Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex
Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex
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DOI：
10.1074/jbc.m611316200
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发表时间：
2007-07-27
影响因子：
4.8
通讯作者：
Rane, Madhavi J.
中科院分区：
文献类型：
--
作者：
Wu, Rui;Kausar, Hina;Rane, Madhavi J.
我们之前已经证明Akt存在于p38 MAPK、MAPK活化蛋白激酶-2 （MK2）和热休克蛋白27 （Hsp27）的信号复合物中，MK2磷酸化Akt的Ser-473位点。此外，在Akt激活之前，Hsp27与Akt分离可诱导多形核白细胞（PMN）凋亡。然而，没有研究Hsp27在调节Akt激活中的作用。本研究验证了Hsp27通过架空MK2介导Akt信号复合物调控Akt活化并促进细胞存活的假说。本研究表明，通过抗Hsp27抗体处理，Akt/Hsp27相互作用的丧失导致Akt/MK2相互作用的丧失，Akt- ser -473磷酸化的丧失，并诱导PMN凋亡。在HK-11细胞中转染肉豆肉酰化Akt （AktCA）可诱导Akt- ser -473磷酸化、活化和Hsp27-Ser-82磷酸化。在HK-11细胞中，将Hsp27短干扰RNA（而非打乱短干扰RNA）共转染AktCA，可以抑制Hsp27的表达，而不改变Akt的表达。沉默Hsp27表达抑制Akt/MK2相互作用，抑制Akt磷酸化和Akt活化，诱导HK-11细胞死亡。缺失诱变研究发现Akt上的酸性连接区（氨基酸117-128）是Hsp27的结合区。免疫沉淀和谷胱甘肽s -转移酶下调研究表明，Akt上氨基酸117-128的缺失导致其与Hsp27和MK2的相互作用丧失，但与Hsp90的相互作用不存在。共转染研究表明，组成型activeMK2（MK2EE）磷酸化Ser-473上的Aktwt（野生型），但在固定细胞中不能磷酸化Akt（Delta 117-128）突变体。这些研究共同确定了Hsp27通过介导Akt与其上游激活因子MK2之间的相互作用，在调节Akt活化和细胞凋亡中的新作用。
We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Co-transfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117-128) on Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively activeMK2(MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta 117-128) mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.