Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.     

The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors.

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.     

C3G mediated suppression of malignant transformation involves activation of PP2A phosphatases at the subcortical actin cytoskeleton

In previous work, we demonstrated that C3G suppresses Ras oncogenic transformation by a mechanism involving inhibition of ERK phosphorylation. Here we present evidences indicating that this suppression mechanism is mediated, at least in part, by serine/threonine phosphatases of the PP2A family. Thus: (i) ectopic expression of C3G or C3GDeltaCat (mutant lacking the GEF activity) increases specific ERK-associated PP2A phosphatase activities; (ii) C3G and PP2A interact, as demonstrated by immunofluorescence and co-immunoprecipitation experiments; (iii) association between PP2A and MEK or ERK increases in C3G overexpressing cells; (iv) phosphorylated-inactive PP2A level decreases in C3G expressing clones and, most importantly, (v) okadaic acid reverts the inhibitory effect of C3G on ERK phosphorylation. Moreover, C3G interacts with Ksr-1, a scaffold protein of the Ras-ERK pathway that also associates with PP2A. The fraction of C3G involved in transformation suppression is restricted to the subcortical actin cytoskeleton where it interacts with actin. Furthermore, the association between C3G and PP2A remains stable even after cytoskeleton disruption with cytochalasin D, suggesting that the three proteins form a complex at this subcellular compartment. Finally, C3G- and C3GDeltaCat-mediated inhibition of ERK phosphorylation is reverted by incubation with cytochalasin D. We hypothesize that C3G triggers PP2A activation and binding to MEK and ERK at the subcortical actin cytoskeleton, thus favouring ERK dephosphorylation.     

Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.     

Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites

The endothelial isoform of nitric oxide synthase (eNOS) is a calcium/calmodulin-dependent enzyme that catalyzes the synthesis of nitric oxide, a key mediator of vascular homeostasis. eNOS undergoes a variety of posttranslational modifications, including phosphorylation on at least three residues: serines 116 and 1179 and threonine 497. Although the agonist-modulated protein kinase pathways that lead to eNOS phosphorylation have been studied in detail, the signaling pathways governing eNOS dephosphorylation remain less well characterized. The present study identifies protein phosphatase 2A (PP2A) as a key determinant of eNOS dephosphorylation and enzyme activity. We transfected bovine aortic endothelial cells (BAEC) with epitope-tagged cDNAs encoding wild-type eNOS or a series of phosphorylation-deficient eNOS mutants, immunoprecipitated [(32)P(i)] biosynthetically labeled recombinant proteins using antibodies directed against the epitope tag and treated the [(32)P(i)]-phosphorylated eNOS with protein phosphatases. We found that PP2A dephosphorylates eNOS residues threonine 497 and serine 1179 but not serine 116 and that an eNOS mutant lacking these three established phosphorylation sites is robustly labeled when expressed in BAEC and is dephosphorylated by PP2A. An inhibitor of PP2A increases eNOS enzymatic activity and augments overall levels of eNOS phosphorylation, specifically increasing phosphorylation of serines 116 and 1179. When transfected into BAEC or COS-7 cells, a "phospho-mimetic" eNOS mutant in which threonine 497 is changed to aspartate shows attenuated phosphorylation at serine 1179 as well as reduced enzyme activity in COS-7 cells. Our results indicate that regulation of eNOS dephosphorylation may be a key point for control of nitric oxide-dependent signaling pathways in vascular endothelial cells.     

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.     

PP2A: unveiling a reluctant tumor suppressor

Although evidence has suggested that the serine/threonine protein phosphatase 2A (PP2A) might be a tumor suppressor protein, it has been difficult to pin down its role in tumor suppression because it acts in a wide variety of signaling pathways. Recent findings, including work in this issue by Junttila et al. (2007), provide convincing evidence that suppression of PP2A activity cooperates with other oncogenic changes to cause transformation of multiple cell types.     

