Activation of serine/threonine protein phosphatase-1 is required for ceramide-induced survival of sympathetic neurons

• Published in: Biochemical journal 2005-02, Vol.385 (Pt 3), p.685-693
• Main Authors: Plummer, Greg, Perreault, Kathleen R, Holmes, Charles F B, Posse De Chaves, Elena I
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Apoptosis - drug effects; Cell Survival - drug effects; Cells, Cultured; Ceramides - pharmacology; Dose-Response Relationship, Drug; Enzyme Activation; Membrane Potentials - drug effects; Mitochondria - drug effects; Mitochondria - metabolism; Nerve Growth Factor - deficiency; Nerve Growth Factor - metabolism; Neurons - cytology; Neurons - drug effects; Neurons - enzymology; Neurons - metabolism; Phosphatidic Acids - pharmacology; Phosphoprotein Phosphatases - metabolism; Phosphorylation - drug effects; Protein Phosphatase 1; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein - metabolism; Serine - metabolism; Sympathetic Nervous System - cytology; Threonine - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20040929

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.