Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A

Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.     

Identification of calcineurin regulated phosphorylation sites on CRHSP-24

CRHSP-24 is a prominently regulated phosphoprotein in pancreatic acinar cells where it is the major substrate for the serine/threonine protein phosphatase, calcineurin, in response to secretagogues. We now identify the four regulated sites of CRHSP-24 phosphorylation as serines 30, 32, 41, and 52 and show that Ser³⁰ and Ser³² are directly dephosphorylated by calcineurin. Coordinate phosphorylation/dephosphorylation of these four serines explains the multiple phosphorylated isoforms of CRHSP-24 present in acinar cells and provides a molecular framework to study CRHSP-24 regulation by secretagogues and growth factor-induced kinases and phosphatases in vivo.     

Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).     

Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP

cAMP-mediated stimulation of hepatic bile acid uptake is associated with dephosphorylation and translocation of Na+-taurocholate (TC) cotransporting peptide (NTCP) to the plasma membrane. Although translocation of NTCP may be facilitated by dephosphorylation, the mechanism of dephosphorylation is unknown. The ability of cAMP to translocate and dephosphorylate NTCP is, in part, dependent on cAMP-mediated increases in cytosolic Ca2+ concentration ([Ca2+]), indicating that a Ca2+/calmodulin-dependent protein phosphatase (PP2B) may be involved. Thus we studied the role of PP2B using the inhibitor cypermethrin (CM). Freshly isolated hepatocytes were pretreated with 1-5 nM CM for 30 min followed by 15 min incubation with 10 microM 8-(4-chlorophenylthio)cAMP. CM (5 nM) and FK-506 (5 microM) inhibited cAMP-stimulated TC uptake by 80 and 75%, respectively, without affecting basal TC uptake. CM also reversed cAMP-mediated NTCP dephosphorylation and translocation to 80 and 15% of the basal level, respectively. cAMP stimulated PP2B activity by 60%, and this effect was completely inhibited by 5 nM CM. PP2B dephosphorylated NTCP immunoprecipitated from control but not from cAMP-treated hepatocytes. The effect of CM was not due to any changes in cAMP-mediated increases in cytosolic [Ca2+] or decreases in mitogen-activated protein kinase (extracellular regulated kinases 1 and 2) activity. Taken together, these results suggest that cAMP dephosphorylates NTCP by activating PP2B in hepatocytes, and PP2B-mediated dephosphorylation of NTCP may be involved in cAMP-mediated NTCP translocation to the plasma membrane.     

Structure-functional analyses of CRHSP-24 plasticity and dynamics in oxidative stress response

The cold shock domain (CSD) is an evolutionarily conserved nucleic acid binding domain that exhibits binding activity to RNA, ssDNA, and dsDNA. Mammalian CRHSP-24 contains CSD, but its structure-functional relationship has remained elusive. Here we report the crystal structure of human CRHSP-24 and characterization of the molecular trafficking of CRHSP-24 between stress granules and processing bodies in response to oxidative stress. The structure of CRHSP-24 determined by single-wavelength anomalous dispersion exhibits an α-helix and a compact β-barrel formed by five curved anti-parallel β strands. Ligand binding activity of the CSD is orchestrated by residues Ser(41) to Leu(43). Interestingly, a phosphomimetic S41D mutant abolishes the ssDNA binding in vitro and causes CRHSP-24 liberated from stress granules in vivo without apparent alternation of its localization to the processing bodies. This new class of phosphorylation-regulated interaction between the CSD and nucleic acids is unique in stress granule plasticity. Importantly, the association of CRHSP-24 with stress granules is blocked by PP4/PP2A inhibitor calyculin A as PP2A catalyzes the dephosphorylation of Ser(41) of CRHSP-24. Therefore, we speculate that CRHSP-24 participates in oxidative stress response via a dynamic and temporal association between stress granules and processing bodies.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Selective Dephosphorylation of the Subunits of Skeletal Muscle Calcium Channels by Purified Phosphoprotein Phosphatases

