Phosphatase Type 2A Research Articles

Deregulation of the phosphatase, PP2A is a common event in breast cancer, predicting sensitivity to FTY720

BackgroundThe most commonly used biomarkers to predict the response of breast cancer patients to therapy are the oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as endocrine treatment in the event of ER and PgR positivity, and the monoclonal antibody, trastuzumab, in the case of HER2-positive patients. Patients who are negative for these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. Deregulation of the protein serine/threonine phosphatase type 2A (PP2A) and its regulatory subunits is a common event in breast cancer, providing a possible target for therapy.MethodsThe data portal, cBioPortal for Cancer Genomics was used to investigate the incidence of conditions that are associated with low phosphatase activity. Four (4) adherent human breast cancer cell lines, MDA-MB-468, MDA-MB-436, Hs578T and BT-20 were cultured to assess their viability when exposed to various dosages of rapamycin or FTY720. In addition, RNA was extracted and cDNA was synthesised to amplify the coding sequence of PPP2CA. Amplification was followed by high-resolution melting to identify variations.Results and conclusionThe sequence of PPP2CA was found to be conserved across a diverse panel of solid tumour and haematological cell lines, suggesting that low expression of PPP2CA and differential binding of inhibitory PPP2CA regulators are the main mechanisms of PP2A deregulation. Interestingly, the cBioPortal for Cancer Genomics shows that PP2A is deregulated in 59.6% of basal breast tumours. Viability assays performed to determine the sensitivity of a panel of breast cancer cell lines to FTY720, a PP2A activator, indicated that cell lines associated with ER loss are sensitive to lower doses of FTY720. The subset of patients with suppressed PP2A activity is potentially eligible for treatment using therapies which target the PI3K/AKT/mTOR pathway, such as phosphatase activators.

Variation in Cyanobacterial Hepatotoxin (Microcystin) Content of Water Samples and Two Species of Fishes Collected from a Shallow Lake in Algeria

Microcystins (MCs) produced from cyanobacteria can accumulate in freshwater fish tissues. In this study, variations in these toxins content were examined monthly in water samples and two species of fish in Lake Oubeira, Algeria, from April 2010 to March 2011. During the study period, MCs were analyzed using protein phosphatase type 2A (PP2A) inhibition assay. In lake water, total (dissolved and intracellular toxins) MC concentrations by PP2A ranged from 0.028 to 13.4μg equivalent MC-LR/l, with a peak in September 2010. MC-LR was the dominant variant (90% of the total) in water samples, followed by MC-YR and MC-(H4)YR. The highest MC concentration in the omnivorous common carp (Cyprinus carpio) was found in the order intestine>hepatopancreas>muscle; however, in the carnivorous European eel (Anguilla anguilla) the order was liver>intestine>muscle. Highest MC concentrations in the intestine tissue of the common carp were found between August and November 2010 where high MC concentrations were detected in water samples, whereas high levels of MCs in the liver of the European eel were found later between January and February 2011. During the entire period of study, the World Health Organization (WHO) lifetime limit for tolerable daily intake was exceeded only in common carp muscle.

Over expression of PPP2R2C inhibits human glioma cells growth through the suppression of mTOR pathway

PPP2R2C encodes a gamma isoform of the subunit B55 subfamily, which is a regulatory subunit of Protein phosphatase type 2A (PP2A). Our study shows that PPP2R2C is downregulated in glioma cells and human brain cancer patient samples. Overexpression of PPP2R2C inhibited cancer cell proliferation both in vitro and in vivo through the suppression of the activity of S6K in the mTOR pathway. Moreover, exogenous expression of PPP2R2C promoted the formation of a complex with the PP2A-C subunit to further enhance the binding of PP2A-C with S6K. Our results suggest that PPP2R2C is a potential tumor suppressor gene in human brain cancers. This study will provide novel insight into the development of therapeutic strategies in the treatment of human brain tumors.

The secret life of CFTR as a calcium-activated chloride channel.

cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca(2+)-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca(2+)-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations.

