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N. Pasupuleti, S. Matsuyama, O. Voss, A. Doseff, K. Song, D. Danielpour, R. H. Nagaraj; Alpha A-Crystallin Inhibits Apoptosis by Inactivating PTEN. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4619.
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© ARVO (1962-2015); The Authors (2016-present)
Alpha-crystallin functions as a molecular chaperone as well as an anti-apoptotic protein. In this study we investigated the mechanism by which αA-crystallin functions as an anti-apoptotic protein using two mutant proteins, R21A and R49A, which exhibit higher and lower chaperone activity relative to the wild type protein, respectively.
We generated Chinese Hamster Ovary (CHO) cell lines that stably overexpress either the wild type (Wt) or R21A or R49A mutant protein. The same three proteins were transiently overexpressed in HeLa cells. Apoptosis was induced by either staurosporine or etoposide or doxorubicin. Phosphorylation of Akt, PDK1 and PTEN was assessed by western blotting. PI3K activity was measured using a commercial kit. PI3kinase downregulation was achieved by expression of a dominant negative mutant.
R21A cells showed enhanced protection and R49A cells showed reduced protection against apoptotic agents, relative Wt cells. This effect was due to higher PI3kinase activity in R21A cells, although the PI3kinase levels were similar in all cell lines. Akt phosphorylation was higher in R21A cells when compared to either R49A or Wt cells. Downregulation of PI3kinase by a dominant negative mutant or inhibition by LY294002 abrogated the enhanced ability of R21A to phosphorylate Akt, suggesting that αA-crystallin inhibits apoptosis through activation of PI3kinase. PDK1 and PTEN phosphorylation were increased in R21A cells relative to Wt cells.
Our data reveal that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating PTEN and that the anti-apoptotic function is directly related to its chaperone activity.
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