Lead Dysregulates Serine/Threonine Protein Phosphatases in Human Neurons

It is well established that lead (Pb) exposure in humans leads to learning and memory impairment. However, the biological and molecular mechanisms are still not clearly understood. When over activated, serine/threonine protein phosphatases are known to function as a constraint on learning and memory. Activation of these phosphatases can also result in cytoskeletal changes that will adversely affect learning and memory. We investigated the effects of Pb exposure on these phosphatases in primary cultures of human neurons. Neurons were exposed to physiologically relevant concentrations of Pb (5, 10, 20 and 40 μg/dL) and total phosphatase and PP2A activities were determined in neuronal lysate using para-nitrophenyl phosphate (pNPP), and a PP2A-specific phosphopeptide as substrates. Expression of various serine/threonine phosphatases, tau and its phosphorylation state were determined by Western blot (WB) and immunocytochemistry (ICC). We found that the total phosphatase activity in the neuronal lysate was increased by 30–50% by all the concentrations of Pb tested. PP2A activity was increased by 5 μg/dL Pb only. PP1 expression was increased (ranging from 25–50%) by 10, 20 and 40 μg/dL of Pb. PP2B expression was increased substantially (up to 2.5-fold) by 10 μg/dL Pb, whereas, higher concentrations did not show any effect. On the other hand, Pb (at all concentrations used) decreased expression of PP2A and PP5. Pb exposure induced substantial hyperphosphorylation of tau at serine 199/202 by 5 and 10 μg/dL Pb, and Threonine 231 at higher doses. Expression of total tau was mostly unaffected by lead. Immunocytochemistry data confirmed the WB results of expression of PP1, PP2A, tau protein and the phosphorylation of tau. These results support our hypothesis that Pb exposure up regulates some of the serine/threonine phosphatases (PP1 and PP2B) that are known to impair memory formation, and suggest a novel mechanism of Pb neurotoxicity.     

Role of mTOR in the degradation of IRS-1: regulation of PP2A activity

We have investigated the role of PI 3-kinase and mTOR in the degradation of IRS-1 induced by insulin. Inhibition of mTOR with rapamycin resulted in approximately 50% inhibition of the insulin-induced degradation of IRS-1. In contrast, inhibition of PI-3 kinase, an upstream activator of mTOR, leads to a complete block of the insulin-induced degradation. Inhibition of either PI-3 kinase or mTOR prevented the mobility shift in IRS-1 in response to insulin, a shift that is caused by Ser/Thr phosphorylation. These results indicate that insulin stimulates PI 3-kinase-mediated degradation of IRS-1 via both mTOR-dependent and -independent pathways. Platelet-derived growth factor (PDGF) stimulation leads to a lower level of degradation, but significant phosphorylation of IRS-1. Both the degradation and phosphorylation of IRS-1 in response to PDGF are completely inhibited by rapamycin, suggesting that PDGF stimulates IRS-1 degradation principally via the mTOR-dependent pathway. Inhibition of the serine/threonine phosphatase PP2A with okadaic acid also induced the phosphorylation and degradation of IRS-1. IRS-1 phosphorylation and degradation in response to okadaic acid were not inhibited by rapamycin, suggesting that the action of mTOR in the degradation of IRS-1 results from inhibition of PP2A. Consistent with this, treatment of cells with rapamycin stimulated PP2A activity. While the role of mTOR in the phosphorylation of IRS-1 appears to proceed primarily through the regulation of PP2A, we also provide evidence that the regulation of p70S6 kinase phosphorylation requires the direct activity of mTOR.     

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.     

Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta

Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin alpha 2 beta 1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, alpha 2 beta 1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3 beta (GSK3 beta) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the alpha 2 cytoplasmic domain and by a function-blocking anti-alpha 2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin alpha 2 beta 1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the alpha 2 beta 1-induced activation of PP2A.     

Protein phosphatase 2A, a negative regulator of the ERK signaling pathway, is activated by tyrosine phosphorylation of putative HLA class II-associated protein I (PHAPI)/pp32 in response to the antiproliferative lectin, jacalin

Protein phosphatase 2A (PP2A) is a family of mammalian serine/threonine phosphatases that is involved in the control of many cellular functions including those mediated by extracellular signal-regulated kinase (ERK) signaling. While investigating the reversible antiproliferative effect of the dietary lectin, jacalin, which binds the Thomsen-Friedenreich antigen (galactose beta1-3 N-acetylgalactosamine alpha-), we have found that this lectin (30 microg/ml) induces rapid, transient, tyrosine phosphorylation of putative human HLA-DR-associated protein I (PHAPI, also known as the tumor suppressor pp32) in HT29 human colon cancer cells. This is accompanied by the release of PP2A from association with PHAPI, allowing increased phosphatase activity of PP2A (by 42 +/- 10% at 10 min) and consequent complete dephosphorylation of the ERK kinase, MEK1/2, by 10 min and of ERK1/2 by 60 min. PHAPI knockdown by RNA interference abolished the effects of jacalin on PP2A activation and MEK inhibition. Thus phosphorylation of PHAPI/pp32 is a critical regulatory step in PP2A activation and ERK signaling.     

