PP2A and differentiation

A regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling (Sieburth, 1999).

KSR proteins positively regulate Ras signaling in C. elegans and Drosophila, as well as in Xenopus oocytes and certain mammalian cells. Murine KSR associates with several proteins in vivo, including Raf, MEK, and MAP kinase, and has been proposed to function as a scaffold protein involved in signal propagation through the Raf/MEK/MAP kinase cascade. Murine KSR is a phosphoprotein; although the role of phosphorylation in KSR regulation is unclear. Thus, KSR-1 is a potential target for regulation by PP2A during vulval induction. Alternatively, KSR-1 may act to regulate PP2A-B function. Another potential sur-6 PP2A-B-dependent PP2A target is LIN-45 Raf. The mechanism of Raf activation is still poorly understood, but there is evidence for both inhibitory and activating phosphates on Raf. Whereas in vitro studies suggest that PP2A can dephosphorylate Raf, it is probably not the major phosphatase to remove activating phosphates. However, a role for PP2A in removing inhibitory phosphates has not been ruled out. The placement of a B regulatory subunit of PP2A as a positive regulator of the Ras pathway, and the unexpected finding that it acts together with KSR-1, should lead to a better understanding of PP2A regulation and its physiological substrates (Sieburth, 1999).

Neurofilament-L (NF-L) mRNA and protein levels in mouse P19 embryonal carcinoma cells are enhanced by retinoic acid-induced differentiation. Okadaic acid (OA) treatment represses this differentiation-dependent expression and neurite outgrowth. OA treatment does not affect NF-L gene transcription level, but it does reduce the stability of NF-L mRNAs. The expression and activity of PP2A and PP2B increases in accordance with the enhanced NF-L gene expression. The presence of OA during the course of neural differentiation reduces PP2A activity. These results demonstrate that the OA treatment inhibits the differentiation-dependent increase in NF-L gene expression by destabilizing its mRNAs and suggest that PP2A plays key roles in the differentiation-dependent enhanced expression of the NF-L gene (Sasahara, 1996).

Three families of cellular B subunits have been identified to date: B55, B56 and PR72/130. Five genes encode human B56 isoforms. Most code for phosphoproteins. There are distinct patterns of intracellular targeting by different B56 isoforms. Specifically, B56alpha, B56beta and B56epsilon complexed with A and C subunits localize to the cytoplasm, whereas B56delta, B56gamma1 and B56gamma3 are concentrated in the nucleus. Two isoforms are expressed in adult brain. mRNA of the brain isoforms increases when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment (McCright, 1996).

Angiotensin receptors elicit a stimulation of PP2A. Ang II elicits significant increases in MAP kinase (ERK) activity in cultured neurons, mediated via Ang II type 1 (AT1) receptors. This stimulatory effect of Ang II on MAPK activity is potentiated by blockade of AT2 receptors. An AT2 receptor agonist causes a significant decrease in neural MAPK activity, which is abolished by the PP2A inhibitor okadaic acid. This indicates that AT1 and AT2 receptors have opposite actions an MAPK activity in neonatal neurons. Since MAP kinases are involved in the regulation of growth/differentiation and apoptosis, these data may provide an intracellular basis for the modulatory effects of Ang II receptors on these processes (Huang, 1996).

The BR beta regulatory subunit of rat PP2A is specifically expressed in rat brain and testis, the lengths of mRNAs in these two organs being different. In the testis, the BR beta mRNA is first detected 40 days after birth, increasing gradually thereafter, and is expressed specifically in elongated spermatids, while mRNA of the alpha-isotype (BR alpha) is expressed equally in all spermatogenic cells. After meiosis, round spermatids change morphologically to elongated spermatids. BR beta may regulate the activity of the PP2A catalytic subunit in spermatids, and be involved in spermatogenic maturation, especially spermatid elongation (Hatano, 1993).

