sh mast cells lacking endogenous c-kit expression, and the effects of mutations on KL-induc ed internalization and ubiquitination/degradation of Kit were studied. Upon binding of KL, KL-Kit receptor complexes were rapidly internaliz ed, and the turnover was accelerated by ubiquitin-mediated degradation . Inactivation of the Kit kinase resulted in a reduced rate of interna lization of KL-Kit complexes, degradation of kinase-inactive receptor complexes was relatively slow, and receptor ubiquitination was absent. But abolishment of Kt induced receptor association and activation of phosphatidylinositol 3'-kinase and of tyrosine 821 autophosphorylation did not affect KL-induced internalization and ubiquitination/degradat ion of Kit. Furthermore, Kit receptors can be down-regulated by proteo lytic cleavage induced by either activation of protein kinase C or by isopropyl alcohol. In summary, KL-induced internalization of ICL Kit c omplexes and ubiquitination/degradation require an active kinase. By c ontrast, proteolytic cleavage of Kit mediated by protein kinase C acti vation is independent of kinase activity.