A Novel Screening Method to Establish Tumor-targeting Antibodies Reliable for Drug Delivery System

Establishment of a system that allows selective drug delivery and gene silencing to a tumor is expected to enable targeted therapy. We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33 (Adv-FZ33). By cross-linking the Adv-FZ33 virus and the surface antigen molecules with the targeting monoclonal antibodies (mAbs), we attained highly enhanced gene deliveries into the respective antigen-positive cancer cells. Therefore, we aimed to establish a systematic screening method to search for antibody and cell surface target candidates that would provide highly selective anti-cancer reagents to malignant tumors. Using an Adv-FZ33, hybridoma libraries producing a variety of mAbs for human pancreatic, prostate, lung or ovarian carcinoma cells were screened, and we were able to selectively obtain several mAbs which had potent high affinity and recognized antigens of high structure. Within these mAbs, we have identified tumor cell target molecules including not only carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), prostate specific membrane antigen (PSMA) but also novel tumor surface target molecules such as phosphatidic acid phosphatase type 2a (PAP2a) and interleukin-13 receptor variant α2 (IL-13Rα2) as tumor antigens. Overall, these results indicate that this type of inductive method approach is a reliable strategy for screening in antibody therapy on par with antibody-dependent drug-delivery system.

GW24-e2244 Ginsenosides-Rbl improves adriamycin-induced heart failure by adjusting connexin 43

ObjectivesOn the basis of adriamycin (Adr)-induced heart failure (HF), the present study elucidated whether the effect of Ginsenosides-Rbl (Gs-Rb1), improving cardiac function, was performed by connexin 43 (CX43) and other...

Notch1 Receptor Regulates AKT Protein Activation Loop (Thr308) Dephosphorylation through Modulation of the PP2A Phosphatase in Phosphatase and Tensin Homolog (PTEN)-null T-cell Acute Lymphoblastic Leukemia Cells

Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

Effects of hibernation on regulation of mammalian protein phosphatase type-2-A

Hibernation is used by many small mammals for winter survival. Animals undergo cycles of deep torpor characterized by strong reductions in metabolic rate, body temperature, and all physiological activities. One prominent biochemical mechanism involved in regulating coordinated transitions to and from the hypometabolic state is reversible protein phosphorylation, a dynamic covalent modification maintained by the actions of protein kinases and protein phosphatases. The present study characterizes protein phosphatase type 2A (PP2A) from liver and skeletal muscle of Richardson’s ground squirrels, Urocitellus richardsonii, to determine if the properties of this enzyme are modified between euthermic and hibernating states to provide differential control of the enzyme or its target proteins. Kinetic analysis showed a significant 1.5-fold increase in the Km for the synthetic phosphorylated peptide substrate (RRApTVA) for PP2A from skeletal muscle (but not liver) of torpid ground squirrels, as compared with euthermic controls, when measured at both 25°C and 5°C. This correlated with enhanced phosphorylation of PP2Aα Tyr-307 (a 2.3-fold increase) during hibernation in skeletal muscle (but not liver), a modification known to inhibit PP2A action. Hence, both kinetic and phosphorylation data suggest a suppression of PP2A function in the torpid state in skeletal muscle. However, total PP2A protein levels (assessed by immunoblotting) and maximal activities did not change in either tissue between euthermic and hibernating states. Liver PP2A from hibernating animals showed a broader pH profile with high activity retained at lower pH values (6.0–6.5) than the euthermic enzyme which might aid enzyme function in vivo under conditions of respiratory acidosis during torpor.

Protein phosphatase 2A regulatory subunits perform distinct functional roles in the maize pathogen Fusarium verticillioides

Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is still incomplete. We have demonstrated previously that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. In general, three PP2A subunits, structural A, regulatory B and catalytic C, make up a heterotrimer complex to perform regulatory functions. Significantly, we identified two PP2A regulatory subunits in the F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. In this study, we hypothesized that Ppr1 and Ppr2 are involved in the regulation of fumonisin biosynthesis and/or cell development in F. verticillioides, and generated a series of mutants to determine the functional role of Ppr1 and Ppr2. The PPR1 deletion strain (Δppr1) resulted in drastic growth defects, but increased microconidia production. The PPR2 deletion mutant strain (Δppr2) showed elevated fumonisin production, similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cells with a central septation site, whereas Δppr2 showed early hyphal branching during conidia germination. A kernel rot assay showed that the mutants were slow to colonize kernels, but this is probably a result of growth defects rather than a virulence defect. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out distinct roles in the regulation of fumonisin biosynthesis and fungal development.