Hox11 codes for a homeobox protein that controls genesis of the spleen. Hox 11 is also oncogenic, having been isolated from a chromosomal breakpoint in human T-cell leukemia. Transgenic mice that redirect Hox11 expression to the thymus demonstrate cell-cycle aberrations and progression to malignancy. In order to understand the cell cycle disruptions caused by HOX11 protein, the protein was tested for interaction partners. HOX11 directly interacts with the PP2A catalytic subunit and the related PP1C. The physical interaction domain of HOX11 is outside the homeodomain. PP2A can regulate the cell cycle of Xenopus oocytes by maintaining G2 meiotic arrest preventing activation of maturation-promoting factor. Microinjection of HOX11 into Xenopus oocytes arrested at the G2 phase of the cell cycle promotes progression to M phase. The interaction of HOX11 with PP2a suggests a mechanism by which a homeobox protein can alter the cell cycle (Kawabe, 1997).

Protein phosphatase type-2A (PP2A) is a highly conserved serine/threonine phosphatase known to play a key role in cell proliferation and differentiation in vitro, but the role of PP2A in mammalian embryogenesis remains unexplored. No particular information exists as to the tissue or cell specific expression of PP2A or the relevance of PP2A expression to mammalian development in vivo. To examine expression of PP2A during mammalian lung development, fetal rats were studied from day 14 of gestation (the lung bud is formed on day 12 of gestation) to parturition. Western analysis with a specific PP2A catalytic subunit antibody identifies a single 36 kDa protein, with protein levels two-fold higher in the 17 and 19 day embryonic lung, as compared to the adult. Both mRNA and protein for PP2A are localized equally to the epithelial lining of the embryonic lung airway and the surrounding mesenchyme in the 14 day embryonic lung. With maturation of the lung, PP2A becomes highly expressed in respiratory epithelium. The highest level of expression is in the earliest developing airways with columnar epithelium (the pseudoglandular stage, 15-18 days of gestation). There is a decrease in expression with the transformation to cuboidal epithelium by day 20 of gestation. This is most noticeable in the developing bronchial epithelium of the 19 and 20 day gestation lungs, where only an occasional cell continues to express PP2A. Mesenchymal hybridization is most obvious in early endothelial cells of forming vascular channels at 17-19 days of gestation. PP2A respiratory epithelial expression mimics the centrifugal development of the respiratory tree where the highest expression is in the peripheral columnar epithelium (15-18 days gestation) with only an occasional central bronchiolar cell continuing to express PP2A at 19 and 20 days gestation. Endothelial hybridization decreases with muscularization of large pulmonary arteries with low levels of expression detected in bronchial and vascular smooth muscle. In the newborn lung PP2A expression is decreased, but detectable in alveolar epithelium and vascular endothelium. In summary: (1) PP2A mRNA and protein exhibit cell specific expression during rat lung development; (2) PP2A is highly expressed in the respiratory epithelium of the fetal rat lung and is temporally related to the maturation of the bronchial epithelium, and (3) the PP2A subunit is highly expressed in early vascular endothelium, but not smooth muscle of the rat lung (Xue, 1998).

PP2A and signal transduction

A Sonic hedgehog (see Drosophila Hedgehog) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter (see Drosophila Seven-up). The Shh response element binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway. The phosphatase that mediates this dephosphorylation in response to Shh-N treatment is PP2A or is like PP2A. This particular response is channeled through a protein with DNA binding activity apparently unrelated to that of the Ci/Gli family. A similar protein phosphatase activity is also required in the Ci/Gli-mediate branch of the Drosophila Hh signaling pathway (Krishnan, unpublished result). Thus, activation of specific protein phosphatase activity appears to be a general feature of Hh signal transduction (Krishnan, 1997).

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) binds to PP2A in vitro and in mitogen-starved cells; binding requires the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha results in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibits the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway (Heriche, 1997).

