Catalytic Subunit Of Protein Phosphatase Research Articles

C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

PP1A-Mediated Dephosphorylation Positively Regulates YAP2 Activity

BackgroundThe Hippo/MST1 signaling pathway plays an important role in the regulation of cell proliferation and apoptosis. As a major downstream target of the Hippo/MST1 pathway, YAP2 (Yes-associated protein 2) functions as a transcriptional cofactor that has been implicated in many biological processes, including organ size control and cancer development. MST1/Lats kinase inhibits YAP2's nuclear accumulation and transcriptional activity through inducing the phosphorylation at serine 127 and the sequential association with 14-3-3 proteins. However, the dephosphorylation of YAP2 is not fully appreciated.Methodology/Principal FindingsIn the present study, we demonstrate that PP1A (catalytic subunit of protein phosphatase-1) interacts with and dephosphorylates YAP2 in vitro and in vivo, and PP1A-mediated dephosphorylation induces the nuclear accumulation and transcriptional activation of YAP2. Inhibition of PP1 by okadiac acid (OA) increases the phosphorylation at serine 127 and cytoplasmic translocation of YAP2 proteins, thereby mitigating its transcription activity. PP1A expression enhances YAP2's pro-survival capability and YAP2 knockdown sensitizes ovarian cancer cells to cisplatin treatment.Conclusions/SignificanceOur findings define a novel molecular mechanism that YAP2 is positively regulated by PP1-mediated dephosphorylation in the cell survival.

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein.

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.

Fasting-Induced Protein Phosphatase 1 Regulatory Subunit Contributes to Postprandial Blood Glucose Homeostasis via Regulation of Hepatic Glycogenesis

OBJECTIVEMost animals experience fasting–feeding cycles throughout their lives. It is well known that the liver plays a central role in regulating glycogen metabolism. However, how hepatic glycogenesis is coordinated with the fasting–feeding cycle to control postprandial glucose homeostasis remains largely unknown. This study determines the molecular mechanism underlying the coupling of hepatic glycogenesis with the fasting–feeding cycle.RESEARCH DESIGN AND METHODSThrough a series of molecular, cellular, and animal studies, we investigated how PPP1R3G, a glycogen-targeting regulatory subunit of protein phosphatase 1 (PP1), is implicated in regulating hepatic glycogenesis and glucose homeostasis in a manner tightly orchestrated with the fasting–feeding cycle.RESULTSPPP1R3G in the liver is upregulated during fasting and downregulated after feeding. PPP1R3G associates with glycogen pellet, interacts with the catalytic subunit of PP1, and regulates glycogen synthase (GS) activity. Fasting glucose level is reduced when PPP1R3G is overexpressed in the liver. Hepatic knockdown of PPP1R3G reduces postprandial elevation of GS activity, decreases postprandial accumulation of liver glycogen, and decelerates postprandial clearance of blood glucose. Other glycogen-targeting regulatory subunits of PP1, such as PPP1R3B, PPP1R3C, and PPP1R3D, are downregulated by fasting and increased by feeding in the liver.CONCLUSIONSWe propose that the opposite expression pattern of PPP1R3G versus other PP1 regulatory subunits comprise an intricate regulatory machinery to control hepatic glycogenesis during the fasting–feeding cycle. Because of its unique expression pattern, PPP1R3G plays a major role to control postprandial glucose homeostasis during the fasting–feeding transition via its regulation on liver glycogenesis.

Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1

TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin–radixin–moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction ( K a = 1.80 × 10 6 M −1) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cβ is inhibited to different extent in the presence of non- (∼60% inhibition), mono- (∼50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3β inhibitor it is shown here that PKA activation is followed by GSK3β activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/GSK3β activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.

The Intrinsically Disordered Nuclear Localization Signal and Phosphorylation Segments Distinguish the Membrane Affinity of Two Cytidylyltransferase Isoforms

Membrane phosphatidylcholine homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP:phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane phosphatidylcholine content by reversible membrane binding and activation. Two prominent isoforms, CCTα and -β2, have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains but have divergent and structurally disordered N-terminal (N) and C-terminal phosphorylation (P) regions. We found that the binding affinity of purified CCTβ2 for anionic membranes was weaker than CCTα by more than an order of magnitude. Using chimeric CCTs, insertion/deletion mutants, and truncated CCTs, we show that the stronger affinity of CCTα can be attributed in large part to the electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured N-terminal segment of CCTα but lacking in CCTβ2. The membrane partitioning of CCTβ2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCTα and was elevated by incorporation of the NLS motif. Thus, the polybasic NLS can function as a secondary membrane binding motif not only in vitro but in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P-truncated forms showed that the in vitro membrane affinity of CCTβ2 is more sensitive than CCTα to phosphorylation status, which antagonizes membrane binding of both isoforms. These data provide a model wherein the primary membrane binding motif, an amphipathic helical domain, works in collaboration with other intrinsically disordered segments that modulate membrane binding strength. The NLS reinforces, whereas the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.