PP2A mediates lateral root development under NaCl-induced osmotic stress throughout auxin redistribution in Arabidopsis thaliana

Auxin plays an important role in modulating root system architecture. The effect of salinity on root development has been extensively studied; however, evidence on how salinity affects lateral root development and its underlying molecular mechanism is scarce. Here, we analyzed the role of protein phosphatase PP2A activity in auxin redistribution during Arabidopsis root system adaptation under NaCl-induced osmotic stress. Arabidopsis Col-0 and DR5::UidA seedlings were grown in MS media containing NaCl alone or in combination with the auxin transport inhibitor naphthylphthalamic acid, the synthetic auxin α-Naphthaleneacetic acid or the phosphatase inhibitor Okadaic acid. After 8 days, primary root length and lateral root number in seedlings were quantified and the auxin distribution was analyzed. Promotion of primary root shortening and lateral root development induced by osmotic stress correlated with an increase in active auxin content and a >50 % reduction in protein phosphatase type 2A (PP2A) activity. Moreover, the observed effects on seedlings under osmotic stress are more pronounced with the PP2A inhibitor Okadaic acid. Our data suggest PP2A is a positive regulator of osmotic stress-induced root system architecture modulation, involving auxin redistribution in Arabidopsis thaliana.

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.

Comparative analysis of the corps en cerise in several species of Laurencia (Ceramiales, Rhodophyta) from the Atlantic Ocean

Different species of Laurencia have proven to be a rich source of natural products yielding interesting bioactive halogenated secondary metabolites, such as terpenoids and acetogenins. It is shown that such compounds are accumulated in the spherical, reniform to claviform refractive inclusions called corps en cerise (CC), which are intensively osmiophilic and located mainly in the cortical cells of the thalli and also in trichoblast cells. Up to now, it was believed that CC were present only in these two kinds of cells. Recently, however, a species of Laurencia, L. marilzae, with CC in all cells of the thallus, i.e., cortical, medullary, including the pericentral and axial cells, as well as in the trichoblasts, was described from the Canary Islands, and subsequently also reported to Brazil and Mexico. Within the Laurencia complex, only Laurencia species produce CC. Since the species of Laurencia are targets of interest for the prospection of bioactive substances due to their potential antibacterial, antifungal, anticholinesterasic, antileishmanial, cytotoxic, and antioxidant activities, the present paper carries out a comparative analysis of the corps en cerise in several species of Laurencia from the Atlantic Ocean to obtain basic information that can support natural product bioprospection projects. Our results show that the number and size of the CC are constant within a species, independent of the geographical distribution, corroborating their use for taxonomical purposes to differentiate groups of species that present a lower number from those that have a higher number. In this regard, there was a tendency for the number of CC to be higher in some species of Laurencia from the Canary Islands. The presence of CC can also be used to distinguish species in which these organelles are present in all cells of the thallus from those in which CC are restricted to the cortical cells. Among the species analyzed, L. viridis displayed the most varied secondary metabolites composition, such as sesquiterpenes, diterpenes, triterpenes, all of which showed potent antiviral, cytotoxic, and antitumoral activities, including protein phosphatase type 2A (PP2A) inhibitory effects.

Inhibition of MKK7–JNK by the TOR Signaling Pathway Regulator-Like Protein Contributes to Resistance of HCC Cells to TRAIL-Induced Apoptosis

The TOR signaling pathway regulator-like (TIPRL) protein, the mammalian ortholog of yeast TIP41, was identified in an expression profiling screen for factors that regulate human liver carcinogenesis. We investigated the role of human TIPRL protein in hepatocellular carcinoma (HCC). We measured the level of TIPRL in HCC and adjacent nontumor tissues from patients. We used small interfering RNAs and zebrafish to study the function of TIPRL. We used annexin V propidium iodide staining and immunoblot analyses to measure apoptosis and activation of apoptotic signaling pathways. We used confocal microscopy, coimmunoprecipitation, and glutathione-S transferase pull-down analyses to determine interactions among mitogen-activated protein kinase kinase 7 (MKK7 or MAP2K7), TIPRL, and the protein phosphatase type 2A (PP2Ac). We studied the effects of TIPRL in tumor xenografts in mice. Levels of TIPRL were higher in HCC tissues and cell lines than nontumor tissues and primary hepatocytes. Knockdown of tiprl expression in zebrafish led to large amounts of apoptosis throughout the embryos. Incubation of HCC cells, but not primary human hepatocytes, with small interfering RNA against TIPRL (siTIPRL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) caused prolonged activation (phosphorylation) of MKK7 and c-Jun N-terminal kinase (JNK) and led to apoptosis, indicated by cleavage of procaspase-8,-3 and of poly-(adenosine diphosphate-ribose) polymerase. TIPRL bound to MKK7 and PP2Ac and promoted the interaction between MKK7 and PP2Ac. In mice, injection of HCC xenograft tumors with siTIPRL and TRAIL led to tumor apoptosis and regression. TIPRL is highly up-regulated in human HCC samples and cell lines, compared with noncancerous liver tissues. TIPRL prevents prolonged activation of MKK7 and JNK and TRAIL-induced apoptosis by mediating the interaction between MKK7 and PP2Ac.

