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 analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [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 involved. 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 in 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; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.
Stewart
C E
CE
University Department of Surgery, Level 7, Bristol Royal Infirmary, Bristol, BS2 8 HW, England, UK. C.Stewart-Paton@bristol.ac.uk
Mihai
R
R
Holly
J M
JM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002518:Ceramides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts; D019833:Genistein / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008246:Lysophospholipids; D051379:Mice; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D002518:Ceramides; D007328:Insulin; D008246:Lysophospholipids; D019000:Phosphotyrosine; C060506:sphingosine 1-phosphate; D000001:Calcimycin; D014638:Vanadates; D004533:Egtazic Acid; D019284:Thapsigargin; D007334:Insulin-Like Growth Factor I; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D010744:Phosphoric Monoester Hydrolases; D013110:Sphingosine; D002118:Calcium