Protein Phosphatase 1 (PP-1)-Dependent Inhibition of Insulin Secretion by Leptin in INS-1 Pancreatic β-Cells and Human Pancreatic Islets

Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca²(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.

Regulation of Platelet Survival by Protein Phosphatase 1 Gamma

AbstractAbstract 2026Platelets are key players in hemostasis and their senescence is intrinsically associated with the activation of apoptotic pathways that shows similarities to the apoptosis of nucleated cells. Anti-apoptotic protein Bcl-xl restrains the pro-apoptotic Bak activity and maintains platelet survival in circulation. In nucleated cells, serine phosphorylation of a pro-apoptotic protein Bad can also promote cell survival. Serine phosphorylation of Bad is regulated by the action of serine/threonine (Ser/Thr) protein kinases and Ser/Thr protein phosphatases. Although alterations in the activities of either enzyme can change the rate of apoptosis, whether Ser/Thr phosphatases participate in regulating platelet apoptosis is unknown. In this study, we report that the mice lacking the catalytic subunit of protein phosphatase 1 gamma (PP1cγ) exhibit a moderate but significant increase in the number of circulating platelets [596 × 103/μL ± 17.9 in WT compared with 694 × 103/μL ± 19.8 in PP1cγ–/– mice; P = .0003]. Examination of the bone marrow from the PP1cγ–/– mice revealed a non-significant increase in the number of morphologically matured megakaryocytes. A trend towards decreased plasma thrombopoietin levels were also noticed in PP1cγ–/– mice. These observations suggest that an increased megakaryopoiesis/thrombopoiesis may not fully account for the increased platelet numbers in PP1cγ–/– mice. In vivo platelet survival studies revealed that the loss of PP1cγ modestly increased platelet half life in circulation (t1/2 ∼69 hours in WT compared to ∼78 hours in PP1cγ–/– mice). PP1cγ–/– platelets had decreased mean platelet volume, suggesting the PP1cγ–/– mice may harbor a greater proportion of older circulating platelets. These studies are consistent with the delayed clearance of platelets from PP1cγ–/– mice. Pro-apoptotic Bad possess PP1c binding motif and mechanistically, PP1cγ interacts with Bad protein in platelets. Phosphorylation of Bad Ser112, which promotes cell survival, was enhanced ∼50% in PP1cγ–/– platelets. Consistent with the increased BAD phosphorylation, co-immunoprecipitation studies revealed increased BAD-14-3-3 protein complexes from the PP1cγ–/– platelets. It is reported that in the phosphorylated state, BAD can interact with 14-3-3 and is sequestered in the cytoplasm, thereby preventing the binding of Bad with the mitochondrial anti-apoptotic Bcl-xl. Interaction of Bad with Bcl-xl has the potential to displace the pro-apoptotic Bak from the Bcl-xl-Bak protein complex to trigger apoptosis. Finally, immunoblotting with anti-caspase 9 antibody revealed decreased caspase 9 cleavage product (∼37 kDa) in the PP1cγ–/– platelets, suggesting decreased activation of caspase 9 dependent apoptotic pathway. These data indicate that the increased platelet counts in PP1cγ–/– mice could be in part due to delayed platelet apoptosis. Loss of PP1cγ leads to the hyperphosphorylation of BAD, which via an interaction with 14-3-3, delays caspase mediated apoptosis to prolong the life span of platelets. Disclosures:No relevant conflicts of interest to declare.

Protein Phosphatase 1β is Critical to Determine Cardiac Systolic and Diastolic Function in Cardiomyopathic Mice

Background: Protein phosphatase 1 (PP1) is the negative regulator of cardiac contractility and its activity was shown to be aberrantly upregulated in failing hearts. PP1 catalytic subunits consist of three different genes, PP1α, PP1β, and PP1γ. We previously reported that in vivo knockdown of PP1β was sufficient to augment left ventricular systolic function in normal mice. We herein investigated whether PP1β knockdown halts progression of heart failure in muscle LIM protein deficient cardiomyopathic mice (MLPKO).

