Phosphatase 2A Regulatory Subunit Research Articles

Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes

BackgroundPROTEIN PHOSPHATASE 2A (PP2A) expression is crucial for the symbiotic association between plants and various microbes, and knowledge on these symbiotic processes is important for sustainable agriculture. Here we tested the hypothesis that PP2A regulatory subunits, especially B’φ and B’θ, are involved in signalling between plants and mycorrhizal fungi or plant-growth promoting bacteria.ResultsTreatment of tomato plants (Solanum lycopersicum) with the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Pseudomonas simiae indicated a role for the PP2A B’θ subunit in responses to PGPR. Arbuscular mycorrhizal fungi influenced B’θ transcript levels in soil-grown plants with canonical arbuscular mycorrhizae. In plant roots, transcripts of B’φ were scarce under all conditions tested and at a lower level than all other PP2A subunit transcripts. In transformed tomato plants with 10-fold enhanced B’φ expression, mycorrhization frequency was decreased in vermiculite-grown plants. Furthermore, the high B’φ expression was related to abscisic acid and gibberellic acid responses known to be involved in plant growth and mycorrhization. B’φ overexpressor plants showed less vigorous growth, and although fruits were normal size, the number of seeds per fruit was reduced by 60% compared to the original cultivar.ConclusionsExpression of the B’θ gene in tomato roots is strongly influenced by beneficial microbes. Analysis of B’φ overexpressor tomato plants and established tomato cultivars substantiated a function of B’φ in growth and development in addition to a role in mycorrhization.

Novel B55α-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest.

Activation of the Protein Kinase B (PKB), or AKT pathway has been shown to correlate with acute myeloid leukemia (AML) prognosis. B55α-Protein Phosphatase 2A (PP2A) has been shown to dephosphorylate AKT at Thr-308 rendering it inactive. In fact, low expression of the PP2A regulatory subunit B55α was associated with activated phospho-AKT and correlated with inferior outcomes in AML. Despite this fact, no studies have specifically demonstrated a mechanism whereby B55α expression is regulated in AML. In this study, we demonstrate novel loss of function mutations in the PPP2R2A gene identified in leukemic blasts from three AML patients. These mutations eliminate B55α protein expression thereby allowing constitutive AKT activation. In addition, leukemic blasts with PPP2R2A gene mutation were more sensitive to treatment with the AKT inhibitor MK2206, but less responsive to the PP2A activator FTY720. Using leukemia cell lines, we further demonstrate that B55α expression correlates with AKT Thr-308 phosphorylation and predicts responsiveness to AKT inhibition and PP2A activation. Together our data illustrate the importance of the B55α-PP2A-AKT pathway in leukemogenesis. Screening for disruptions in this pathway at initial AML diagnosis may predict response to targeted therapies against AKT and PP2A.

Abstract 3685: Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55

Abstract Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that has critical roles in numerous cellular pathways. The role of PP2A as an essential tumor suppressor in numerous death signaling pathways is well documented. In this regard, recent work from our laboratory has highlighted a novel mechanism of apoptosis inhibition via post-translational redox-modification of PP2A. Increase in intracellular superoxide, either by pharmacological inhibition or siRNA-mediated silencing of superoxide dismutase 1 (SOD1), results in the dissociation of PP2A-AC catalytic core from its B regulatory subunit. Dissociation of the PP2A heterotrimer is a result of the selective nitration [by peroxynitrite derived from the reaction of superoxide with nitric oxide] of a conserved tyrosine residue on B56δ which we identified as Y289 (B56δY289). Importantly, we showed in lymphoma cells that PP2A-AC catalytic core is typically targeted to Bcl-2 by its B56δ regulatory subunit. When O2- is elevated, it signals for the sustained phosphorylation of Bcl-2 at S70 by inducing the release of PP2A-AC catalytic core from B56δ-bound Bcl-2. This results in the sustained phospho-activation of anti-apoptotic Bcl-2 and resistance to apoptotic stimuli. Notably, Bcl-2 is not the only substrate of PP2A, nor is B56δ the only PP2A regulatory subunit susceptible to redox modification. Thus, implications from redox-modification of PP2A may extend beyond the phospho-activation of Bcl-2. In the context of carcinogenesis, another notable substrate of PP2A is the tumor suppressor, p53. p53 stability is negatively influenced by its phosphorylation status at threonine-55 (T55). Phosphorylation of T55, on the other hand, is countered by PP2A harboring the B56γ3 subunit. Here we provide evidence that the B56γ3 sub-unit of PP2A complexes with p53 and this complex is sustained upon an increase in intracellular superoxide. Importantly, redox-mediated inhibition of B56γ3-containing PP2A (PP2AB56γ3) prevents the dephosphorylation of p53 at T55, thereby negatively impacting the stability of p53 in tumor cells with elevated superoxide level. This, we hypothesize, may be a novel death inhibitory mechanism that renders tumor cells resistant to conventional DNA-damaging therapeutic agents that rely heavily on the activation of p53 for death induction. Citation Format: Stephen Chong, Shazib Pervaiz. Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3685.

