Protein phosphatase 2A in stretch-induced endothelial cell proliferation

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459–466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.     

Protein phosphatase 2A regulatory subunits and cancer

The serine/threonine protein phosphatase (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of a number of major signaling pathways whose deregulation can contribute to cancer. The specificity and activity of PP2A are highly regulated through the interaction of a family of regulatory B subunits with the substrates. Accumulating evidence indicates that PP2A acts as a tumor suppressor. In this review we summarize the known effects of specific PP2A holoenzymes and their roles in cancer relevant pathways. In particular we highlight PP2A function in the regulation of MAPK and Wnt signaling.     

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.     

Protein phosphatase 2A activity associated with Golgi membranes during the G2/M phase may regulate phosphorylation of ERK2

The extracellular signal-regulated kinase (ERK) 1 and 2 proteins are mitogen-activated protein kinase (MAPK) members that regulate cell proliferation and differentiation. ERK proteins are activated exclusively by MAPK kinase 1 and 2 phosphorylation of threonine and tyrosine residues located within the conserved TXY MAPK activation motif. Although dual phosphorylation of Thr and Tyr residues confers full activation of ERK, in vitro studies suggest that a single phosphorylation on either Thr or Tyr may yield partial ERK activity. Previously, we have demonstrated that phosphorylation of the tyrosine residue (Tyr(P) ERK) may be involved in regulating the Golgi complex structure during the G2 and M phases of the cell cycle (Cha, H., and Shapiro, P. (2001) J. Cell Biol. 153, 1355-1368). In the present study, we examined mechanisms for generating Tyr(P) ERK by determining cell cycle-dependent changes in localized phosphatase activity. Using fractionated nuclei-free cell lysates, we find increased serine/threonine phosphatase activity associated with Golgi-enriched membranes in cells synchronized in the late G2/early M phase as compared with G1 phase cells. The addition of phosphatase inhibitors in combination with immunodepletion assays identified this activity to be related to protein phosphatase 2A (PP2A). The increased activity was accounted for by elevated PP2A association with mitotic Golgi membranes as well as increased catalytic activity after normalization of PP2A protein levels in the phosphatase assays. These data indicate that localized changes in PP2A activity may be involved in regulating proteins involved in Golgi disassembly as cells enter mitosis.     

Protein phosphatase 2A: a panoply of enzymes

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.     

Elevated levels of protein phosphatase 1 and phosphatase 2A may contribute to cardiac dysfunction in diabetes

Although protein phosphorylation and dephosphorylation are known to regulate the activities of different enzymes, sufficient information on the role of dephosphorylation in cardiac function is not available. Since protein phosphatases mediate dephosphorylation, it is possible that cardiac dysfunction induced by diabetes may be due to alterations in the activities of these enzymes. We therefore determined cardiac protein phosphatase activity as well as protein contents of phosphatase 1 and phosphatase 2A in diabetic animals. For this purpose, rats were made diabetic by administering a single intravenous injection of streptozotocin (65 mg/kg body weight) and hearts were examined after 1, 2, 3, 4 and 8 weeks. Some of the 4-week diabetic animals received subcutaneous injections of insulin (3 U/day) for a further period of 4 weeks. Cardiac dysfunction was evident after 2 weeks of inducing diabetes and deteriorated further with time. A significant increase in protein phosphatase activity appeared after 1 week and persisted until 8 weeks. Increased protein phosphatase activity in the diabetic heart was associated with a corresponding increase in the protein contents of both phosphatase 1 and phosphatase 2A. Insulin treatment partly prevented the changes observed in diabetic animals. The results suggest that increased protein phosphatase activities and subsequent enhanced protein dephosphorylation may play a role in diabetes-induced cardiac dysfunction.     

Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.     

Protein phosphatase 2A on track for nutrient-induced signalling in yeast

Early studies identified two bona fide protein phosphatase 2A (PP2A)-encoding genes in Saccharomyces cerevisiae, designated PPH21 and PPH22. In addition, three PP2A-related phosphatases, encoded by PPH3, SIT4 and PPG1, have been identified. All share as much as 86% sequence similarity at the amino acid level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4 regulation will also be discussed. Whereas a role for PP2A in yeast morphology and cell cycle has been readily recognized, uncovering its function in yeast signal transduction is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP-PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient-induced signalling in baker's yeast.     