The PP1-R6 protein phosphatase holoenzyme is involved in the glucose-induced dephosphorylation and inactivation of AMP-activated protein kinase, a key regulator of insulin secretion, in MIN6 β cells

Mammalian AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status. It is activated by phosphorylation of the catalytic subunit on Thr172. The main objective of this study was the identification of a phosphatase involved in the regulation of AMPK activity. Mouse MIN6 β cells were used to study the glucose-induced regulation of the phosphorylation of AMPK. Small interfering RNA (siRNA) technology was used to deplete putative phosphatase candidate genes that could affect AMPK regulation. The effect of the siRNAs used in the study was compared with the effect observed using a negative control siRNA. A protein phosphatase complex composed of the catalytic subunit of protein phosphatase-1 (PP1) and the regulatory subunit R6 participates in the glucose-induced dephosphorylation of AMPK. R6 interacts physically with the β-subunit of the AMPK complex and recruits PP1 to dephosphorylate the catalytic α-subunit on Thr172. siRNA depletion of R6 decreases glucose-induced insulin secretion due to the presence of a constitutively active AMPK complex. The characterization of the PP1-R6 complex identifies this holoenzyme as a possible target for therapeutic intervention with the aim of regulating the activity of AMPK in pancreatic β cells.

DNA Methylation Regulates Cocaine-Induced Behavioral Sensitization in Mice

The behavioral sensitization produced by repeated cocaine treatment represents the neural adaptations underlying some of the features of addiction in humans. Cocaine administrations induce neural adaptations through regulation of gene expression. Several studies suggest that epigenetic modifications, including DNA methylation, are the critical regulators of gene expression in the adult central nervous system. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) and consequent promoter region hypermethylation is associated with transcriptional silencing. In this study a potential role for DNA methylation in a cocaine-induced behavioral sensitization model in mice was explored. We report that acute cocaine treatment caused an upregulation of DNMT3A and DNMT3B gene expression in the nucleus accumbens (NAc). Using methylated DNA immunoprecipitation, DNA bisulfite modification, and chromatin immunoprecipitation assays, we observed that cocaine treatment resulted in DNA hypermethylation and increased binding of methyl CpG binding protein 2 (MeCP2) at the protein phosphatase-1 catalytic subunit (PP1c) promoter. These changes are associated with transcriptional downregulation of PP1c in NAc. In contrast, acute and repeated cocaine administrations induced hypomethylation and decreased binding of MeCP2 at the fosB promoter, and these are associated with transcriptional upregulation of fosB in NAc. We also found that pharmacological inhibition of DNMT by zebularine treatment decreased cocaine-induced DNA hypermethylation at the PP1c promoter and attenuated PP1c mRNA downregulation in NAc. Finally, zebularine and cocaine co-treatment delayed the development of cocaine-induced behavioral sensitization. Together, these results suggest that dynamic changes of DNA methylation may be an important gene regulation mechanism underlying cocaine-induced behavioral sensitization.

An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme-docking site in the Rb C-terminal domain that is required for efficient PP1c activity towards Rb. The phosphatase-docking site overlaps with the known docking site for Cyclin dependent kinase, and PP1 competition with Cdk-Cyclins for Rb binding is sufficient to retain Rb activity and block cell cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.

Identification and Characterization of a Novel Human PP1 Phosphatase Complex

Mammalian Wdr82 is a regulatory component of the Setd1a and Setd1b histone H3-lysine 4 methyltransferase complexes and is implicated in the tethering of Setd1 complexes to transcriptional start sites of active genes. In the studies reported here, immunoprecipitation and mass spectrometry analyses reveal that Wdr82 additionally associates with multiple protein complexes, including an RNA polymerase II complex, four distinct histone H3-Lys(4) methyltransferase complexes, protein phosphatase 1 (PP1)-associated proteins, a chaperonin-containing Tcp1 complex, and other uncharacterized proteins. Further characterization of the PP1-associated proteins identified a stable multimeric complex composed of regulatory subunits PNUTS, Tox4, and Wdr82 and a PP1 catalytic subunit (denoted as the PTW/PP1 phosphatase complex). The PTW/PP1 complex exhibits in vitro phosphatase activity in a PP1-dependent manner. Analysis of protein-protein interactions reveals that PNUTS mediates phosphatase complex formation by providing a binding platform to each component. The PNUTS and Tox4 subunits are predominantly associated with the PTW/PP1 phosphatase complex in HEK293 cells, and the integrity of this complex remains intact throughout cell cycle progression. Inducible expression of a PP1 interaction-defective form of PNUTS (W401A) or small interfering RNA-mediated depletion of PNUTS in HEK293 cells causes cell cycle arrest at mitotic exit and apoptotic cell death. PNUTS (W401A) shows normal association with chromosomes but causes defects in the process of chromosome decondensation at late telophase. These data reveal that mammalian Wdr82 functions in a variety of cellular processes and reveal a potential role of the PTW/PP1 phosphatase complex in the regulation of chromatin structure during the transition from mitosis into interphase.

