Truncating mutations of PPM1D are found in blood DNA samples of lung cancer patients.

BACKGROUND: PPM1D (WIP1) negatively regulates by dephosphorylation many proteins including p53 tumour suppressor. The truncating mutations (nonsense and frameshift) in exon 6 of PPM1D were found recently in blood cells of patients with breast, ovarian or colorectal cancer. These mutants code for gain-of-function PPM1D with retained phosphatase activity. Their significance in carcinogenesis is unknown. METHODS: The exon 6 of PPM1D was sequenced in blood DNA of 543 non-small-cell lung cancer patients (NSCLC). The functional significance of selected PPM1D alterations (Arg458X, Lys469Glu) was compared with the wild-type gene and examined by recombinant DNA techniques, immunoblotting and luciferase reporter assays. RESULTS: The frameshift mutations were found in five NSCLC patients (5/543; 0.92%), all of them had squamous cell carcinomas (5/328; 1.5%). All patients with the mutations were exposed, before the blood collection, to the DNA damaging agents as a part of chemotherapeutic regimen. Functional tests demonstrated that truncating mutation Arg458X causes enhancement of dephosphorylation activity of PPM1D toward serine 15 of p53, whereas Lys469Glu version is equivalent to the wild-type. Neither version of PPM1D (wild-type, Arg458X, Lys469Glu) significantly modulated the ability of p53 to transactivate promoters of the examined p53-target genes (BAX and MDM2). CONCLUSIONS: The truncating mutations of PPM1D are present in blood DNA of NSCLC patients at frequency similar to percentage determined for ovarian cancer patients. Our findings raise a question if the detected lesions are a result of chemotherapy.

Cardiac contractile reserve parameters are related to prognosis in septic shock.

INTRODUCTION: Cardiac reserve could be defined as the spontaneous magnitude from basal to maximal cardiac power under stress conditions. The aim of this study was to evaluate the prognostic value of cardiac reserve parameters in resuscitated septic shock patients. METHODS: Seventy patients with septic shock were included in a prospective and observational study. Prior to inclusion, patients were resuscitated to reach a mean arterial pressure of 65-75 mmHg with an euvolemic status. General, hemodynamic, and cardiac reserve-related parameters (cardiac index, double product, and cardiac power index) were collected at inclusion and at day 1. RESULTS: Seventy patients were included with 28-day mortality at 38.5%. Ten of the 70 patients died during the first day. In multivariate analysis, independent predictors of death were SAPS II ≥ 58 (OR: 3.36 [1.11-10.17]; P = 0.032), a high double product at inclusion (OR [95% IC]: 1.20 [1.00-1.45] per 10(3) mmHg · min; P = 0.047), and at day 1, a decrease in cardiac index (1.30 [1.08-1.56] per 0.5 L/min/m(2); P = 0.007) or cardiac power index (1.84 [1.18-2.87] per 0.1 W/m(2), P = 0.008). CONCLUSION: In the first 24 hours, parameters related to cardiac reserve, such as double product and cardiac index evolution, provide crucial and easy to achieve hemodynamic physiological information, which may impact the outcome.

von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function.

The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF-dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF-dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF-dependent platelet activation was compared to their ability to inhibit thrombin-dependent activation. The results demonstrate that VWF-dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin-sensitive phosphatidylinositide 3-kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin-dependent platelet activation. In addition, thrombin-dependent but not VWF-dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin- and VWF-dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin-dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF-dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF-dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.

P38 mitogen-activated protein kinase dephosphorylation is regulated by protein phosphatase 2A in human platelets activated by collagen.

Collagen and the cross-linked collagen-related peptide (CRP-XL) each induced platelet p38 mitogen-activated protein kinase (p38) phosphorylation after 2 min. Subsequent dephosphorylation occurred in platelets activated with collagen, but not with CRP-XL, demonstrating glycoprotein VI-independent regulation of p38. Okadaic acid and fostriecin, inhibitors specific for protein phosphatase 2A (PP2A), blocked p38 dephosphorylation, and PP2A co-immunoprecipitated with phospho-p38. In addition, use of phenylarsine oxide suggested that tyrosine phosphatases and PP2A may act in concert to dephosphorylate p38. Finally, regulation of p38 in collagen-stimulated Glanzmann's platelets was indistinguishable from that in normal platelets, showing that p38 regulation is independent of integrin alphaIIbbeta3.

Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.

The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5'-[gamma-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was not affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1(667-1004)). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.

Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++.

An increase in the activity of membrane-associated protein phosphatase type 1 (mb-PP1) is associated with stimulation of erythrocyte K-Cl cotransport (KCC). We have recently proposed that membrane-associated protein phosphatase type 2A (mb-PP2A) is also involved in KCC regulation by cell swelling (Bize et al., 1999. Am. J. Physiol. 277:C899-C912). We used two protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CalA), and two KCC activating treatments, n-ethylmaleimide (NEM) and Mg(i)++-depletion, and determined KCC transport activity and mb-PP1 and mb-PP2A activities. OA, an inhibitor of erythrocyte mb-PP2A, partially prevents stimulation of KCC activity by NEM but not by Mg(i)++-depletion. CalA, an inhibitor of both mb-PP1 and mb-PP2A prevents stimulation of KCC activity by both treatments. NEM and Mg(i)++-depletion inhibit mb-PP1 activity, suggesting that activation of KCC can take place in the absence of mb-PP1 activation. Mb-PP2A activity is stimulated in NEM-treated cells but not in Mg(i)++-depleted cells. In NEM-treated cells, Mg(i)++-depletion inhibits both KCC and mb-PP2A. In Mg(i)++-depleted cells, NEM does not stimulate KCC or mb-PP2A. The strong correlation between KCC stimulation and mb-PP2A stimulation provides further support to the idea that mb-PP2A plays an important role in KCC regulation. Our results are consistent with the hypothesis that KCC regulation involves at least two distinguishable phosphorylation sites.

Agonist-induced regulation of myosin phosphatase activity in human platelets through activation of Rho-kinase.

Human platelets contained about 15 times lower amounts of Rho-kinase than Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Anti-myosin-binding subunit (MBS) antibody coimmunoprecipitated Rho-kinase of human platelets, and addition of GTPgammaS-RhoA stimulated phosphorylation of the 130-kD MBS of myosin phosphatase and consequently inactivated myosin phosphatase. Two kinds of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both GTPgammaS-RhoA-dependent MBS phosphorylation and inactivation of the phosphatase activity. Activation of human platelets with thrombin, a stable thromboxane A2 analog STA2, epinephrine, and serotonin resulted in an increase in MBS phosphorylation, and the agonist-induced MBS phosphorylation was prevented by pretreatment with the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS phosphorylation in platelets stimulated with these agonists. These compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas these compounds did not affect MLC phosphorylation or ATP secretion when platelets were stimulated with more than 0.1 U/mL thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet activation induced by various agonists. These results also suggest that Rho-kinase-mediated MLC phosphorylation contributes to a greater extent to the platelet secretion induced by relatively weak agonists.

Volume-sensitive transport systems and volume homeostasis in vertebrate red blood cells.

Animal cells regulate their volume in the short term by controlling solute movements into and out of the cell. A quite of dissipative transport systems are involved which allow either regulatory volume increase (RVI) or decrease (RVD) responses depending upon the direction of the electrochemical gradients of the solutes. Many of these transporters have been identified at the molecular level and structure-function studies have identified transmembrane transport domains and cytoplasmic regulatory domains. In vertebrate red blood cells, protein phosphorylation appears to be central to the coordinated regulation of transporter activity. Inhibitors of protein phosphatases (PPs) cause inhibition of the K+/Cl- cotransporter (a transporter mediating RVD), whilst some inhibitors of protein kinases (PKs) cause activation. A sequence of potential phosphorylation sites appears to constitute a cascade of reactions leading to transporter regulation. PP and PK inhibitors have opposite effects on transporters mediating RVI responses, which is consistent with the coordinated but reciprocal regulation of transporters activated during both RVI and RVD using some common phosphorylation reactions. The transporters are sensitive to other stimuli including, in red blood cells, changes in PO2 and pH. These responses are also sensitive to PK/PP inhibitors and may involve elements of the volume-sensitive transduction pathway.