Protein phosphatase 1 dephosphorylates Orc2

Phosphorylation of Thr¹¹⁶ and Thr²²⁶ on Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, β and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.     

A novel glycosphingolipid that may participate in embryo inversion in Volvox carteri

Mouse monoclonal antibody 4-I-244 detects a developmentally regulated antigen in embryos of Volvox carteri and inhibits specifically the morphogenetic process of inversion (the process by which the embryo turns inside out). Antigen 4-I-244 was chemically characterized as a complex phytosphingolipid containing the neutral sugars xylose, galactose, and glucose as well as inositol and phosphate.     

Protein phosphatase type 2C dephosphorylates BAD

Reversible phosphorylation modulates a cells’ susceptibility to apoptosis. The phosphorylation status of BAD, a member of the Bcl-2 protein family, is an important checkpoint governing life-or-death decisions: Phosphorylation of serine residues 112, 136 and 155 on BAD prevents apoptosis. Here we report that BAD is a substrate for PP2C. Ser¹⁵⁵ is involved in heterodimerization with Bcl-X_L. We could demonstrate that PP1, PP2A and PP2C act on this site in vitro. However, only PP2C gives priority to P-Ser¹⁵⁵ compared to P-Ser¹¹² and P-Ser¹³⁶ on BAD. The results indicate that PP2C is an additional factor triggering the pro-apoptotic function of BAD.     

Protein phosphatase 2A: the Trojan Horse of cellular signaling

Dynamic phosphorylation and dephosphorylation of proteins are fundamental mechanisms utilized by cells to transduce signals. Whereas transduction by protein kinases has been a major focus of studies in the last decade, protein phosphatase 2A (PP2A) enzymes emerge in this millenium as the most fashionable players in cellular signaling. Viral proteins target specific PP2A enzymes in order to deregulate chosen cellular pathways in the host and promote viral progeny. The observation that a variety of viruses utilize PP2A to alienate cellular behavior emphasizes the fundamental importance of PP2A in signal transduction. This review will primarily focus on discussing the uniqueness of PP2A regulation and uncovering the critical role played by protein-protein interactions in the modulation of PP2A signaling. Moreover, the place of PP2A in signaling pathways and its functional significance for human diseases will be discussed.     

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.     

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa

We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A B subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.     

Protein phosphatase 2A: a definite player in viral and parasitic regulation

Cells use phosphorylation/dephosphorylation mechanisms to regulate the activity of several proteins required to transmit information from the cell surface to the nucleus. Recent studies have significantly increased our knowledge regarding the structure/function of one major regulator of cell phosphorylation: protein phosphatase 2A (PP2A). This review will discuss the role of PP2A in virology and parasitology.     

Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes

In adipocytes, protein kinase B (PKB) has been suggested to be the enzyme that phosphorylates phosphodiesterase 3B (PDE3B), a key enzyme in insulin's antilipolytic signalling pathway. In order to screen for PKB phosphatases, adipocyte homogenates were fractionated using ion-exchange chromatography and analysed for PKB phosphatase activities. PKB phosphatase activity eluted as one main peak, which coeluted with serine/threonine phosphatases (PP)2A. In addition, adipocytes were incubated with inhibitors of PP. Incubation of adipocytes with 1 μM okadaic acid inhibited PP2A by 75% and PP1 activity by only 17%, while 1 μM tautomycin inhibited PP1 activity by 54% and PP2A by only 7%. Okadaic acid, but not tautomycin, induced the activation of both PKBα and PKBβ. Finally, PP2A subunits were found in several subcellular compartments, including plasma membranes (PM) where the phosphorylation of PKB is thought to occur. In summary, our results suggest that PP2A is the principal phosphatase that dephosphorylates PKB in adipocytes.     

Protein phosphatase type 2A, PP2A, is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp 130 at Ser-782 and degradation of gp 130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp 130. Taken together, present results strongly suggest that degradation of gp 130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp 130 is a potential therapeutic target in cancers.