Inhibition of protein phosphatase 2A (PP2A) by the expression of SV40 small t stimulates the mitogenic MAP kinase cascade. SV40 small t can substitute for either tumor necrosis factor-alpha (TNF-alpha) or serum, and stimulate atypical protein kinase C zeta (PKC zeta) activity, resulting in MEK activation, cell proliferation and NF-kappaB-dependent gene transcriptional activation in CV-1 and NIH 3T3 cells. These effects are abrogated by co-expression of kinase-deficient PKC zeta and inhibition of phosphatidylinositol 3-kinase p85alpha-p110 by wortmannin, LY294002 and a dominant-negative mutant of p85alpha. In contrast, expression of kinase-inactive ERK2 inhibits small t-dependent cell growth but is unable to abolish small t-induced NF-kappaB transactivation. These results provide the first in vivo evidence for a critical regulatory role of PP2A in bifunctional PKC zeta signaling pathways controlled by phosphatidylinositol 3-kinase. Constitutive activation of PKC zeta and NF-kappaB 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-alpha, these findings could explain why PP2A is a primary target for the alteration of cellular behavior, as utilized during SV40 infection (Sontag, 1997).

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacts with APC in the yeast two-hybrid system. Expression of B56 reduces the abundance of beta-catenin and inhibits transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin is blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling. Loss of PP2A function may provide an additional route to activation of Wnt signaling and oncogenesis. Consistent with this, mutations in the gene encoding the beta isoform of the PP2A subunit have been identified in colon and lung cancers (Seeling, 1999).

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. The results are reported of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. These results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. A region of Axin was identified that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, it was demonstrated that the C-terminal 100 amino acids of Axin can bind to this same region (Hsu, 1999).

Wnt signaling increases ß-catenin abundance and transcription of Wnt-responsive genes. The B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, ß-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. The B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 is epistatically positioned downstream of GSK3ß and axin but upstream of ß-catenin, and axin co-immunoprecipitates B56 A and C subunits suggesting that PP2A:B56 is in the ß-catenin degradation complex. PP2A appears to be essential for ß-catenin degradation, since ß-catenin degradation is reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis (Li, 2001).

The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70(s6k) (the 70-kDa S6 kinase: see Drosophila RPS6-p70-protein kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70(s6k) in response to mitogenic stimuli. FRAP controls 4E-BP1 and p70(s6k) phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70(s6k), and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70(s6k) but not with a mutated p70(s6k) that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70(s6k), whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase (Peterson, 1999).

Protein phosphatase 2A (PP2A) plays central roles in development, cell growth and transformation. Inactivation of the murine gene encoding the PP2A catalytic subunit Calpha by gene targeting generates a lethal embryonic phenotype. No mesoderm is formed in Calpha minus embryos. During normal early embryonic development Calpha is predominantly present at the plasma membrane whereas the highly homologous isoform Cbeta is localized to the cytoplasm and nuclei, suggesting the inability of Cbeta to compensate for vital functions of Calpha in Calpha minusembryos. In addition, PP2A is found in a complex containing the PP2A substrates E-cadherin and beta-catenin. In Calpha minus embryos, E-cadherin and beta-catenin are redistributed from the plasma membrane to the cytosol. Cytosolic concentrations of beta-catenin are low. These results suggest that Calpha is required for stabilization of E-cadherin/ beta-catenin complexes at the plasma membrane (Gotz, 2000).

These data suggest that PP2A is involved in wnt signaling. One likely explanation is that PP2A containing the Calpha subunit binds to and stabilizes the E-cadherin/beta-catenin complex within the plasma membrane. PP2A forms a complex with beta-catenin and E-cadherin. It is likely that PP2A regulates the phosphorylation status of E-cadherin and beta-catenin at the plasma membrane. In the cytosol, the activity of PP2A may not be high enough to offset GSK3 beta-mediated phosphorylation of beta-catenin. Wnt signaling increases cytoplasmic concentrations of PP2A by releasing it from the plasma membrane into the cytosol. Increased concentrations of PP2A effectively offset the GSK3 beta-mediated phosphorylation of beta-catenin thus preventing its degradation, and promoting its translocation to the nucleus with subsequent activation of wnt target genes. In the Calpha knockout situation both E-cadherin and beta-catenin are no longer stabilized in the plasma membrane. It is likely that both are phosphorylated and translocated to the cytoplasm. These data show that cytoplasmic E-cadherin is stable, and suggest that the associated beta-catenin is targeted for degradation resulting in reduced beta-catenin levels. It is proposed that in the absence of PP2A Calpha, wnt signaling releases beta-catenin from the axin/APC/GSK3 beta complex; beta-catenin associates with the highly abundant cytoplasmic E-cadherin before it reaches the nucleus, where it is phosphorylated and becomes degraded. Consequently, no target genes of wnt signaling, including Brachyury or goosecoid, are transcribed and embryonic development halts (Gotz, 2000).