Non-receptor protein tyrosine kinases and phosphatases in human platelets.

There is now a large and rapidly growing body of information on the different types of non-receptor tyrosine kinases and phosphatases present within platelets. These enzymes appear to play a critical role in co-ordinating, integrating and amplifying signals from multiple cell surface receptors. Despite considerable progress in this area of research over the last decade, a coherent understanding of how these enzymes fit into the complex communication networks of platelets remains elusive. The challenge ahead will be to define the molecular interactions and hierarchies between tyrosine kinases, phosphatases and other platelet signalling enzymes, and to pinpoint the key phosphorylation reactions required for the induction of specific platelet responses.

Adenosine A2 receptor occupancy regulates stimulated neutrophil function via activation of a serine/threonine protein phosphatase.

Adenosine modulates generation of superoxide anion by neutrophils via occupancy of specific adenosine A2A receptors. However, the intracellular signal transduction pathways by which occupancy of neutrophil adenosine A2A receptors inhibits superoxide anion generation (O2.-) are not well understood. We, therefore, tested the hypothesis that signaling at polymorphonuclear leukocyte (PMN) adenosine receptors proceeds via activation of a serine/threonine protein phosphatase (pp). Both the specific pp1 inhibitor calyculin A (10 nM) and the pp2A inhibitor okadaic acid (10 microM) enhanced O2.- generation (185 +/- 24 and 189 +/- 35% of control, respectively, p < 0.0001 for both, n = 8), as reported previously. Calyculin A, but not okadaic acid, completely reversed inhibition of stimulated O2.- generation by the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; IC50 = 30 nM; p < 0.0001, analysis of variance). Calyculin A also reversed the adenosine receptor-mediated desensitization of bound chemoattractant receptors in neutrophils. Treatment of PMNs with NECA increased the pp1 activity of crude membrane preparations in a time- and dose-dependent fashion (EC50 = 40 nM; p < 0.001, analysis of variance, n = 5). NECA inhibited cytosolic protein phosphatase activity by 78 +/- 12% (p < 0.003, n = 6) but did not shift pp1 catalytic subunit from cytosol to plasma membrane. Similar changes were observed in neutrophil cytoplasts depleted of organelles and nucleus. Moreover, the selective protein kinase A inhibitor KT5720 (10 microM) reversed the capacity of dibutyryl cAMP but not NECA to increase pp1 activity (p < 0.01, n = 5) in keeping with its effects on O2.- generation. Western blot analysis of PMN subcellular fractions demonstrated the presence of pp1alpha and pp1gamma1 but not pp1gamma2 isotypes in both cytosol and plasma membrane but not in azurophil or specific granules. We conclude from these studies that signal transduction by adenosine in PMN proceeds via a novel pathway: cAMP-independent activation of a serine/threonine protein phosphatase in the plasma membrane.

Recovery of blood mononuclear cell calcineurin activity segregates two populations of renal transplant patients with different sensitivities to cyclosporine inhibition.

In vitro studies have shown that the immunosuppressive property of cyclosporine (CsA) depends on its ability to inhibit the phosphatase activity of calcineurin, a critical enzyme for T cell activation. Here we sought to investigate whether measurement of calcineurin activity in peripheral blood mononuclear cells (PBMC) from 30 renal transplant patients given CsA as a part of their immunosuppressive regimen would help in optimizing CsA therapy. We first documented that in PBMC from these patients complete inhibition of calcineurin phosphatase activity by in vitro addition of CsA occurs at concentrations that are easily achieved in vivo for a dose as low as 3 mg/kg/day orally, which corresponds to trough CsA blood levels of 100-150 ng/ml. However, ex vivo, at a blood CsA trough level of 250 ng/ml, calcineurin activity in PBMC was only inhibited from 40% to 70% as compared with controls. Patients on higher doses of CsA had a further inhibition of baseline calcineurin activity, although a complete suppression was never reached. A significant correlation was found between trough CsA concentration and the basal calcineurin activity (r=0.48; P=0.0085). To clarify the relationship between the daily exposure of patients to CsA and changes in the enzyme activity of calcineurin, we then correlated the pharmacokinetic profile of CsA in these patients with different CsA dosing (<4, 4-6, >6-8, >8 mg/kg/day) with the profile of calcineurin activity at different intervals from dosing. Each of the above CsA doses suddenly reduced calcineurin activity, with a nadir at 2 hr after maximum blood concentration. The degree of the inhibition was not a function of peak CsA blood levels. In all patients, CsA blood level returned to basal values 10 hr after dosing. By contrast, only in 50-70% of patients (depending on the dose) did calcineurin activity return to baseline at the same time point after dosing. In summary we have shown that (1) inhibition of calcineurin activity measured ex vivo in PBMC taken from CsA-treated transplanted recipients reflects the blood CsA trough level; (2) after CsA the time-course of inhibition of enzyme activity is relatively independent from CsA pharmacokinetics; (3) the rate of recovery of calcineurin activity 10 hr after CsA dosing segregates two populations of transplanted recipients -- one with complete recovery of the enzyme activity and another that never returns to the baseline calcineurin level.

