Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation

Abstract

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 → Gly (PEDS116G) but not Ser-104 → Gly (PEDS104G) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-ζ and by the expression of a dominant-negative PKC-ζ mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-ζ increased cellular content and phosphorylation of WT-PED/PEA-15 and PEDS104G but not of PED S116G. These events were accompanied by the activation of Ca 2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-ζ and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-ζ overexpression and increased by KN-93 and PKC-ζ block. Furthermore, in HEK293 cells expressing PEDS116G, TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPAincreases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.