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Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.Protein phosphatase 2A (PP2A) 1 is a highly conserved serine/threonine phosphatase involved in the regulation of a wide variety of enzymes, signal transduction pathways, and cellular events (1, 2). Consonant with its diverse roles, subpopulations of PP2A have been found to localize to the nucleus, cytoplasm, cytoskeleton, and membranes (3-6). The smallest functional unit of PP2A thought to exist in vivo consists of a heterodimer between a catalytic 36-kDa subunit, termed C, and a constant regulatory 63-kDa subunit, termed A (7). This A/C heterodimer often further complexes with a member of one of three additional cellular regulatory subunit families termed B (or B55), BЈ (or B56), and BЉ (or PR72/120) (1). In cells stably transformed by the middle tumor antigen (MT) of polyomavirus, MT substitutes for the B subunit in a small portion (ϳ10%) (8) 2 of PP2A complexes (9). MT⅐PP2A complex formation is known to be important for MT-mediated transformation (10 -13), but the precise functional consequences of MT association with PP2A are still being elucidated.Efforts aimed at understanding PP2A regulation have uncovered a complex set of noncovalent and covalent mechanisms. These include association with different regulatory subunits (1), association with heat stable inhibitors (14), action of a phosphotyrosyl activator protein (15), lipid binding (16), phosphorylation (17), and methylation (18 -22). These mechanisms affect the catalytic activity, substrate specificity, and cellular localization of PP2A. However, little is known about the molecular bases of their effects, and even less about how th...

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca 2؉ -dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G 2 nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the postsynaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G 2 phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca 2؉ -dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca 2؉-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA. PP2A,1 an essential serine/threonine protein phosphatase found in all eukaryotic cells, regulates a wide variety of important cellular events, including DNA replication, transcription, translation, development, neuronal signaling and progression of the cell cycle (for reviews see Refs. 1-3). The PP2A heterotrimer consists of a catalytic (C) subunit, a structural (A) subunit, and a regulatory (B-type) subunit (4). Although relatively few C-and A-type subunits have been identified, multiple B-type subunits exist, including B (or B55), BЈ (or B56), and BЈЈ (or PR72/130) classes (5-9). To enable utilization of this phosphatase for numerous substrates in different pathways, PP2A is regulated at multiple levels, including covalent modifications, interaction with inhibitory proteins and lipids, and association with the various B-type subunits. For example, BЈ subunits were recently shown to target PP2A to the adenomatous polyposis coli tumor suppressor scaffolding protein, physically associating PP2A with specific substrates and thus regulating Wnt-␤-catenin signaling (10).PP2A has also been shown to form complexes with CaM-dependent kinase IV (CaMKIV) (11), suggesting a role for PP2A in Ca 2ϩ -dependent signaling. This possibility is further supported by patch clamp experiments with both neuronal (12) and smooth muscle cells (13) that have used both okadaic acid and recombinant PP2A C subunit to implicate PP2A in the regulation of calcium-activated potassium channels and L-type Ca 2ϩ channels (14).To better understand how PP2A is targeted to various microenvironments and signal transduction pathways within the cell, we have looked for additional PP2A targeting subunits. Here we report the identification of two PP2A-associated proteins that may represent a novel ...

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