Activation of serine/threonine protein phosphatase-1 is required for ceramide-induced survival of sympathetic neurons.
In sympathetic neurons, C6-ceramide, as well as endogenous ceramides, blocks apoptosis elicited by NGF (nerve growth factor) deprivation. The mechanism(s) involved in ceramide-induced neuronal survival are poorly understood. Few direct targets for the diverse cellular effects of ceramide have been identified. Amongst those proposed is PP-1c, the catalytic subunit of serine/threonine PP-1 (protein phosphatase-1). Here, we present the first evidence of PP-1c activation by ceramide in live cells, namely NGF-deprived sympathetic neurons. We first determined PP activity in cellular lysates from sympathetic neurons treated with exogenous ceramide and demonstrated a 2-3-fold increase in PP activity. PP activation was completely blocked by the addition of the specific type-1 PP inhibitor protein I-2 as well as by tautomycin, but unaffected by 2 nM okadaic acid, strongly indicating that the ceramide-activated phosphatase activity was PP-1c. Inhibition of PP activity by phosphatidic acid (which has been reported to be a selective inhibitor of PP-1c) and tautomycin (a PP-1 and PP-2A inhibitor), but not by 10 nM okadaic acid, abolished the anti-apoptotic effect of ceramide in NGF-deprived neurons, suggesting that activation of PP-1c is required for ceramide-induced neuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1c activation, suggesting that two consequences of NGF deprivation in sympathetic neurons are inhibition of PP-1c and subsequent hyperphosphorylation of pRb protein. These findings suggest a novel mechanism for ceramide-induced survival, and implicate the involvement of PPs in apoptosis induced by NGF deprivation.
Plummer
Greg
G
Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Perreault
Kathleen R
KR
Holmes
Charles F B
CF
Posse De Chaves
Elena I
EI
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020932:Nerve Growth Factor / Q000172:deficiency / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D010712:Phosphatidic Acids / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1; D051381:Rats; D017207:Rats, Sprague-Dawley; D016160:Retinoblastoma Protein / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013564:Sympathetic Nervous System / Q000166:cytology*; D013912:Threonine / Q000378:metabolism
D002518:Ceramides; D010712:Phosphatidic Acids; D016160:Retinoblastoma Protein; C101954:N-caproylsphingosine; D013912:Threonine; D012694:Serine; D020932:Nerve Growth Factor; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1