Jean-Marie Sontag

Protein phosphatase 2A is a critical regulator of protein kinase C ζ signaling targeted by SV40 small t to promote cell growth and NF‐κB activation

We have reported that inhibition of protein phosphatase 2A (PP2A) by expression of SV40 small t stimulates the mitogenic MAP kinase cascade. Here, we show that SV40 small t can substitute for tumor necrosis factor‐α (TNF‐α) or serum and stimulate atypical protein kinase C ζ (PKC ζ) activity, resulting in MEK activation, cell proliferation and NF‐κB‐dependent gene transcriptional activation in CV‐1 and NIH 3T3 cells. These effects were abrogated by co‐expression of kinase‐deficient PKC ζ and inhibition of phosphatidylinositol 3‐kinase p85α‐p110 by wortmannin, LY294002 and a dominant‐negative mutant of p85α. In contrast, expression of kinase‐inactive ERK2 inhibited small t‐dependent cell growth but was unable to abolish small t‐induced NF‐κB transactivation. Our results provide the first in vivo evidence for a critical regulatory role of PP2A in bifunctional PKC ζ signaling pathways controlled by phosphatidylinositol 3‐kinase. Constitutive activation of PKC ζ and NF‐κB following inhibition of PP2A supports new mechanisms by which SV40 small t promotes cell growth and transformation. By establishing PP2A as a key player in the response of cells to growth factors and stress signals like TNF‐α, our findings could explain why PP2A is a primary target utilized during SV40 infection to alter cellular behavior.

Protein Phosphatase 2A Methyltransferase Links Homocysteine Metabolism with Tau and Amyloid Precursor Protein Regulation

Alzheimers disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-β peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of Bα-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of β-secretase-cleaved APP fragments and amyloid-β peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective α-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-β-synthase +/− mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.

Protein phosphatase 2A dysfunction in Alzheimer’s disease

Protein phosphatase 2A (PP2A) is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD) field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. “PP2A” dysfunction has been linked to tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/Bα holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/Bα isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/Bα-tau protein interactions likely contribute to tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/Bα, enhanced phosphorylation of tau and amyloid precursor protein, tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie “PP2A” dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.

Folate Deficiency Induces In Vitro and Mouse Brain Region-Specific Downregulation of Leucine Carboxyl Methyltransferase-1 and Protein Phosphatase 2A Bα Subunit Expression That Correlate with Enhanced Tau Phosphorylation

Altered folate homeostasis is associated with many clinical and pathological manifestations in the CNS. Notably, folate-mediated one-carbon metabolism is essential for methyltransferase-dependent cellular methylation reactions. Biogenesis of protein phosphatase 2A (PP2A) holoenzyme containing the regulatory Bα subunit, a major brain tau phosphatase, is controlled by methylation. Here, we show that folate deprivation in neuroblastoma cells induces downregulation of PP2A leucine carboxyl methyltransferase-1 (LCMT-1) expression, resulting in progressive accumulation of newly synthesized demethylated PP2A pools, concomitant loss of Bα, and ultimately cell death. These effects are further accentuated by overexpression of PP2A methylesterase (PME-1) but cannot be rescued by PME-1 knockdown. Overexpression of either LCMT-1 or Bα is sufficient to protect cells against the accumulation of demethylated PP2A, increased tau phosphorylation, and cell death induced by folate starvation. Conversely, knockdown of either protein accelerates folate deficiency-evoked cell toxicity. Significantly, mice maintained for 2 months on low-folate or folate-deficient diets have brain-region-specific alterations in metabolites of the methylation pathway. Those are associated with downregulation of LCMT-1, methylated PP2A, and Bα expression and enhanced tau phosphorylation in susceptible brain regions. Our studies provide novel mechanistic insights into the regulation of PP2A methylation and tau. They establish LCMT-1- and Bα-containing PP2A holoenzymes as key mediators of the role of folate in the brain. Our results suggest that counteracting the neuronal loss of LCMT-1 and Bα could be beneficial for all tauopathies and folate-dependent disorders of the CNS.

Expression of protein phosphatase 2A mutants and silencing of the regulatory Bα subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down‐regulation of PP2A methylation and PP2A enzymes containing the Bα regulatory subunit occur in Alzheimer’s disease. In this study, we show that expressed wild‐type and methylation‐ (L309Δ) and phosphorylation‐ (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B′, and B′′‐type regulatory subunits in NIH 3T3 fibroblasts and neuro‐2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild‐type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309Δ, and T304D mutants, which are impaired in their ability to associate with the Bα subunit, induces their loss. Silencing of Bα subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole‐induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal Bα‐containing PP2A heterotrimers contribute to MT destabilization in Alzheimer’s disease.

