Role Of Protein Phosphatases Research Articles

Role of protein phosphatases in the run down of guinea pig cardiac Cav1.2 Ca2+ channels.

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca(2+) channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca(2+) channel currents with the patch-clamp technique. Calmodulin (CaM) and ATP were used to restore channel activity in inside-out patches. Inhibitors of protein phosphatases were applied to investigate the role of phosphatases. The specific protein phosphatase type 1 (PP1) inhibitor (PP1 inhibitor-2) and protein phosphatase type 2A (PP2A) inhibitor (fostriecin) abolished the slow run down of Cav1.2 Ca(2+) channels, which was evident as the time-dependent attenuation of the reversing effect of CaM/ATP on the run down. However, protein phosphatase type 2B (PP2B, calcineurin) inhibitor cyclosporine A together with cyclophilin A had no effect on the channel run down. Furthermore, PP1 inhibitor-2 mainly prolonged the open time constants of the channel, specifically, the slow open time. Fostriecin primarily shortened the slow close time constants. Our data suggest that PP1 and PP2A were involved in the slow phase of Cav1.2 Ca(2+) channel run down. In addition, they exerted different effects on the open-close kinetics of the channel. All above support the view that PP1 and PP2A may dephosphorylate distinct phosphorylation sites on the Cav1.2 Ca(2+) channel.

Protein Phosphatases of Pathogenic Bacteria: Role in Physiology and Virulence.

The role of protein phosphatases in pathogenic bacteria has been studied extensively over the last two decades. Ser/Thr and Tyr phosphatases are associated with growth and virulence of many bacteria. These phosphatases control kinase-mediated functions and return the proteins to their unmodified state. Biochemical, structural, and functional studies, in addition to extensive genetic characterization, have highlighted the importance of phosphatases in bacteria. However, questions remain regarding the mechanisms driving localization of secretory phosphatases to cellular compartments, identification of receptor phosphatase sensory signals, and a possible role of cofactors and ligands in their functions. This review focuses on the role of Ser/Thr- and Tyr-specific phosphatases present in pathogenic bacteria, with an emphasis on the regulation of basic cellular processes and virulence. Furthermore, we highlight their clinical importance and analyze the development of drugs targeting protein phosphatases.

CONTROL OF ATRIAL GAP JUNCTION CONDUCTANCE BY INTRACELLULAR PHOSPHATASES

Myocardial action potential conduction velocity is a function of gap junction (GJ) electrical conductance (Gj). The phosphorylation state of GJ proteins, in particular Cx43 determines Gj and the role of protein phosphatases (PPs) remains unclear. This study aims to investigate the role of serine-threonine PPs in regulating Gj, in particular the Ca2+-dependent PP, calcineurin, and the Ca2+-independent PPs, PP1 and PP2A. Left atrial strips were dissected from male, Dunkin-Hartley guinea pigs to estimate Gj by measuring the frequency-dependent intracellular conductance1 in control solution (Na=149.4 mM), or low-Na solution (Na=29.4 mM) to raise [Ca2+]i. Inhibitors to calcinuerin, (cyclosporine-A; 5 µM; calcineurin auto-inhibitory peptide; CAIP, 50 µM); PPI (tautomycin, 5 nM) or PP2A (okadaic acid, 2 nM; fostreicin 100 nM) were used. Western blots were conducted on inhibitor perfused tissue. Values are mean±SEM, normalised to control. Differences were tested by ANOVA; null hypotheses rejected at p Low-Na significantly and reversibly decreased Gj, and in control conditions individual inhibitors had no effect. Cyclosporine-A and CAIP diminished the effects of low-Na (Gj: low-Na, 55±5.8% low-Na+ cyclosporine-A (n=6): 85±9.7%; low-Na+CAIP (n=3): 104±10.2% control). Fostreicin also reduced the low-Na effect (low-Na: 54±2.5%; low-Na+fostreicin (n=6): 77±6.1% control) but not tautomycin (low-Na: 65±4.9%; low-Na+TTM (n=7): 66±3.9%). Phosphorylation of Cx43 during low-Na was diminished by CysA, CAIP and FST. The phosphatases calcineurin and PP2A mediate the decrease of Gj in the left atrium during elevated [Ca2+]i. Connexin43 is one of the targets for calcineurin and PP2A.

