Trencia Alessandra, Perfetti Anna, Cassese Angela, Vigliotta Giovanni, Miele Claudia, Oriente Francesco, Santopietro Stefania, Giacco Ferdinando, Condorelli Gerolama, Formisano Pietro, Beguinot Francesco
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.
Mol Cell Biol. 2003 Jul;23(13):4511-21. doi: 10.1128/MCB.23.13.4511-4521.2003.
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.
抗凋亡蛋白PED/PEA-15在Ser(116)上游具有一个Akt磷酸化基序。在体外，重组PED/PEA-15被Akt磷酸化，化学计量比接近1。基于用特异性磷酸化Ser(116) PED/PEA-15抗体进行的蛋白质印迹分析，PED/PEA-15的Akt磷酸化主要发生在Ser(116)。此外，将Ser(116)替换为甘氨酸的PED/PEA-15突变体（PED(S116-->G)）显示其被Akt磷酸化的程度降低了10倍。在完整的293细胞中，Akt也诱导PED/PEA-15在Ser(116)处发生磷酸化。基于下拉和共沉淀分析，PED/PEA-15特异性结合Akt，与Akt活性无关。在转染了PED/PEA-15的293细胞和未转染的细胞（不表达内源性PED/PEA-15）中，Akt的血清激活以及Akt介导的BAD磷酸化没有差异。然而，与PED/PEA-15(WT)细胞相比，PED(S116-->G)中PED/PEA-15的抗凋亡作用几乎降低了两倍。在293细胞中，PED/PEA-15的稳定性与血清对Akt的激活密切相关。在这些细胞中，不可磷酸化的PED(S116-->G)突变体的降解速率比野生型PED/PEA-15观察到的降解速率大三倍。在U373MG胶质瘤细胞中，阻断Akt也降低了PED/PEA-15的水平，并诱导对肿瘤坏死因子相关凋亡诱导配体凋亡的敏感性。因此，Akt磷酸化至少部分地通过控制PED/PEA-15的稳定性来调节PED/PEA-15的抗凋亡功能。部分地，Akt存活信号可能由PED/PEA-15介导。
