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 ...

Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine phosphatase known to play important roles in cell cycle regulation. Association of different B-type targeting subunits with the heterodimeric core (A/C) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. We have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approaches were taken. First, S. cerevisiae cells were generated whose survival depended on the expression of different carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PP2A C subunit carboxyl-terminal leucine was identified, and cells deleted for this methyltransferase were utilized for our studies. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p. Loss of Cdc55p association was accompanied in each case by a large reduction in binding of the yeast A subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylation also greatly reduced the association of another yeast B-type subunit, Rts1p. Thus, methylation of Pph21p is important for formation of PP2A trimeric and dimeric complexes, and consequently, for PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.Protein phosphatase 2A (PP2A) 1 is a highly conserved eukaryotic serine/threonine phosphatase known to play important roles in many cellular events including regulation of the cell cycle (reviewed in Refs. 1-3). PP2A often exists as a heterotrimeric enzyme composed of a catalytic (C) subunit, a structural (A) subunit, and a variable regulatory/targeting (B-type) subunit (1, 4). In mammalian cells, three major families of B-type subunits, B (or B55), BЈ (or B56), and BЉ (or PR72/130), have been described (1). Binding of B-type subunits to the A/C core heterodimer modulates PP2A activity (5-11) and localizes PP2A to specific cellular microenvironments and/or signaling pathways (for examples, see Refs. 12-15).Although it is clear that binding of B-type targeting subunits is a major mechanism by which cells regulate PP2A, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. Recently, we showed in mammalian cells that deletion of nine C subunit carboxyl-terminal residues (amino acids 301-309) or nonconservative substitution of threonine 304 or tyrosine 307 a...

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I1 PP2A enhanced E4orf4-induced cell killing and G2/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.

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Supplementary Table 4 from Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56γ Knockdown

<div>Abstract<p>One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56γ subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56γ subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56γ knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor κB activation, while inhibiting cell-cell adhesion pathways.</p></div>

Supplementary Table 4 from Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56γ Knockdown