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Session III : Protein kinase cascades as therapeutic targets Poster III, 47
HSP27 SCAFFOLDS MK2 TO THE AKT SIGNAL COMPLEX
Madhavi J. Rane
Department of Medicine, Molecular Signaling Group, University of Louisville,
Louisville, KY 40202, U.S.A. E-mail:mrane@louisville.edu
The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB) signaling pathway
plays a critical role in cell growth, proliferation, and cell survival. Up regulation of this
pathway has been demonstrated in various carcinomas. Thus, inhibiting Akt activation and
promoting cell death provides a strategy for targeted cancer therapy. We have shown
previously that Akt/Hsp27 interaction regulates neutrophil apoptosis and that MK2 acts as
PDK2 for Akt. The current 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 in neutrophils or by
transfection of Hsp27 siRNA in HK-11 cells resulted in induction of cellular apoptosis.
Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt Ser473
phosphorylation, activation, and Hsp27 Ser82 phosphorylation. Co-transfection of AktCA
and Hsp27siRNA in HK-11 cells specifically silenced Hsp27 expression, without altering
expression of Akt. Silencing Hsp27 expression resulted in the loss of Akt/MK2 interaction,
loss of Akt Ser473 phosphorylation, activation, Hsp27 Ser82 phosphorylation, and induced
HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (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 Hsp90 as demonstrated by immunoprecipitation
and GST pull-down studies. Co-transfection studies in HEK-293 cells demonstrated that
constitutively active MK2 (MK2EE) phosphorylated Aktwt on Ser473 but failed to
phosphorylate Akt&117-128 mutant. In conclusion, Hsp27 scaffolds MK2 to Akt signal
complex and promotes Akt activation and cell survival. Veterans Administration Grant and
AHA 0335278N
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