This model is consistent with the finding that Calpha is mainly plasma membrane-associated in the inner cell mass, but this association becomes lost during differentiation. This model would also explain why the wnt target gene T-brachyury is negatively regulated by E-cadherin: T-brachyury mRNA levels are high in E-cad minus embryonic stem cells (ES) cells, but absent in wild-type ES cells. Basal cytoplasmic levels of non-phosphorylated beta-catenin may be quenched by cytoplasmic or membrane-bound E-cadherin, preventing transcription of T-brachyury in the absence of a wnt signal. In E-cad minus cells, the quencher is missing, allowing low but significant amounts of non-phosphorylated beta-catenin to be translocated into the nucleus, and to activate transcription of T-brachyury. This is consistent with the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, it has been found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1), which targets beta-catenin to the nucleus, form mutually exclusive complexes with beta-catenin. This model is also consistent with the known association of PP2A with axin. Axin is a negative regulator of embryonic axis formation in vertebrates (Gotz, 2000).

The Wnt/ß-catenin pathway plays important roles during embryonic development and growth control. The B56 regulatory subunit of protein phosphatase 2A (PP2A) has been implicated as a regulator of this pathway. Loss-of-function analysis of PP2A:B56epsilon during early Xenopus embryogenesis has been examined. PP2A:B56epsilon is required for Wnt/ß-catenin signaling upstream of Dishevelled and downstream of the Wnt ligand. Maternal PP2A:B56epsilon function is required for dorsal development, and PP2A:B56epsilon function is required later for the expression of the Wnt target gene engrailed, for subsequent midbrain-hindbrain boundary formation, and for closure of the neural tube. These data demonstrate a positive role for PP2A:B56epsilon in the Wnt pathway (Yang, 2003).

Loss-of-function analysis in Drosophila and zebrafish suggests that PP2A:B56 family members play roles in the PCP pathway upstream of dsh. Although Frizzled and Dsh function in both the PCP pathway and the canonical Wnt/ß-catenin pathway, the requirement for PP2A:B56epsilon upstream of Dsh in Wnt/ß-catenin signaling has not been shown previously. The loss-of-function analysis of PP2A:B56epsilon presented here directly supports the hypothesis that PP2A:B56epsilon is required for Wnt/ß-catenin signaling. PP2A:B56epsilon is required for the expression of multiple Wnt/ß-catenin target genes, including Xnr5, Xnr6 during pre-MBT stages; Xnr3, siamois, noggin, goosecoid, cerberus and Xdkk1, during the gastrula stage, and en2 during neurulation. Furthermore, PP2A:B56epsilon is required for ß-catenin accumulation in cells that respond to endogenous Wnt signaling, such as dorsal blastomeres in blastula-stage embryos and in the neural ectoderm of early neurulae. In contrast, the constitutive turnover of ß-catenin in ventral blastomeres and in oocytes is not sensitive to PP2A:B56epsilon depletion. The requirement for PP2A:B56epsilon in canonical Wnt signaling is further supported by epistatic analysis. Dorsal gene expression is rescued by dsh and downstream Wnt signaling components but not by Wnt8b. Based on these observations, it is concluded that PP2A:B56epsilon is required for Wnt/ß-catenin signaling downstream of the Wnt ligand and upstream of dsh (Yang, 2003).