Isoform-specific roles of protein phosphatase 1 catalytic subunits in sarcoplasmic reticulum-mediated Ca2+ cycling

protein phosphatase 1 (PP1) is the major isotype of serine/threonine phosphatase in cardiomyocytes, and its activity has been thought to be important for heart failure progression. The PP1 catalytic subunits consist of three distinct genes, PP1α, PP1β/δ, and PP1γ. To date, the function of each PP1 isoform is not well characterized in cardiomyocytes. We sought to determine the functional contribution of each PP1 isoform to sarcoplasmic reticulum (SR)-mediated Ca(2+) cycling in isolated adult rat cardiomyocytes. adenoviral vectors encoding short hairpin RNA for each PP1 isoform were transfected into isolated rat cardiomyocytes, and this was followed by analysis of cell shortening, Ca(2+) transients, and the phosphorylation levels of Ca(2+) regulatory proteins. Physical interactions between each PP1 isoform and SR Ca(2+) regulatory proteins were characterized in isolated cardiomyocytes expressing green fluorescent protein (GFP)-tagged PP1 catalytic subunits, and also in canine junctional and longitudinal SR preparations. Successful PP1 isoform knockdown was achieved for each isoform without affecting the expression of the other isoforms. PP1β knockdown most significantly enhanced the Ca(2+) transient and cell shortening by augmenting phospholamban (PLN) phosphorylation at baseline and with low-dose isoproterenol stimulation (10 nM). Interestingly, PP1β was preferentially associated with sarco-endoplasmic ATPase and PLN in GFP-PP1-transfected cardiomyocytes, as well as in canine longitudinal SR preparations. these findings indicate that PP1β is the most significant PP1 isoform involved in regulating SR Ca(2+) cycling in rat cardiomyocytes.

Cyanobacterial Cyclopeptides as Lead Compounds to Novel Targeted Cancer Drugs

Cyanobacterial cyclopeptides, including microcystins and nodularins, are considered a health hazard to humans due to the possible toxic effects of high consumption. From a pharmacological standpoint, microcystins are stable hydrophilic cyclic heptapeptides with a potential to cause cellular damage following uptake via organic anion-transporting polypeptides (OATP). Their intracellular biological effects involve inhibition of catalytic subunits of protein phosphatase 1 (PP1) and PP2, glutathione depletion and generation of reactive oxygen species (ROS). Interestingly, certain OATPs are prominently expressed in cancers as compared to normal tissues, qualifying MC as potential candidates for cancer drug development. In the era of targeted cancer therapy, cyanotoxins comprise a rich source of natural cytotoxic compounds with a potential to target cancers expressing specific uptake transporters. Moreover, their structure offers opportunities for combinatorial engineering to enhance the therapeutic index and resolve organ-specific toxicity issues. In this article, we revisit cyanobacterial cyclopeptides as potential novel targets for anticancer drugs by summarizing existing biomedical evidence, presenting structure-activity data and discussing developmental perspectives.

Smad anchor for receptor activation SARA in TGF-beta signaling

Smad anchor for receptor activation (SARA) is known as Smad cofactor that interacts directly with Smad2/3 and functions to recruit Smad2/3 to the TGF-beta receptor. SARA plays an essential role in TGF-beta-induced Smad2 activation and it may modulate TGF-beta signaling through regulating the balance between Smad2 and Smad3. SARA also functions as an anchor for catalytic subunit of protein phosphatase 1 (PP1c) and maybe involved in the dephosphorylation of TGF-beta type I receptor (TbetaR-I) mediated by Smad7. The expression of SARA changes as the development of epithelial to mesenchymal transition (EMT) and fibrosis and it plays a critical role in the maintenance of epithelial cell phenotype. Modulation of SARA may provide a new therapeutic approach to TGF-beta-mediated EMT and fibrosis.

A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg(2+) binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.

NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase

Na+/H+ exchanger 3 (NHE3) is phosphorylated and regulated by multiple kinases, including PKA, SGK1, and CK2; however, the role of phosphatases in the dephosphorylation and regulation of NHE3 remains unknown. The purpose of this study was to determine whether serine/threonine phosphatases alter NHE3 activity and phosphorylation and, if so, at which sites. To this end, we first examined the effects of calyculin A [a combined protein phosphatase 1 (PP1) and PP2A inhibitor] and okadaic acid (a PP2A inhibitor) on general and site-specific NHE3 phosphorylation. Calyculin A induced a phosphorylation-dependent NHE3 gel mobility shift and increased NHE3 phosphorylation at serines 552 and 605. No change in NHE3 phosphorylation was detected after okadaic acid treatment. An NHE3 gel mobility shift was also evident in calyculin A-treated COS-7 cells transfected with either wild-type or mutant (S552A, S605G, S661A, S716A) rat NHE3. Since the NHE3 gel mobility shift occurred despite mutation of known phosphorylation sites, novel sites of phosphorylation must also exist. Next, we assayed NHE3 activity in response to calyculin A and okadaic acid and found that calyculin A induced a 24% inhibition of NHE3 activity, whereas okadaic acid had no effect. When all known NHE3 phosphorylation sites were mutated, calyculin A induced a stimulation of NHE3 activity, demonstrating a functional significance for the novel phosphorylation sites. Finally, we established that the PP1 catalytic subunit can directly dephosphorylate immunopurified NHE3 in vitro. In conclusion, our data demonstrate that a calyculin A-sensitive phosphatase, most likely PP1, is involved in the regulation and dephosphorylation of NHE3 at known and novel sites.

The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

BackgroundHemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear.Methodology/Principal FindingsUsing γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets.Conclusions/SignificanceThese studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

Distinct Roles for the α, β and γ Isoforms of the Catalytic Subunit of Protein Phosphatase 1 in Outside-in Integrin αIIbβ3 Signaling.

Abstract 154Outside-in integrin αIIbγ3 signaling mediated by kinases and phosphatases regulate platelet adhesion and thrombus formation. The catalytic subunit of protein phosphatase 1 (PP1c), expressed as α, β and γ isoforms regulates a variety of cellular functions. We have previously demonstrated that fibrinogen-αIIbβ3 engagement during outside-in signaling dissociated integrin αIIb*β3 anchored PP1c, which was followed by PP1c activation and dephosphorylation of myosin light chain (MLC). However, it is unknown whether any functions regulated by outside-in integrin αIIbβ3 signaling are modulated by PP1c. To overcome the lack of specificity of pharmacological agents towards PP1c, we have pursued a genetic approach to inhibit PP1c in 293 cells overexpressing αIIbβ3. In this study, we demonstrated that PP1cα interacts with αIIbβ3 in platelets and 293 cells by co-immunoprecipitation and GST pull down assays. Knockdown of PP1cα by short interference RNA resulted in a 50-60% enhanced adhesion (p<0.003) of 293 cells to immobilized fibrinogen. Conversely, overexpression of PP1cα inhibited (p<0.002) αIIbβ3 adhesiveness to fibrinogen. Furthermore, the enhanced adhesiveness of PP1cα depleted 293 cells was also observed with von Willebrand factor, indicating that the differential adhesion due to the lack of PP1cα was not ligand specific. Signaling pathways implicated in the reorganization of cellular cytoskeleton, like extracellular-regulated kinase (ERK1/2) and AKT were activated in PP1cα depleted 293 cells. Such observations imply that PP1cα, knockdown of PP1cγ, which also associates with αIIbβ3, did not affect the adhesion to fibrinogen. Indeed, platelets from the PP1cγ null and wild type mice showed comparable adhesion to fibrinogen. Unexpectedly, depletion of PP1cβ isoform in 293 cells significantly (p<0.01) inhibited the ability of these cells to adhere to fibrinogen. Since MLC is a specific substrate of PP1cβ, it is likely that alterations in the MLC phosphorylation may underlie the loss of adhesiveness in PP1cβ depleted cells. Finally, depletion of all the PP1c isoforms also significantly (p<0.001) inhibited the adhesion of 293 cells to fibrinogen. These data illustrate that each isoform of PP1c contributes distinctly to the adhesive phenotype of integrin αIIbβ3 and implies that the control of outside-in integrin αIIbβ3 signaling response might be a composite of all PP1c isoforms. Perhaps, each isoforms have specific substrates that contribute to the process of adhesion, such that alterations in the phosphorylation of these substrates may underlie the distinct functional roles for PP1c. Disclosures:No relevant conflicts of interest to declare.