Role of Protein Phosphatase Magnesium‐Dependent 1A and Anti–Protein Phosphatase Magnesium‐Dependent 1A Autoantibodies in Ankylosing Spondylitis

ObjectiveAlthough ankylosing spondylitis (AS) is driven by immune‐mediated processes, little is known about the presence and role of autoantibodies in this disease. This study was undertaken to investigate whether autoantibodies occur in and are involved in AS.MethodsWe performed human protein microarray analysis of sera derived from patients with AS or other autoimmune disorders to identify autoantibodies associated specifically with AS, and identified autoantibody targeting of protein phosphatase magnesium‐dependent 1A (PPM1A) in AS. We performed enzyme‐linked immunosorbent assay (ELISA) analysis of sera from 2 independent AS cohorts to confirm autoantibody targeting of PPM1A, and to assess associations between levels of anti‐PPM1A antibodies and AS disease severity or response to anti–tumor necrosis factor (anti‐TNF) therapy (as measured by Bath AS Disease Activity Index [BASDAI] score). Levels of anti‐PPM1A antibodies were also evaluated in sera from rats transgenic for HLA–B27 and human β2‐microglobulin. The expression of PPM1A was assessed by immunohistochemistry in synovial tissue samples from patients with AS, rheumatoid arthritis, or osteoarthritis. The role of PPM1A in osteoblast differentiation was investigated by gene knockdown and overexpression.ResultsAS was associated with autoantibody targeting of PPM1A, and levels of anti‐PPM1A autoantibodies were significantly higher in patients with more advanced sacroiliitis and correlated positively with BASDAI score after treatment with anti‐TNF agents. The levels of anti‐PPM1A autoantibodies were also higher in the sera of transgenic rats that are prone to develop spondyloarthritis than in those that are not. PPM1A was expressed in AS synovial tissue, and PPM1A overexpression promoted osteoblast differentiation, whereas PPM1A knockdown suppressed it.ConclusionAnti‐PPM1A autoantibodies are present in AS, and our findings suggest that PPM1A may contribute to the pathogenic bone ankylosis characteristic of AS.

Role of Protein Phosphatase 1 in Dephosphorylation of Ebola Virus VP30 Protein and Its Targeting for the Inhibition of Viral Transcription

The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr(143) and Thr(146) of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species.

Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei

The cell cycle consists of an orderly sequence of events, whose purpose is to faithfully replicate and segregate cellular components. Many events in the cell cycle are triggered by protein kinases and counteracting phosphoprotein phosphatases (PPP). In Trypanosoma brucei, RNAi has been used to characterize numerous regulatory kinases, while the role of protein phosphatases has primarily been deduced with inhibitors such as okadaic acid and calyculin. In the present study, we identify for the first time a protein phosphatase 2A family member (TbPP2A-1) whose knockdown with RNAi phenocopies the effects of okadaic acid (OKA). In bloodstream forms (BF) and insect stage procyclic forms (PF) RNAi of TbPP2A-1 generates a cell population characterized by: an inhibition of cell growth, a block in cytokinesis; continued synthesis of nuclear DNA leading to aneuploidy; continued mitosis leading to cells with N>2, and an unusual phenotype where number of kinetoplasts (and flagella) is less than the number of nuclei. An engineered cell line was constructed to further study TbPP2A-1 and to facilitate the discovery of other cell cycle regulatory genes.