Phosphorylation of membrane proteins in erythrocytes treated with lead.

In immature rat microvessels, endothelial cells and glioma cells, exposure to lead results in an increase in the level of protein kinase C in membranes. In this paper we have extended these studies to human erythrocytes and, in addition, studied the phosphorylation of membrane proteins. A significant increase in the phosphorylation of membrane cytoskeletal proteins of molecular mass 120, 80, 52 and 45 kDa was observed in human erythrocytes treated for 60 min with lead acetate at concentrations greater than 100 nM. These same proteins were phosphorylated when erythrocytes were treated for 10 min with 50 nM phorbol 12-myristate 13-acetate (PMA). Similarly, protein kinase C activity was elevated and an increase in the amount of protein kinase C-alpha was observed in membranes from erythrocytes exposed to concentrations of lead acetate above 100 nM. No changes, however, in the activities of cAMP-dependent protein kinase, protein phosphatases I and IIA or casein kinase were observed. Phosphorylation of these membrane proteins stimulated by lead acetate or by PMA was not observed in erythrocytes depleted of protein kinase C by a 72-h treatment with 500 nM phorbol 12,13-dibutyrate. Finally, no changes in the levels of calcium or diacylglycerol were observed in erythrocytes stimulated with 100 nM lead acetate. These results indicate that, in erythrocytes, lead acetate stimulates the phosphorylation of membrane cytoskeletal proteins by a mechanism dependent on protein kinase C. Since levels of calcium or diacylglycerols did not increase, it appears that lead may activate the enzyme by a direct interaction.

Calcineurin protein phosphatase activity in peripheral blood lymphocytes.

The protein phosphatase activity of peripheral blood T lymphocytes (PBLs) was examined to quantify the contribution of calcineurin and other members of the family of serine/threonine protein phosphatases. Using selective phosphatase inhibitors, the fractional phosphatase activities of calcineurin, protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C) were determined. Okadaic acid was used to inhibit the activity of both PP1 and PP2A while cyclosporin A/cyclophilin or trifluoperazine were used as a specific inhibitors of the calmodulin-dependent phosphatase calcineurin. Using a [32P]labeled 19-residue phosphopeptide substrate, RII peptide, it was found that PP1 and PP2A comprise the majority of the total phosphatase activity in PBLs with okadaic acid inhibiting 80% of the phosphatase activity. The remaining 20% of the phosphatase activity can be attributed primarily to calcineurin since it was Ca2+ dependent, sensitive to inhibition by the calmodulin antagonist trifluoperazine, and inhibited by the complex of cyclosporin A (CsA) and cyclophilin. These results indicate that PBL extracts contain little PP2C activity. In addition, PBLs treated with CsA had measurably lower calcineurin activity in cell lysates. The measurement of calcineurin activity may provide a useful means of assessing the extent of immunosuppression during drug therapy.

Type 1 phosphatase inhibitors reduce the restoration of guanine nucleotide exchange activity of eukaryotic initiation factor 2B inhibited reticulocyte lysates rescued by hemin.