Abstract: Multiple sites on the α1 and β subunits of purified skeletal muscle calcium channels are phosphorylated by cyclic AMP-dependent protein kinase, resulting in three different tryptic phosphopeptides derived from each subunit. Phosphoprotein phosphatases dephosphorylated these sites selectively. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A) dephosphorylated both α1 and β subunits at similar rates, whereas calcineurin dephosphorylated β subunits preferentially. PP1 dephosphorylated phosphopeptides 1 and 2 of the α1 subunit more rapidly than phosphopeptide 3. In contrast, PP2A dephosphorylated phosphopeptide 3 of the α1 subunit preferentially. All three phosphoprotein phosphatases preferentially dephosphorylated phosphopeptide 1 of the β subunit and dephosphorylated phosphopeptides 2 and 3 more slowly. Mn²⁺ increased the rate and extent of dephosphorylation of all sites by calcineurin so that >80% dephosphorylation of both α1 and β sub-units was obtained. The results demonstrate selective dephosphorylation of different phosphorylation sites on the α1 and β subunits of skeletal muscle calcium channels by the three principal serine/threonine phosphoprotein phosphatases.     

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.     

Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases.

The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity.     

Differential Inactivation of Postsynaptic Density-Associated and Soluble Ca2+/Calmodulin-Dependent Protein Kinase II by Protein Phosphatases 1 and 2A

Abstract: Autophosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at Thr²⁸⁶ generates Ca²⁺-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr²⁸⁶ in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (³²P autophosphorylated in the presence of Ca²⁺/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca²⁺-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.     

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.     

Phagocytosis induced by thyrotropin in cultured thyroid cells is associated with myosin light chain dephosphorylation and stress fiber disruption

The actin/myosin II cytoskeleton and its role in phagocytosis were examined in primary cultures of dog thyroid cells. Two (19 and 21 kD) phosphorylated light chains of myosin (P-MLC) were identified by two- dimensional gel electrophoresis of antimyosin immunoprecipitates, and were associated with the Triton X-100 insoluble, F-actin cytoskeletal fraction. Analyses of Triton-insoluble and soluble 32PO4-prelabeled protein fractions indicated that TSH (via cAMP) or TPA treatment of intact cells decreases the MLC phosphorylation state. Phosphoamino acid and tryptic peptide analyses of 32P-MLCs from basal cells showed phosphorylation primarily at threonine and serine residues; most of the [32P] appeared associated with a peptide containing sites typically phosphorylated by MLC kinase. Even in the presence of the agents which induced dephosphorylation, the phosphatase inhibitor, calyculin A, caused a severalfold increase in MLC phosphorylation at several distinct serine and threonine sites which was also associated with actomyosin and cell contraction. Phosphorylation of cell homogenate proteins or the cytoskeletal fraction with [gamma-32P]ATP indicated that Ca2+, EGTA, or trifluoperazine (TFP) has little effect on the phosphorylation of MLC. Both fluorescent phalloidin and antimyosin staining of cells showed distinct dorsal and ventral stress fiber complexes which were disrupted within 30 min by TSH and cAMP; TPA appeared to cause disruption of dorsal, and rearrangement of ventral complexes. Concomitant with MLC dephosphorylation and stress fiber disruption, TSH/cAMP, but not TPA, induced dorsal phagocytosis of latex beads. While stimulation of either A or C-kinase disrupts dorsal stress fibers and rearranges actomyosin, another event(s) mediated by A-kinase appears necessary for phagocytic activity.     

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Protein phosphatase 1, but not protein phosphatase 2A, dephosphorylates DNA-damaging stress-induced phospho-serine 15 of p53

Okadaic acid (OA) is a protein phosphatase (PP) inhibitor and induces hyperphosphorylation of p53. We investigated whether the inhibition of PP1 by OA promotes the phosphorylation of the serine 15 of p53. In vitro dephosphorylation assay showed that PP1 dephosphorylated ultraviolet C (UVC)-induced phospho-ser15 of p53, and that OA treatment inhibited it. One of the PP1 regulators, growth arrest and DNA damage 34 (GADD34), disturbed PP1 binding with p53, interfered with the dephosphorylation of p53 and increased the amount of phospho-p53 after UVC-treatment. This report provides the first evidence that PP1, but not PP2A, dephosphorylates phospho-serine 15 of p53.     

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 microM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.     

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).     

Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.