Revisiting the Evolutionary History and Roles of Protein Phosphatases with Kelch-Like Domains in Plants

Protein phosphatases with Kelch-like domains (PPKL) are members of the phosphoprotein phosphatases family present only in plants and alveolates. PPKL have been described as positive effectors of brassinosteroid (BR) signaling in plants. Most of the evidence supporting this role has been gathered using one of the four homologs in Arabidopsis (Arabidopsis thaliana), brassinosteroid-insensitive1 suppressor (BSU1). We reappraised the roles of the other three members of the family, BSL1, BSL2, and BSL3, through phylogenetic, functional, and genetic analyses. We show that BSL1 and BSL2/BSL3 belong to two ancient evolutionary clades that have been highly conserved in land plants. In contrast, BSU1-type genes are exclusively found in the Brassicaceae and display a remarkable sequence divergence, even among closely related species. Simultaneous loss of function of the close paralogs BSL2 and BSL3 brings about a peculiar array of phenotypic alterations, but with marginal effects on BR signaling; loss of function of BSL1 is, in turn, phenotypically silent. Still, the products of these three genes account for the bulk of PPKL-related activity in Arabidopsis and together have an essential role in the early stages of development that BSU1 is unable to supplement. Our results underline the functional relevance of BSL phosphatases in plants and suggest that BSL2/BSL3 and BSU1 may have contrasting effects on BR signaling. Given that BSU1-type genes have likely undergone a functional shift and are phylogenetically restricted, we caution that inferences based on these genes to the whole family or to other species may be misleading.

Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.

Protein-tyrosine Phosphatase 1B Antagonized Signaling by Insulin-like Growth Factor-1 Receptor and Kinase BRK/PTK6 in Ovarian Cancer Cells*♦

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R β-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R β-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rβ signaling in ovarian cancer cells.

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.

Pyruvate restores β-adrenergic sensitivity of L-type Ca2+channels in failing rat heart: role of protein phosphatase

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

Protein Phosphatase 2A Dephosphorylates CaBP4 and Regulates CaBP4 Function

CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCζ) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca(v)1 Ca(2+) channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation. The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca(v)1.3 currents. PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCζ. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca(v)1.3 Ca(2+) channels in HEK293T cells. This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca(2+) signals at the photoreceptor synapse.

PP2A-dependent control of transcriptionally active FOXO3a in CD8+ central memory lymphocyte survival requires p47phox

Forkhead box O3a (FOXO3a) transcription factor is regulated by complex post-translational modifications that allow for transcriptional control of various apoptosis factors including pro-apoptotic Bim. Although it has been shown that kinases phosphorylate FOXO3a in memory T cells, the role of protein phosphatases in the control of memory T lymphocyte FOXO3a function is less clear. Here, we report that FOXO3a is dephosphorylated (activated) by a protein phosphatase 2A (PP2A)-dependent mechanism in CD8+ memory lymphocytes (Tm) during Listeria monocytogenes (Lm) infection, which allows for enhanced Bim transcription in nicotinamide adenine dinucleotide phosphate-oxidase p47phox-deficient (p47phox−/−) Tm. Consequently, CD8+ Tm from Lm-infected p47phox−/− mice express significantly higher levels of each pro-apoptotic Bim protein isoform. Furthermore, there was a profound reduction in the accumulation of CD8+ T central memory (Tcm) cells in infected p47phox−/− spleens, and 65% p47phox−/− mouse moribundity following secondary Lm reinfection compared with 25% in wild-type mice. Notably, blocking PP2A activity attenuated FOXO3 activation and Bim transcription in p47phox−/− CD8+ memory lymphocytes. Our findings indicate a critical role for p47phox in a dynamic interplay between PP2A and FOXO3a that regulates pro-apoptotic Bim transcription in CD8+ memory lymphocytes during infection.

Kinases, phosphatases and proteases during sperm capacitation

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca(2+). Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60 s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca(2+) might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other's activities.

