Phosphorylation of Raf by ceramide-activated protein kinase

Abstract

THE sphingomyelin pathway, initiated by hydrolysis of sphingo-myelin to ceramide and stimulation of a Ser/Thr ceramide-activated protein (CAP) kinase, mediates tumour necrosis factor-α (TNF-α) and interleukin-1β action1–4. CAP kinase is membrane-bound and proline-directed, recognizing the minimal substrate motif Thr-Leu-Pro (ref. 5). TNF may use the sphingomyelin pathway to signal Raf1 to activate the MAP kinase cascade6–8. Evidence shows that cytoplasmic Rafl binds to GTP-ras upon cellular stimulation, is recruited to the plasma membrane, and activated9,11. How membrane-bound Rafl is activated is uncertain, but regulation of its kinase activity may involve its phosphorylation12–19. Specific Raf kinases, however, have not hitherto been identified. Here we report that CAP kinase phosphorylates Rafl on Thr 269, increasing its activity towards MEK (MAP kinase or ERK kinase). Moreover, in intact HL-60 cells, CAP kinase complexes with Rafl and, in response to TNF and ceramide analogues, phosphorylates and activates Rafl, implicating CAP kinase as a link between the TNF receptor and Rafl.