In contrast to observations with depletion of PP2A:B56epsilon, overexpression of PP2A:B56 family members inhibits Wnt/ß-catenin signaling. Other B56 family members, such as B56alpha, may have different functions in the Wnt/ß-catenin pathway. For example, overexpression of PP2A:B56epsilon in dorsal blastomeres leads to anterior truncation of embryos without altering dorsal gene expression, a phenotype that is distinct from the strong ventralization caused by PP2A:B56alpha overexpression. These observations are consistent with the suggestion that the two isoforms have different functions in the Wnt pathway. However, overexpressed PP2A:B56epsilon also inhibits secondary dorsal axis induction by positive modulators of Wnt/ß-catenin pathway. To explain this, PP2A:B56epsilon could regulate multiple steps in Wnt signaling, and loss-of-function at an upstream step may obscure an inhibitory role at a downstream step in the pathway. Alternatively, overexpression of PP2A:B56 or other subunits that interact with the PP2A complex could sequester essential components of the phosphatase complex and interfere with its normal function. It also remains unclear whether PP2A:B56epsilon regulates Wnt signaling through modulation of PP2A activity. Further experiments are needed in order to understand the mechanism by which PP2A:B56epsilon regulates the Wnt/ß-catenin pathway (Yang, 2003).

Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. Using mass spectrometry to analyze the KSR1-scaffolding complex, the serine/threonine protein phosphatase PP2A has been identified as a KSR1-associated protein; PP2A is a critical regulator of KSR1 activity. The enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites (Ory, 2003).

PP2A, Receptors, and Memory

Calcineurin is a calcium-sensitive serine/threonine phosphatase that is present at high levels in the hippocampus and enriched at synapses. Once activated, calcineurin can act both directly and indirectly on protein substrates, including CREB. (1) It can dephosphorylate target proteins directly and thereby regulate specific cellular functions. (2) It can modulate an even larger variety of substrates indirectly by its ability to dephosphorylate inhibitor 1 (I-1). I-1, when phosphorylated, inhibits the function of protein phosphatase 1 (PP1). Dephosphorylation of I-1 by calcineurin activates PP1 and leads to the dephosphorylation of a large and independent set of target proteins. One interesting feature of the regulatory actions of calcineurin comes from its interactions with the cAMP cascade. Calcineurin inhibits the action of I-1 by dephosphorylating the site on I-1 phosphorylated by the cAMP-dependent kinase, PKA (see Drosophila PKA). Indeed, calcineurin and PKA antagonistically regulate the function of several proteins, including NMDA and GluR6 glutamate receptors (Winder, 1998 and references).

The interactions of Protein kinase and calcineurin are of particular interest in the context of LTP. Based on the requirement for macromolecular synthesis, LTP can be divided into at least two components: an early component (E-LTP) and a late component (L-LTP). Delivery to the Schaffer collateral-CA1 pyramidal cell (SC-CA1) synapse of a single 100 Hz train lasting 1s elicits E-LTP, a relatively short-lived and weak enhancement of synaptic transmission that does not require protein and RNA synthesis and is not dependent on PKA. By contrast, administration of three or four trains of 100 Hz elicits L-LTP, a more robust and stable form of LTP lasting many hours that is dependent on the activation of PKA as well as the synthesis of both RNA and protein. Recent experiments with inhibitors of phosphatases suggest that one role of PKA in LTP in area CA1 may be to suppress the actions of PP1 or PP2A. In particular, when LTP in area CA1 is induced by strong stimuli it can be blocked by inhibitors of PKA. However, this effect of PKA inhibitors is removed by preincubation of slices with PP1/PP2A inhibitors. This has led to the suggestion that under certain circumstances, PKA may "gate" LTP by suppressing a phosphatase cascade (Winder, 1998 and references).