Analysis of differential gene expression profile in peripheral blood of patients with chronic hepatitis B and syndromes of dual deficiency of liver and kidney yin and accumulation of dampness heat

To investigate the differential gene expression profile in two typical traditional Chinese medicine (TCM) syndromes of patients with chronic hepatitis B (CHB), and to find the relationship between TCM syndromes and gene expressions. Patients with CHB were collected from Department of Liver Diseases of Longhua Hospital in Shanghai and diagnosed as dual deficiency of liver and kidney yin syndrome (n=3) or accumulation of dampness heat syndrome (n=3). Three healthy volunteers were used as a control. Blood samples were collected before treatment. After total RNA was isolated from leukocytes, the gene expression profiles were detected by microarray. For confirming the gene expressions, a further 10 patients with dual deficiency of liver and kidney yin syndrome or accumulation of dampness heat syndrome and 10 healthy volunteers were enrolled. Their peripheral blood was used to test the gene expression by real-time reverse transcription-polymerase chain reaction (RT-PCR) which was differently expressed by microarray. Microarray analysis identified that there were significant differences in gene expression between patients with dual deficiency of liver and kidney yin syndrome and patients with accumulation of dampness heat syndrome, as well as between CHB patients and healthy volunteers. Patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome were fully distinguished by clustering of 403 differentially expressed genes (P<0.05). A total of 239 genes were significantly differentially expressed (absolute value of foldchange≥2, P<0.05), among which 142 were up-regulated and 97 were down-regulated. Genes specially regulated by the dual deficiency of liver and kidney yin syndrome were mainly related to peroxide activity and stem cell maintenance; genes specially regulated by the accumulation of dampness heat syndrome were mainly related to cytokines, immune and inflammatory responses. ATP-binding cassette, sub-family C (CFTR/MRP), member 3, peroxisome proliferator-activated receptor α and phosphatidic acid phosphatase type 2A mRNA expressions detected by RT-PCR were similar to the results of microarray. Differential gene expressions are observed in CHB patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome, indicating that there is molecular foundation in the classification of TCM syndrome. The preliminary findings of this study suggest that TCM syndrome types are related to gene expression.

Regulation of Protein Kinase C Inactivation by Fas-associated Protein with Death Domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.

The relationship between the MMP system, adrenoceptors and phosphoprotein phosphatases

The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs)] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although α- and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system.

The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity

Protein phosphatase type 2A (PP2A) enzymes constitute a large family of Ser/Thr phosphatases with multiple functions in cellular signaling and physiology. The composition of heterotrimeric PP2A holoenzymes, resulting from the combinatorial assembly of a catalytic C subunit, a structural A subunit, and regulatory B-type subunit, provides the essential determinants for substrate specificity, subcellular targeting, and fine-tuning of phosphatase activity, largely explaining why PP2A is functionally involved in so many diverse physiological processes, sometimes in seemingly opposing ways. In this review, we highlight how PP2A holoenzyme biogenesis and enzymatic activity are controlled by a sophisticatedly coordinated network of five PP2A modulators, consisting of α4, phosphatase 2A phosphatase activator (PTPA), leucine carboxyl methyl transferase 1 (LCMT1), PP2A methyl esterase 1 (PME-1) and, potentially, target of rapamycin signaling pathway regulator-like 1 (TIPRL1), which serve to prevent promiscuous phosphatase activity until the holoenzyme is completely assembled. Likewise, these modulators may come into play when PP2A holoenzymes are disassembled following particular cellular stresses. Malfunctioning of these cellular control mechanisms contributes to human disease. The potential therapeutic benefits or pitfalls of interfering with these regulatory mechanisms will be briefly discussed.

Characterization of ypa1 and ypa2, the Schizosaccharomyces pombe Orthologs of the Peptidyl Proyl Isomerases That Activate PP2A, Reveals a Role for Ypa2p in the Regulation of Cytokinesis

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis. Cdc7p is the first kinase in the core SIN; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologs of mammalian phosphotyrosyl phosphatase activator. We have characterized this gene and its ortholog SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of protein phosphatase type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major protein phosphatase type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.

Regulation of Fission Yeast Morphogenesis by PP2A Activator pta2

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity.