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J. Biol. Chem., Vol. 279, Issue 46, 47732-47739, November 12, 2004
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Critical Role for Protein Phosphatase 2A Heterotrimers in Mammalian Cell Survival*
From the Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (
/
), whereas three gene families (B, B', and B'') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding A subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. A RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B'' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of A triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant A cDNAs rescued RNAi-induced loss of the C subunit, and A point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type A and A mutants capable of binding to at least one family of regulatory subunits were able to delay A RNAi-induced death of PC12 cells. However, only the expression of wild-type A restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the A subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.
Received for publication, July 15, 2004 , and in revised form, August 31, 2004.
* This work was supported by National Institutes of Health Grant NS43254 (to S. S.). 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.
To whom correspondence should be addressed: Dept. of Pharmacology, University of Iowa Carver College of Medicine, 2-432 BSB, 51 Newton Rd., Iowa City, IA 52242. Tel.: 319-384-4439; Fax: 319-335-8930; E-mail: stefan-strack{at}uiowa.edu.
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