In heme-deficient reticulocyte lysates, the alpha-subunit of eukaryotic initiation factor-2 (eIF-2alpha) is phosphorylated due to the activation of the heme-regulated eIF-2alpha kinase (HRI). Phosphorylation of eIF-2alpha impairs the guanine nucleotide exchange activity of eIF-2B and thereby inhibits or shuts off protein synthesis. Delayed addition of hemin to shut-off lysates inhibits the eIF-2alpha kinase activity of HRI and restores protein synthesis; under those conditions, the endogenous phosphatase of the lysate dephosphorylates phosphorylated eIF-2alpha and restores eIF-2B activity. In this report we present evidence that the restoration of eIF-2B activity is dependent on the concentration of added hemin and is related to HRI activity in lysates. The recovery of eIF-2B activity is not affected by protein synthesis inhibitors such as cycloheximide, pactamycin and puromycin, which do not affect the eIF-2alpha phosphorylation. Also, the functional eIF-2B activity that is available in hemin-supplemented lysates is not affected by phosphatase inhibitors such as okadaic acid and heat-stable inhibitor-2. However, the recovery of eIF-2B activity that is observed by the delayed addition of hemin to inhibited heme-deficient lysates is reduced by inhibitor-2 and high concentrations of okadaic acid. These findings suggest that a type 1 phosphatase is involved in the recovery of eIF-2B activity and protein synthesis upon delayed addition of hemin to heme-deficient lysates.

Cyclosporine inhibition of calcineurin activity in human leukocytes in vivo is rapidly reversible.

Despite increasing information about the mechanism of action of cyclosporine A (CsA), little is known about the way lymphocytes recover from CsA. Recovery is central to understanding the pharmacodynamics of CsA in vivo. We studied the recovery of calcineurin phosphatase (CN) activity in CsA-treated cells. Single dose kinetics in renal transplant patients showed that inhibition of CN activity in PBL increased and fell concomitant with CsA blood vessels. In vitro, control PBL treated with CsA 100 micrograms/l, washed, and resuspended in CsA-free medium showed little recovery (0-20%) after 24 h. Erythrocytes or anti-CsA Ab added to the recovery medium increased recovery to 50% within 4 h. Similar recovery was seen in the ability of cells to produce IFN-gamma after OKT3 stimulation. Recovery of CN activity was associated with the efflux of [3H]CsA, was not blocked by cycloheximide and was temperature sensitive. A cell line with high expression of surface P glycoprotein (PGP), showed rapid recovery. However, PGP blockade did not prevent recovery in PBL, indicating a different PGP-independent mechanism. In PBL, recovery from CsA is slow and limited in vitro, but rapid in vivo, where CsA equilibrates among a complex set of extralymphocytic binding sites.

Calcineurin activity is only partially inhibited in leukocytes of cyclosporine-treated patients.

Measurement of the degree of immunosuppression induced clinically by drugs such as cyclosporine is an important but elusive goal. In lymphocytes in vitro, cyclosporine (CsA) blocks the phosphatase activity of the enzyme calcineurin, preventing cytokine induction. We sought to measure the degree of calcineurin blockade in patients on CsA. Calcineurin activity was measured in peripheral blood mononuclear cells (PBL) from stable CsA-treated renal transplant patients, compared with controls. Cytokine expression was assessed by challenging ex vivo PBL with calcium ionophore A23187 (5 microM) for 60 min and measuring interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) mRNA induction. In vitro, CsA inhibited both calcineurin activity and cytokine induction with an IC50 of 10-20 micrograms/L. In CsA-treated patients with therapeutic CsA levels (mean trough CsA blood level = 180 +/- 55 micrograms/L), calcineurin activity was detectable but reduced by 50% compared with controls (P < or = 0.001) and correlated with CsA trough levels (r = -0.390, P < or = 0.01). The induction of cytokine mRNA in such patients was not blocked, but was sensitive to CsA in vitro, suggesting that CsA is much less available in vivo in body fluids than it is for isolated cells in vitro. In lymphocytes of patients on CsA, calcineurin activity is reduced but 50% of the activity persists, permitting strong signals to trigger cytokine expression. Partial calcineurin inhibition may explain why the immune responsiveness of patients on CsA is reduced but still sufficient for host defense.