Increased tyrosine kinase activity but not calcium mobilization is required for ceramide-induced apoptosis

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid an alogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -pos itive murine fibroblasts, indicating a protective feedback loop in the latt er and corroborating evidence that the IGF-IR functions as a survival recep tor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms inv olved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that i n the presence of sodium orthovanadate, ceramide-induced death is increased , and the phosphorylation of a 75-kDa protein which associates with the IGF -I receptor is enhanced. Although the identity of this protein is not known , we speculate that it may link into the Raf kinase signaling pathway; inde ed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiati ng this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptos is. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphat e was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apo ptosis. (C) 1999 Academic Press.