Abstract 3955: Identification of RACK1 as a novel regulatory subunit of PP2A

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a key role to play in the regulation of cell spreading and cell migration in tumour cells. We have identified PP2A as a RACK1 interacting protein downstream of the IGF-IR (1). In tumour cells PP2A and β1 integrin compete for binding to the C-terminus of RACK1. The IGF-I-mediated release of PP2A from RACK1 results in a decrease in PP2A activity, and promotes cell migration (2). This is coupled with the IGF-I-mediated dephosphorylation of FAK and focal adhesion turnover in MCF-7 cells (3). PP2A enzymes exist as heterotrimers comprising of the catalytic C-, the structural A- and the regulatory/variable B-type subunits. The B-type subunits have a critical role in targeting the PP2A protein to specific areas of the cell and also in regulating the overall activity and substrate specificity of PP2A. To date, at least 15 known PP2A B-type subunits have been identified but their detection has proven difficult because of the transient nature of their interaction with the PP2A holoenzyme. We used in vitro PP2A activity assays to determine that RACK1 stabilizes PP2A activity. This led us to predict that RACK1 may function as a regulatory subunit of PP2A. A comparative study between RACK1 and the well studied B56 family of PP2A regulatory subunits reveals strong homology. Both of these proteins are characterized by the presence of 7 WD repeat sequences which in the case of B56 gamma facilitates PP2A holoenzyme assembly. We used immobilized peptide arrays representing the entire PP2A-catalytic protein to identify a series of amino acids on the catalytic subunit of PP2A that are required for the binding of RACK1. Mutation analysis of these residues reveals the binding site for RACK1 on PP2A. Suppression of RACK1 expression in MCF-7 and DU145 cells results in a decrease in the activity and an altered location of PP2A-C suggesting that RACK1 has an essential role to play in the overall activity and substrate specificity of PP2A. Taken together the data supports our hypothesis that RACK1 functions as a B-type subunit of PP2A which is required for the regulation of PP2A activity. Our data also indicate that RACK1 targets PP2A to specific locations to promote IGF-I-mediated cell migration and proliferation in tumour cells. 1. Kiely, P. A., O'Gorman, D., Luong, K., Ron, D., and O'Connor, R. (2006) Mol Cell Biol 26, 4041-4051 2. Kiely, P. A., Baillie, G. S., Lynch, M. J., Houslay, M. D., and O'Connor, R. (2008) J Biol Chem 3. Kiely, P. A., Baillie, G. S., Barrett, R., Buckley, D. A., Adams, D. R., Houslay, M. D., and O'Connor, R. (2009) J Biol Chem 284, 20263-20274 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3955.

Adenovirus Protein E4orf4 Induces Premature APC Cdc20 Activation in Saccharomyces cerevisiae by a Protein Phosphatase 2A-Dependent Mechanism

Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G(2)/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2A(Cdc55) in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2A(Cdc55) in regulating APC(Cdc20) activity.

Differential Expression of the B′β Regulatory Subunit of Protein Phosphatase 2A Modulates Tyrosine Hydroxylase Phosphorylation and Catecholamine Synthesis

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

Protein phosphatase 2A negatively regulates insulin's metabolic signaling pathway by inhibiting Akt (protein kinase B) activity in 3T3-L1 adipocytes.

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C lambda (PKC lambda) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC lambda activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC lambda expression or PKC lambda siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC lambda and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.

Overexpression of the Protein Phosphatase 2A Regulatory Subunit Bγ Promotes Neuronal Differentiation by Activating the MAP Kinase (MAPK) Cascade

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous Bbeta, Bgamma, and B'beta protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of Bgamma, but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of Bgamma caused growth arrest and neurofilament expression, further evidence that PP2A/Bgamma can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, Bgamma specifically promoted long lasting activation of the mitogen-activated protein (MAP) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that Bgamma promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the TrkA NGF receptor but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N terminus is critical for Bgamma activity. These studies implicate PP2A/Bgamma as a positive regulator of MAP kinase signaling in neurons.

Protein Phosphatase 2A Forms a Molecular Complex with Shc and Regulates Shc Tyrosine Phosphorylation and Downstream Mitogenic Signaling

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.