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J. Biol. Chem., Vol. 282, Issue 30, 21598-21608, July 27, 2007

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Hsp27 Regulates Akt Activation and Polymorphonuclear Leukocyte Apoptosis by Scaffolding MK2 to Akt Signal Complex^*

Rui Wu¹, Hina Kausar¹, Paul Johnson, Diego E. Montoya-Durango, Michael Merchant, and Madhavi J. Rane²

From the Departments of Medicine and Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40202

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_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.

Received for publication, December 11, 2006 , and in revised form, May 15, 2007.

^* This work was supported by American Heart Association-Scientist Development Grant 0335278N (to M. J. R.) and National Institutes of Health Grant 1R56AI059165-01A2 (to M. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: University of Louisville, 570 S. Preston St., Baxter I Bldg. South, 102C, Louisville, KY 40202. Tel.: 502-852-0014; Fax: 502-852-4384; E-mail: mjrane01{at}gwise.louisville.edu.

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