Wu Rui, Kausar Hina, Johnson Paul, Montoya-Durango Diego E, Merchant Michael, Rane Madhavi J
Department of Medicine, University of Louisville, Louisville, KY 40202, USA.
J Biol Chem. 2007 Jul 27;282(30):21598-608. doi: 10.1074/jbc.M611316200. Epub 2007 May 17.
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. Cotransfection 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 active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta117-128) 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.
我们之前已经表明，Akt存在于与p38丝裂原活化蛋白激酶（MAPK）、MAPK活化蛋白激酶-2（MK2）以及热休克蛋白27（Hsp27）组成的信号复合物中，且MK2使Akt的丝氨酸473位点发生磷酸化。此外，在Akt激活之前，Hsp27从Akt上解离会诱导多形核白细胞（PMN）凋亡。然而，Hsp27在调节Akt激活过程中的作用尚未得到研究。本研究检验了这样一个假说，即Hsp27通过将MK2搭建到Akt信号复合物上来调节Akt激活并促进细胞存活。在此我们表明，用抗Hsp27抗体处理导致Akt/Hsp27相互作用丧失，进而导致Akt/MK2相互作用丧失、Akt丝氨酸473位点磷酸化缺失，并诱导PMN凋亡。在HK - 11细胞中转染肉豆蔻酰化的Akt（AktCA）可诱导Akt丝氨酸473位点磷酸化、激活以及Hsp27丝氨酸82位点磷酸化。将AktCA与Hsp27小干扰RNA共转染，但不与乱序小干扰RNA共转染，可使HK - 11细胞中的Hsp27表达沉默，而不改变Akt表达。使Hsp27表达沉默会抑制Akt/MK2相互作用、抑制Akt磷酸化和Akt激活，并诱导HK - 11细胞死亡。缺失诱变研究确定Akt上的酸性连接区（氨基酸117 - 128）为Hsp27结合区域。如免疫沉淀和谷胱甘肽S - 转移酶下拉实验所示，缺失Akt上的氨基酸117 - 128会导致其与Hsp27和MK2的相互作用丧失，但与Hsp90的相互作用未丧失。共转染实验表明，组成型活性MK2（MK2EE）可使固定细胞中的野生型Akt（Aktwt）丝氨酸473位点发生磷酸化，但不能使Akt（Delta117 - 128）突变体发生磷酸化。这些研究共同确定了Hsp27在通过介导Akt与其上游激活剂MK2之间的相互作用来调节Akt激活和细胞凋亡方面的新作用。
