An In Vivo Assay to Quantify Stable Protein Phosphatase 2A (PP2A) Heterotrimeric Species
Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. The enzyme is, in fact, largely a collection of varied heterotrimeric species composed of a catalytic (C) subunit and regulatory (B-type) subunit bound together by a structural (A) subunit. One important feature of the C subunit is that its carboxy-terminus can be modified by phosphorylation and methylation. The mechanisms that trigger such posttranslational modifications, as well as their consequences, are still under investigation. However, data collected thus far indicate that these modifications alter the binding to B subunits for an AC dimer, thereby affecting the makeup of the PP2A species in the cell. In this chapter, we describe an in vivo assay for assessing stable PP2A heterotrimer formation that is based on specific subcellular localizations of PP2A heterotrimers. This assay can be used to study the impact of a wide variety of alterations (such as mutations and covalent modifications) on PP2A heterotrimer formation. We specifically describe the use of this assay to quantify the effects of methylation on the stable formation of PP2ARts1p and PP2ACdc55p heterotrimers.
Key WordsProtein phosphatase 2A PP2A GFP methylation phosphorylation
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