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,blocksapoptosiselicitedbyNGF(nervegrowthfactor) deprivation. The mechanism(s) involved in ceramide-induced neuronalsurvivalarepoorlyunderstood.Fewdirecttargetsforthe diversecellulareffectsofceramidehavebeenidentified.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 NGFdeprived 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 antiapoptoticeffectofceramideinNGF-deprivedneurons,suggesting thatactivationofPP-1cisrequiredforceramide-inducedneuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1cactivation,suggestingthattwoconsequencesofNGFdeprivation 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.