Protein phosphatase 2A effectively modulates basal L-type Ca2+ current by dephosphorylating Cav1.2 at serine 1866 in mouse cardiac myocytes

Calcium (Ca2+) influx through Cav1.2 L-type Ca2+ channels is an important event for cardiac excitation–contraction (E–C) coupling. The functional regulation of Cav1.2 is controlled by multiple kinases and phosphatases. It has been well documented that phosphorylation of Cav1.2 by PKA or other kinases is sufficient for the upregulation of channel activity. However, little is known about the role of protein phosphatases in counterbalancing the phosphorylation of Cav1.2, especially the degree to which protein phosphatase 2A (PP2A)-mediated dephosphorylation is involved in the regulation of Cav1.2 in the mouse heart. Here, we report a physical interaction between PP2A and the C-terminus of Cav1.2 in mouse heart extracts as revealed by coimmunoprecipitation. This interaction was further confirmed by the observation that PP2A and Cav1.2 are colocalized in isolated mouse cardiomyocytes. Specifically, PP2A was bound at serine 1866 in the C-terminus of Cav1.2, and PP2A-induced Cav1.2 dephosphorylation at serine 1866 was observed in mouse cardiomyocytes. Importantly, the density of L-type calcium current increased in line with the increase in the phosphorylation at serine 1866 of Cav1.2 in cardiac-specific PP2A Cα knockout mice. These phenomena were reproduced by treatment with okadaic acid, a PP2A inhibitor, in H9c2 cells. In summary, our data reveal the functional role of PP2A in cardiac Cav1.2 regulation.

Human Sperm Capacitation Requires the Inhibition of the Activity of the Serine/Threonine Phosphatase PP2A.

Capacitation is a complex process that involves a series of morphological and biochemical changes in the sperm and that allow them to acquire fertilizing capacity. Some of the changes described that take place during the early stages of capacitation includes increase in the concentration of intracellular calcium, the generation of reactive oxygen species, changes in the membrane potential, and increase in the AMPc intracellular concentration. It has also been reported that during capacitation the phosphorylation pattern in tyrosine and threonine proteins residues changes. The role of protein kinases, including tyrosine kinases and PKA, in this process is well documented. However, little is known about the role of protein phosphatases (PPs) in this event. PP2A is serine threonine phosphatase that has been detected in human and primate sperm extracts, but its role during sperm capacitation is not known. The aim of this work was to study the role of PP2A during human sperm capacitation.To this end, we have used inhibitors and measured the activity of PP2A during this process. Semen samples were obtained from normal donors according to the WHO guidelines. Highly motile sperm were selected by a Percoll gradient using a modified Tyrode's medium (without BSA and bicarbonate). Subsequently, sperm aliquots were resuspended in the same medium and incubated at 37 °C and 5% CO2 in the presence or absence of PP2A inhibitors (okadaic acid and Endothal with IC50= 0.1 nM and IC50= 90 nM respectively). Other sperm aliquots were treated by adding 2.6% BSA and 25 nM bicarbonate to the culture medium. The capacitation process was evaluated at different times (0, 15, 30, 60, and 300 min) by using the chlortetracycline (CTC) fluorescence assay. The activity of PP2A was determined in human sperm extracts by using the "RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit" of Invitrogen.The results indicate that treatment with the inhibitors blocked the activity of PP2A and rapidly increased the percentage of capacitated sperm. Moreover, upon addition of BSA and bicarbonate to the culture medium the sperm became capacitated, as evidenced by CTC assay, and the activity of PP2A rapidly decreased and remained low thereafter.In conclusion, the onset of capacitation in human sperm is associated with a rapid decrease in the activity of PP2A and inhibition of this phosphatase rapidly triggers capacitation. Fondecyt 1080028. (poster)

Protein phosphatases and the regulation of mitosis

Dynamic control of protein phosphorylation is necessary for the regulation of many cellular processes, including mitosis and cytokinesis. Indeed, although the central role of protein kinases is widely appreciated and intensely studied, the importance of protein phosphatases is often overlooked. Recent studies, however, have highlighted the considerable role of protein phosphatases in both the spatial and temporal control of protein kinase activity, and the modulation of substrate phosphorylation. Here, we will focus on recent advances in our understanding of phosphatase structure, and the importance of phosphatase function in the control of mitotic spindle formation, chromosome architecture and cohesion, and cell division.

