Protein kinase B/Akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action.
The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser(116). In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser(116) PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser(116). In addition, a mutant of PED/PEA-15 featuring the substitution of Ser(116)-->Gly (PED(S116-->G)) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also induced phosphorylation of PED/PEA-15 at Ser(116). Based on pull-down and coprecipitation assays, PED/PEA-15 specifically bound Akt, independently of Akt activity. Serum activation of Akt as well as BAD phosphorylation by Akt showed no difference in 293 cells transfected with PED/PEA-15 and in untransfected cells (which express no endogenous PED/PEA-15). However, the antiapoptotic action of PED/PEA-15 was almost twofold reduced in PED(S116-->G) compared to that in PED/PEA-15(WT) cells. PED/PEA-15 stability closely paralleled Akt activation by serum in 293 cells. In these cells, the nonphosphorylatable PED(S116-->G) mutant exhibited a degradation rate threefold greater than that observed with wild-type PED/PEA-15. In the U373MG glioma cells, blocking Akt also reduced PED/PEA-15 levels and induced sensitivity to tumor necrosis factor-related apoptosis-inducing ligand apoptosis. Thus, phosphorylation by Akt regulates the antiapoptotic function of PED/PEA-15 at least in part by controlling the stability of PED/PEA-15. In part, Akt survival signaling may be mediated by PED/PEA-15.
Trencia
Alessandra
A
Dipartimento di Biologia e Patologia Cellulare e Molecolare and Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R., Federico II University of Naples, Naples, Italy.
Perfetti
Anna
A
Cassese
Angela
A
Vigliotta
Giovanni
G
Miele
Claudia
C
Oriente
Francesco
F
Santopietro
Stefania
S
Giacco
Ferdinando
F
Condorelli
Gerolama
G
Formisano
Pietro
P
Beguinot
Francesco
F
D000730:Androstadienes / Q000494:pharmacology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D051017:Apoptosis Regulatory Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C092039:PEA15 protein, human; D010455:Peptides; D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012694:Serine; D003513:Cycloheximide; D005982:Glutathione Transferase; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin