Abstract
Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases all composed of a catalytic C, a structural A, and a regulatory B subunit. Assembly of the complex with the appropriate B subunit forms the key to the functional specificity and regulation of PP2A. Emerging evidence suggests a crucial role for methylation and phosphorylation of the PP2A C subunit in this process. In this study, we show that PP2A C subunit methylation was not absolutely required for binding the PR61/B' and PR72/B'' subunit families, whereas binding of the PR55/B subunit family was determined by methylation and the nature of the C-terminal amino acid side chain. Moreover mutation of the phosphorylatable Tyr(307) or Thr(304) residues differentially affected binding of distinct B subunit family members. Down-regulation of the PP2A methyltransferase LCMT1 by RNA interference gradually reduced the cellular amount of methylated C subunit and induced a dynamic redistribution of the remaining methylated PP2A(C) between different PP2A trimers consistent with their methylation requirements. Persistent knockdown of LCMT1 eventually resulted in specific degradation of the PR55/B subunit and apoptotic cell death. Together these results establish a crucial foundation for understanding PP2A regulatory subunit selection.
Highlights
The mammalian regulatory B subunits can be divided into three distinct groups: 1) PR55/B, 2) PR61/ BЈ␣,1,␥,⑀, and 3) PR61/BЈ␦1 and PR72/BЉ, each with specific intrinsic requirements or constraints for trimer formation
The authors basically came to the same conclusions that C-terminal methylation of PP2AC and phosphorylation of Tyr307 and Thr304 are differentially important for formation of the different holoenzyme forms
Whereas in our study the pulldowns of the individual GST fusions with the “third” subunits would reveal rather the intrinsic affinity of a specific subunit for the mutant forms of PP2AC, the use of an IP approach with the mutant catalytic subunits would largely reflect the presence of the relative amounts and affinities of the different subunits in the cellular conditions used
Summary
Plasmids and Antibodies—Plasmids used for the expression of GST- or HA-tagged fusion proteins were made by subcloning the appropriate human cDNAs in the pGMEX-T1,2,3 (Amersham Biosciences) and pMB001 vectors (25). This suspension was briefly vortexed, and after 15 min of incubation on ice, it was centrifuged at 16,000 ϫ g for 15 min at 4 °C. The immunoprecipitates were analyzed by Western blotting for the presence of endogenous PR65 and expressed HA-tagged PP2AC subunits
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