The Molecular Mechanism Underlying Morphine-Induced Akt Activation: Roles of Protein Phosphatases and Reactive Oxygen Species

Although Akt is reported to play a role in morphine's cardioprotection, little is known about the mechanism underlying morphine-induced Akt activation. This study aimed to define the molecular mechanism underlying morphine-induced Akt activation and to determine if the mechanism contributes to the protective effect of morphine on ischemia/reperfusion injury. In cardiac H9c2 cells, morphine increased Akt phosphorylation at Ser(473), indicating that morphine upregulates Akt activity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling, was not involved in the action of morphine on Akt activity. Morphine decreased the activity of PP2A, a major protein Ser/Thr phosphatase, and inhibition of PP2A with okadaic acid (OA) mimicked the effect of morphine on Akt activity. The effects of morphine on PP2A and Akt activities were inhibited by the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)glycine (MPG) and the mitochondrial K(ATP) channel closer 5-hydroxydecanoate (5HD). In support, morphine could produce ROS and this was reversed by 5HD. Finally, the cardioprotective effect of morphine on ischemia-reperfusion injury was mimicked by OA but was suppressed by 5HD or MPG, indicating that protein phosphatases and ROS are involved in morphine's protection. In conclusion, morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via ROS, which may contribute to the cardioprotective effect of morphine.

Protein Phosphatase 2A as a Potential Target for Anticancer Therapy

The kinase oncogenes are well-characterized drivers of cancer development, and several targeted therapies focused on both specific and selectively nonselective kinase inhibitors have now been approved for clinical use. In contrast, much less is known about the role of protein phosphatases, although modulation of their activities might form the foundation for an effective anti-cancer approach. The serine-threonine protein phosphatase 2A (PP2A) is implicated in the regulation of numerous signaling pathways and may function as a tumor suppressor. Recently pharmacological modulation of PP2A activity has been showed to have a potent anti-tumor activity in both in vitro and in vivo cancer models. These studies implicate PP2A as a promising therapeutic target for the treatment of cancer.

Forced Expression of Methionine Adenosyltransferase 1A in Human Hepatoma Cells Suppresses in Vivo Tumorigenicity in Mice

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the principal methyl donor, and is encoded by MAT1A and MAT2A in mammals. Normal liver expresses MAT1A, which is silenced in hepatocellular carcinoma. We have shown that hepatoma cells overexpressing MAT1A grew slower, but whether this is also true in vivo remains unknown. To investigate the effect of overexpressing MAT1A on in vivo tumorigenesis, we generated stable transfectants of Huh7 cells overexpressing either MAT1A or empty vector. Real-time PCR and Western blotting were used to measure expression, and BALB/c nude mice were injected subcutaneously with untransfected or Huh7 cells transfected with empty or MAT1A expression vector to establish tumors. Tumor properties such as proliferation, angiogenesis, and apoptosis were compared, and microarray analysis was performed. Huh7 cells overexpressing MAT1A had higher S-adenosylmethionine levels but lower bromodeoxyuridine incorporation than control cells. Tumor growth rates and weights were lower in MAT1A transfected tumors. In addition, microvessel density and CD31 and Ki-67 staining were lower in MAT1A transfected tumors than control tumors, whereas the apoptosis index was higher in MAT1A-transfected tumors. Forced expression of MAT1A induced genes related to apoptosis and tumor suppression and lowered expression of cell growth and angiogenesis proteins. Our data demonstrate in vivo overexpression of MAT1A in liver cancer cells can suppress tumor growth. They also suggest inducing MAT1A expression might be a strategy to treat hepatocellular carcinoma.