Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation.
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 (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-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-zeta 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-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), 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 TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.
Perfetti
Anna
A
Dipartimento di Biologia e Patologia cellulare e Molecolare (DBPCM), Federico II University of Naples, Via Pansini 5, 80131 Naples, Italy.
Oriente
Francesco
F
Iovino
Salvatore
S
Alberobello
A Teresa
AT
Barbagallo
Alessia P M
AP
Esposito
Iolanda
I
Fiory
Francesca
F
Teperino
Raffaele
R
Ungaro
Paola
P
Miele
Claudia
C
Formisano
Pietro
P
Beguinot
Francesco
F
D051017:Apoptosis Regulatory Proteins; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010703:Phorbol Esters / Q000737:chemistry* / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D000099221:Protein Kinase C zeta; D000096926:Benzenesulfonamides
D051017:Apoptosis Regulatory Proteins; D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047908:Intracellular Signaling Peptides and Proteins; D010703:Phorbol Esters; D010750:Phosphoproteins; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate; D025801:Ubiquitin; D000099221:Protein Kinase C zeta; C092039:PEA15 protein, human; C072105:KN 93; D000096926:Benzenesulfonamides