To investigate the role of phosphatases in synaptic plasticity using genetic approaches, transgenic mice were generated that overexpress a truncated form of calcineurin under the control of the CaMKIIalpha promoter. Mice expressing this transgene show increased calcium-dependent phosphatase activity in the hippocampus. Physiological studies of these mice and parallel pharmacological experiments in wild-type mice reveal a novel, intermediate phase of LTP (I-LTP) in the CA1 region of the hippocampus. This intermediate phase differs from E-LTP by requiring multiple trains for induction and by being dependent on PKA. It differs from L-LTP by not requiring new protein synthesis. These data suggest that calcineurin acts as an inhibitory constraint on I-LTP, one which is relieved by PKA. This inhibitory constraint acts as a gate to regulate the synaptic induction of L-LTP (Winder, 1998 ).

Since phosphatases impose an inhibitory constraint on LTP, these results suggest that PKA is required to suppress phosphatase activity sufficiently to elicit LTP fully. Activation of NMDA receptors increases cAMP levels and PKA activity in CA1 through a calmodulin-dependent process. Therefore, while calcium directly regulates the balance of kinase and phosphatase activity, the generation of cAMP by NMDA-receptor-dependent activation of calcium-sensitive adenylyl cyclases can favor kinases further by inducing a PKA-dependent inactivation of the activation of PP1 by calcineurin, through phosphorylation of I-1. It should be noted, however, that although physiological studies suggest the presence of I-1 or an I-1-like protein in CA1, the histological localization of I-1 in CA1 is somewhat controversial. Because I-1 is a member of a family of proteins that modulate phosphatase function, it is possible that another protein from this family mediates the effects reported here (Winder, 1998 and references).

Hippocampal-dependent memory in mice that express a truncated form of calcineurin was assessed. Mutant mice have normal short-term memory but exhibit a profound and specific defect in long-term memory on both the spatial version of the Barnes maze and on a task requiring the visual recognition of a novel object. To determine whether mutant mice have the capacity for long-term memory, the training protocol was intensified on the spatial version of the Barnes maze by increasing the number of daily training trials. The memory defect is fully reversed, indicating that these mice are capable of forming long-term memory. This rescue experiment suggests that mice overexpressing calcineurin have impaired long-term memory possibly due to a specific defect in the transition between short-term and long-term memory. The memory defect observed was not the result of a developmental abnormality due to the genetic manipulation. In mice in which the expression of the calcineurin transgene is regulated by the tetracycline-controlled transactivator (tTA) system, the spatial memory defect is reversed when the expression of the transgene is repressed by doxycycline. Thus calcineurin has a role in the transition from short- to long-term memory, which correlates with a novel intermediate phase of LTP (Mansuy, 1998).

Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked. The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits. It is hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS. A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait. The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain. Immunoprecipitation studies indicate that the NR3A subunit forms a stable complex with PP2A in the rat brain in vivo. Association of PP2A with NMDARs lead to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1. Stimulation of NMDARs leads to the dissociation of PP2A from the complex and the reduction of PP2A activity. A peptide corresponding to the PP2A-NR3A binding domain functions as a negative regulator of PP2A activity. These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state. The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism. It is possible to speculate that protein kinase A, PP2A, and NMDARs are part of a signaling mechanism linking the history of NMDAR stimulation to the phosphorylation of state of serine 897 of NR1. Along these lines, the finding that NR3A knock-out mice show an increased density of spines in cortical neurons might indicate that such a signaling pathway could be involved in the regulation of the growth of dendritic spines. In fact, NMDAR activity has been implicated in the modulation of dendritic arbors and spines, whereas PP2A has been shown to influence the modification of synaptic structures (Chan, 2001).

PP2A and Apoptosis

The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types, regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, a single cell approach was used to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to microtubule-stabilizing protein known as tau, it is shown that tau becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-li10ke phosphatase is responsible. Transfecting tau into CHO cells to act as a 'reporter' protein shows a similar dephosphorylation of tau by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis; this is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit (tau) dephosphorylation suggest that microtubule disassembly is necessary for (tau) dephosphorylation to occur. Thus, it is propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly, which frees or activates PP2A to dephosphorylate tau as well as other substrates (Mills, 1998).

PP2A and malignancy

Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, a cellular inhibitor of PP2A with oncogenic activity is described. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, these data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies (Junttila, 2007).

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