The Bα and Bδ regulatory subunits of PP2A are necessary for assembly of the CaMKIV·PP2A signaling complex

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that is important in synaptic plasticity and T cell maturation. Activation of CaMKIV requires calcium/calmodulin binding and phosphorylation at T200 by CaMK kinase. Our previous work has shown that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV and negatively regulates its activity. Here we demonstrate that PP2A tightly regulates T200 phosphorylation of endogenous CaMKIV, but has little effect on the phosphorylation of the ectopically-expressed kinase. This differential regulation of endogenous versus exogenous CaMKIV is due to differences in their ability to associate with PP2A, as exogenous CaMKIV associates poorly with PP2A in comparison to endogenous CaMKIV. The inability of exogenous CaMKIV to associate with PP2A appears to be due to limiting amounts of endogenous PP2A regulatory B subunits, since coexpression of Bα or Bδ causes the recruitment of PP2Ac to ectopic CaMKIV, leading to formation of a CaMKIV·PP2A complex. Together, these data indicate that the B subunits are essential for the interaction of PP2A with CaMKIV.     

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.     

Glycogen synthase kinase-3β regulates Tyr307 phosphorylation of protein phosphatase-2A via protein tyrosine phosphatase 1B but not Src

GSK-3β (glycogen synthase kinase-3β), a crucial tau kinase, negatively regulates PP2A (protein phosphatase 2A), the most active tau phosphatase that is suppressed in the brain in AD (Alzheimer's disease). However, the molecular mechanism is not understood. In the present study we found that activation of GSK-3β stimulates the inhibitory phosphorylation of PP2A at Tyr307 (pY307-PP2A), whereas inhibition of GSK-3β decreased the level of pY307-PP2A both in vitro and in vivo. GSK-3β is a serine/threonine kinase that can not phosphorylate tyrosine directly, therefore we measured PTP1B (protein tyrosine phosphatase 1B) and Src (a tyrosine kinase) activities. We found that GSK-3β can modulate both PTP1B and Src protein levels, but it only inhibits PTP1B activity, with no effect on Src. Furthermore, only knockdown of PTP1B but not Src by siRNA (small interfering RNA) eliminates the effects of GSK-3β on PP2A. GSK-3β phosphorylates PTP1B at serine residues, and activation of GSK-3β reduces the mRNA level of PTP1B. Additionally, we also observed that GSK-3 negatively regulates the protein and mRNA levels of PP2A, and knockdown of CREB (cAMP-response-element-binding protein) abolishes the increase in PP2A induced by GSK-3 inhibition. The results of the present study suggest that GSK-3β inhibits PP2A by increasing the inhibitory Tyr307 phosphorylation and decreasing the expression of PP2A, and the mechanism involves inhibition of PTP1B and CREB.     

Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis

The reversible phosphorylation of proteins controlled by protein kinases and protein phosphatases is a major mechanism that regulates a wide variety of cellular processes. In contrast to C. elegans, recent studies in mammalian cells have highlighted a major role of serine/threonine protein phosphorylation in apoptosis. To illustrate the importance of dephosphorylation processes in apoptosis, this review will focus on recent studies suggesting that the interaction of the serine/threonine protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with certain regulators of the Bcl-2 family is critically involved in the control of apoptosis.     

Human T-lymphotropic virus type I tax activates I-kappa B kinase by inhibiting I-kappa B kinase-associated serine/threonine protein phosphatase 2A

I-kappa B kinase (IKK) is a serine/threonine kinase that phosphorylates I-kappa B alpha and I-kappa B beta and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, alpha and beta, and a regulatory gamma subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKK gamma and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKK gamma, and PP2A form a stable ternary complex. In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKK gamma, PP2A-interaction-defective Tax mutants failed to activate NF-kappa B. Our data support the notion that IKK gamma-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK x PP2A x Tax ternary complex leads to constitutive IKK and NF-kappa B activation.