07-250 Sigma-AldrichAnti-PP2A Antibody, A subunit

The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A-C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.

Adenovirus E4orf4 protein induces the death of human cancer cells and Saccharomyces cerevisiae. Binding of E4orf4 to the B/B55/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) is required, and such binding inhibits PP2A(B55) activity leading to dose-dependent cell death. We found that E4orf4 binds across the putative substrate binding groove predicted from the crystal structure of B55α such that the substrate p107 can no longer interact with PP2A(B55α). We propose that E4orf4 inhibits PP2A(B55) activity by preventing access of substrates and that at high E4orf4 levels this inhibition results in cell death through the failure to dephosphorylate substrates required for cell cycle progression. However, E4orf4 is expressed at much lower and less toxic levels during a normal adenovirus infection. We suggest that in this context E4orf4 largely serves to recruit novel substrates such as ASF/SF2/SRSF1 to PP2A(B55) to enhance adenovirus replication. Thus E4orf4 toxicity probably represents an artifact of overexpression and does not reflect the evolutionary function of this viral product.

Concurrent activation of RAS/ERK and PI3K/AKT pathways is implicated in prostate cancer progression. The negative regulators of these pathways, including sprouty2 (SPRY2), protein phosphatase 2A (PP2A), and phosphatase and tensin homolog (PTEN), are commonly inactivated in prostate cancer. The molecular basis of cooperation between these genetic alterations is unknown. Here, we show that SPRY2 deficiency alone triggers activation of AKT and ERK, but this is insufficient to drive tumorigenesis. In addition to AKT and ERK activation, SPRY2 loss also activates a PP2A-dependent tumor suppressor checkpoint. Mechanistically, the PP2A-mediated growth arrest depends on GSK3β and is ultimately mediated by nuclear PTEN. In murine prostate cancer models, Pten haploinsufficiency synergized with Spry2 deficiency to drive tumorigenesis, including metastasis. Together, these results show that loss of Pten cooperates with Spry2 deficiency by bypassing a novel tumor suppressor checkpoint. Furthermore, loss of SPRY2 expression correlates strongly with loss of PTEN and/or PP2A subunits in human prostate cancer. This underlines the cooperation between SPRY2 deficiency and PTEN or PP2A inactivation in promoting tumorigenesis. Overall, we propose SPRY2, PTEN, and PP2A status as an important determinant of prostate cancer progression. Characterization of this trio may facilitate patient stratification for targeted therapies and chemopreventive interventions.

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.

Cerebral cavernous malformations (CCMs) are alterations in brain capillary architecture that can result in neurological deficits, seizures, or stroke. We recently demonstrated that CCM3, a protein mutated in familial CCMs, resides predominantly within the STRIPAK complex (striatin interacting phosphatase and kinase). Along with CCM3, STRIPAK contains the Ser/Thr phosphatase PP2A. The PP2A holoenzyme consists of a core catalytic subunit along with variable scaffolding and regulatory subunits. Within STRIPAK, striatin family members act as PP2A regulatory subunits. STRIPAK also contains all three members of a subfamily of Sterile 20 kinases called the GCKIII proteins (MST4, STK24, and STK25). Here, we report that striatins and CCM3 bridge the phosphatase and kinase components of STRIPAK and map the interacting regions on each protein. We show that striatins and CCM3 regulate the Golgi localization of MST4 in an opposite manner. Consistent with a previously described function for MST4 and CCM3 in Golgi positioning, depletion of CCM3 or striatins affects Golgi polarization, also in an opposite manner. We propose that STRIPAK regulates the balance between MST4 localization at the Golgi and in the cytosol to control Golgi positioning.

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B''' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.

Evidence from genetic linkage analysis indicates that a gene located at 19q13.4, FWT2, is responsible for predisposition to Wilms tumor in many Wilms tumor families. This region has also been implicated in the etiology of sporadic Wilms tumor through loss of heterozygosity analyses. The PPP2R1A gene, encoding the alpha isoform of the heterotrimeric serine/threonine protein phosphatase 2A (PP2A), is located within the FWT2 candidate region and is altered in breast and lung carcinomas. PPP2R1B, encoding the beta isoform, is mutated in lung, colon, and breast cancers. These findings suggested that both PPP2R1A and PPP2R1B may be tumor suppressor genes. Additionally, PP2A is important in fetal kidney growth and differentiation and has an expression pattern similar to that of the Wilms tumor suppressor gene WT1. Since PPP2R1A was therefore a compelling candidate for the FWT2 gene, we analysed the coding region of PPP2R1A in DNA and RNA samples from affected members of four Wilms tumor families and 30 sporadic tumors and identified no mutations in PPP2R1A in any of these 34 samples. We conclude that PPP2R1A is not the 19q familial Wilms tumor gene and that mutation of PPP2R1A is not a common event in the etiology of sporadic Wilms tumor.

Protein phosphatase 2A (PP2A) consists of three subunits, A, B and C. The A and B subunits have regulatory functions while C is the catalytic subunit. PP2A core enzyme is composed of subunits A and C, and the holoenzyme of subunits A, B and C. All subunits exist as multiple isoforms or splice variants. The A subunit exists as two isoforms, A alpha and A beta. Here we report about the properties of eight A beta mutants, which were found in human lung and colon cancer. These mutants were reconstructed by site-directed mutagenesis and assayed for their ability to bind B and C subunits. Two mutants showed decreased binding of PR72, a member of the B" family of B subunits, but normal C subunit binding; two mutants exhibited decreased binding of the C subunit and of B"/PR72; and one mutant showed increased binding of both the C subunit and B"/PR72. Of three mutants that behaved like the wild-type A beta subunit, one is a polymorphic variant and another one is altered outside the binding region for B and C subunits. Importantly, we also found that the wild-type A alpha and A beta isoforms, although 85% identical, are remarkably different in their ability to bind B and C subunits. Our findings may have important implications in regard to the role of PP2A as a tumor suppressor.