Regulation of cell adhesion by PP2A and SV40 small tumor antigen: An important link to cell transformation

Abstract.The serine/threonine protein phosphatase 2A (PP2A) represents a large family of highly conserved heterotrimeric enzymes. Their critical importance in cell homeostasis is underlined by the fact that they are targets of natural toxins like the tumor promoter okadaic acid, and of simian virus 40 small tumor antigen (SV40 small t), a viral protein known to promote cell transformation. Furthermore, mutated or lower expression levels of PP2A subunits have been found in certain cancers. One major known event in PP2A-dependent cell transformation is the alteration of key signaling pathways that control cell growth and survival. In this review, we focus on how PP2A enzymes also affect cell adhesion and cytoskeletal dynamics, the disruption of which is linked to loss of cell polarity, increased cell motility and invasiveness. We also examine how those various pathways participate in the transforming activity of SV40 small t.

Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.

J. Neurochem. (2010) 115, 1455–1465.

Acute administration of L-DOPA induces changes in methylation metabolites, reduced protein phosphatase 2A methylation, and hyperphosphorylation of Tau protein in mouse brain.

Folate deficiency and hypomethylation have been implicated in a number of age-related neurodegenerative disorders including dementia and Parkinsons disease (PD). Levodopa (l-dopa) therapy in PD patients has been shown to cause an increase in plasma total homocysteine as well as depleting cellular concentrations of the methyl donor, S-adenosylmethionine (SAM), and increasing the demethylated product S-adenosylhomocysteine (SAH). Modulation of the cellular SAM/SAH ratio can influence activity of methyltransferase enzymes, including leucine carboxyl methyltransferase that specifically methylates Ser/Thr protein phosphatase 2A (PP2A), a major Tau phosphatase. Here we show in human SH-SY5Y cells, in dopaminergic neurons, and in wild-type mice that l-dopa results in a reduced SAM/SAH ratio that is associated with hypomethylation of PP2A and increased phosphorylation of Tau (p-Tau) at the Alzheimers disease-like PHF-1 phospho-epitope. The effect of l-dopa on PP2A and p-Tau was exacerbated in cells exposed to folate deficiency. In the folate-deficient mouse model, l-dopa resulted in a marked depletion of SAM and an increase in SAH in various brain regions with parallel downregulation of PP2A methylation and increased Tau phosphorylation. l-Dopa also enhanced demethylated PP2A amounts in the liver. These findings reveal a novel mechanism involving methylation-dependent pathways in l-dopa induces PP2A hypomethylation and increases Tau phosphorylation, which may be potentially detrimental to neuronal cells.

Leucine Carboxyl Methyltransferase 1 (LCMT1)-dependent Methylation Regulates the Association of Protein Phosphatase 2A and Tau Protein with Plasma Membrane Microdomains in Neuroblastoma Cells

Background: Altered methylation of PP2A, a major Tau phosphatase, occurs in Alzheimer disease. Results: PP2A methylation regulates the association of PP2A with membrane rafts and plasma membrane-bound Tau levels. Conclusion: Altered PP2A methylation inhibits targeting of PP2A and Tau to the plasma membrane. Significance: Altered PP2A and Tau membrane distribution could promote neuronal dysfunction and phospho-Tau pathology in Alzheimer disease. Down-regulation of protein phosphatase 2A (PP2A) methylation occurs in Alzheimer disease (AD). However, the regulation of PP2A methylation remains poorly understood. We have reported that altered leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down-regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in AD. Here, we show that pools of LCMT1, methylated PP2A, and PP2A/Bα are co-enriched in cholesterol-rich plasma membrane microdomains/rafts purified from N2a cells. In contrast, demethylated PP2A is preferentially distributed in non-rafts wherein small amounts of the PP2A methylesterase PME-1 are exclusively present. A methylation-incompetent PP2A mutant is excluded from rafts. Enhanced methylation of PP2A promotes the association of PP2A and Tau with the plasma membrane. Altered PP2A methylation following expression of a catalytically inactive LCMT1 mutant, knockdown of LCMT1, or alterations in one-carbon metabolism all result in a loss of plasma membrane-associated PP2A and Tau in N2a cells. This correlates with accumulation of soluble phosphorylated Tau, a hallmark of AD and other tauopathies. Thus, our findings reveal a distinct compartmentalization of PP2A and PP2A regulatory enzymes in plasma membrane microdomains and identify a novel methylation-dependent mechanism involved in modulating the targeting of PP2A, and its substrate Tau, to the plasma membrane. We propose that alterations in the membrane localization of PP2A and Tau following down-regulation of LCMT1 may lead to PP2A and Tau dysfunction in AD.

Altered protein phosphatase 2A methylation and Tau phosphorylation in the young and aged brain of methylenetetrahydrofolate reductase (MTHFR) deficient mice

Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr−/− and aged heterozygous Mthfr+/− mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr+/+ mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr+/− mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer’s disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD.