Mutation of BAD within the BH3 domain impairs its phosphorylation-mediated regulation
Mutation of BAD within the BH3 domain impairs its phosphorylation-mediated regulation
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DOI：
10.1016/s0014-5793(03)00915-3
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发表时间：
2003-09-11
期刊：
影响因子：
3.5
通讯作者：
Imai, K
中科院分区：
文献类型：
--
作者：
Adachi, M;Zhang, YB;Imai, K
仅BH3蛋白BAD的促凋亡功能受存活信号介导的几个丝氨酸残基的磷酸化的负调控。最近，我们发现突变的BAD(BADD119G)与BH3区的天冬氨酸(Asp119到Gly)取代，在血清饥饿的COS-7细胞中显示出很强的促凋亡活性，但它不能与Bcl-2相互作用。在这里，我们证明了BADD119G失去了磷酸化介导的负调控。重要的是，野生型BAD(BADwt)与结构活性Akt(CA-Akt)共转染组的促凋亡活性被强烈抑制，而BADD119G的促凋亡活性不受抑制。在这些转染体中，丝氨酸残基(S75和S99)上几乎检测不到BADD119G的磷酸化，但CA-Akt明显促进了BADwt的磷酸化。此外，在COS-7细胞中，UV、TPA和Forskolin不能使S75、S99和S118处的BADD119G磷酸化。然而，体外蛋白激酶分析显示，催化蛋白激酶A(PKA)在S75和S118强烈磷酸化这两个BADS，排除了PKA的靶序列在S119被突变破坏的可能性。此外，由于磷酸化中断，BADD119G不能与143-3发生物理作用。综上所述，干扰磷酸化介导的负调控可能至少部分解释了BADD119G强大的促凋亡功能，并提示BH3结构域在磷酸化事件中发挥作用。(C)2003年，由Elsevier B.V.代表欧洲生化学会联合会出版。
Pro-apoptotic functions of the BH3-only protein BAD are negatively regulated by survival signal-mediated phosphorylation at several serine residues. Recently, we found that the mutant BAD (BADD119G) with an amino acid substitution of Asp (Asp119 to Gly) within the BH3 domain displays strong pro-apoptotic activity in serum-starved COS-7 cells, although it cannot interact with Bcl-2. Here, we demonstrate that the BADD119G loses phosphorylation-mediated negative regulation. Importantly, pro-apoptotic activity of wild-type BAD (BADwt) was strongly suppressed by co-transfection with constitutively active Akt (CA-Akt) cDNA, whereas that of BADD119G was not. In these transfectants, BADD119G phosphorylation was barely detectable at serine residues (S75 and S99), although BADwt phosphorylation was clearly increased by CA-Akt. In addition, various external stimuli UV, TPA and forskolin could not phosphorylate BADD119G neither at S75, S99 nor S118 in COS-7 cells. However, in vitro kinase assay revealed that catalytic protein kinase A (PKA) strongly phosphorylated both BADs at S75 and S118, excluding the possibility that the target sequence of PKA was disrupted by mutation at S119. Furthermore, as a result of disrupted phosphorylation, BADD119G could not physically interact with 143-3. Taken together, disruption of phosphorylation-mediated negative regulation may explain, at least in part, the strong pro-apoptotic functions of BADD119G, and suggest a role for the BH3 domain in phosphorylation events. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.