Increase In Basal Phosphorylation Research Articles

Sarcoplasmic reticulum Ca2 + cycling protein phosphorylation in a physiologic Ca2 + milieu unleashes a high-power, rhythmic Ca2 + clock in ventricular myocytes: Relevance to arrhythmias and bio-pacemaker design

Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2+], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.

Dysregulation in p38 signalling contributes to altered phenotype and function in Nrf2 deficient dendritic cells (P5025)

Abstract Nrf2 is a redox sensitive transcription factor vital for the maintenance of cellular homeostasis. Dendritic cells (DCs) are potent APCs responsible for the initiation of an immune response. Our previous study highlights the role of Nrf2 in DC function (increased costimulatory receptors expression and T cell stimulatory capacity) and intracellular signaling (such as ROS, NFκB and MAPKs). The p38 MAPK pathway plays a pivotal role in the up regulation of costimulatory receptors (CD86 & MHC II). However, it is unclear how Nrf2 affects the integrity of this pathway. In the current study using bone marrow-derived immature DCs from Nrf2 deficient (KO) and wild type mice, we investigate the effect of Nrf2 on various signaling events in the p38 pathway and associated functional responses. Our results indicate that the loss of Nrf2 in DCs resulted in elevated basal p38 phosphorylation. Furthermore, an increase in basal phosphorylation of CREB and ATF-1 - downstream targets of p38, was also observed. This was coupled with increased IL-10 cytokine production at basal conditions and also upon exposure to LPS. Increased costimulatory receptor expression, antigen specific T cell activation propensity and LPS stimulated IL-10 production observed in the KO DCs can be reversed by pharmacological inhibition of p38. These results suggest that deficiency of Nrf2 in DCs leads to dysregulation of the p38 signaling pathway contributing to the altered phenotype and function of these DCs.

Abstract 4736: Amplification of ERK2 mediates resistance to the novel irreversible EGFR inhibitor WZ4002

Abstract Background: EGFR T790M causes clinical resistance to the quinazoline EGFR kinase inhibitors gefitinib and erlotinib in non-small cell lung cancer. Amplification of EGFR T790M causes resistance to the irreversible quinazoline EGFR inhibitor PF00299804 (Ercan etl al. Oncogene 2010). A novel T790M selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002, is effective in NSCLC models harboring EGFR T790M (Zhou, W, Ercan, D et al., Nature 2009). The mechanism(s) of resistance to this class of irreversible EGFR inhibitors have not been explored. Methods and Results: We exposed the Gefitinib resistant PC9 GR cells (EGFR del E746_A750/T790M; IC50 to WZ4002: ∼30nM) to increasing concentrations of WZ4002 to generate PC9 GW resistant cells (IC50 to WZ4002 ∼10µM) and isolated several resistant clones. The PC9 GWR cells contain EGFR del E746_A750/T790M but do not harbor additional EGFR mutations and are cross resistant to the irreversible quinazoline EGFR inhibitor BIBW2992. The PC9 GWR cells demonstrate an increase in basal phosphorylation of ERK2 but not ERK1 as well as total protein compared to parental PC9 GR cells. WZ4002 effectively inhibits EGFR phosphorylation but not ERK 2 phosphorylation in the PC9 GWR cells whereas in the PC9 GR cells WZ4002 inhibits both EGFR and ERK 1/2 phosphorylation. Furthermore, unlike in the PC9 GR cells, EGFR inhibition does not lead to BIM induction or apoptosis in the PC9 GWR cells. Genomic analyses of the PC9 GWR compared with the PC9 GR cells, using single nucleotide polymorphism (SNP) arrays, identify a focal amplification in the PC9 GWR cells on chromosome 22 which includes ERK2 (MAP2K). Using fluorescence in situ hybridization (FISH), we confirm amplified intra-chromosomal regions mapping to MAP2K. Inhibition of ERK signaling by either an ERK2 specific shRNA or by the MEK inhibitor CI-1040, restores sensitivity to WZ4002 and BIBW2992. In addition, combination of WZ4002 and CI-1040 results in BIM induction and apoptosis in the PC9 GWR cells. In contrast inhibition of PI3K signaling using the PI3K inhibitor PI-103 does not restore WZ4002 sensitivity. Ectopic activation of ERK signaling using an activated MEK1 allele (MEK K57N) found in lung cancer, in different lung cancer cell lines (PC9 (EGFR del E746_A750), PC9 GR (EGFR del E746_A750), and H1975 (L858R/T790M), is sufficient to cause resistance to WZ4002 and block WZ4002 mediated apoptosis. Conclusions: We identify amplification of ERK2, a component downstream component of EGFR signaling, as a mechanism of resistance to structurally distinct irreversible EGFR kinase inhibitors. The combination of an EGFR inhibitor and a MEK inhibitor is effective in against these drug resistant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2011-4736

Compound K produces cardiac protection by activating Akt phosphorylation

Ginseng root is one of the most popular herbs throughout the world and it has been used as a medicine to enhance stamina and capacity to cope with fatigue and physical stress. In this study, we focused the cardiac protective effects of Compound K (C‐K; 20‐O‐D‐glucopyranosyl‐20(S)‐protopanaxadiol), which is a protopanaxadiol metabolite derived from ginsenoside Rb1, Rb2, and Rc by the metabolic actions of intestinal bacteria.Mice were exposed to 30 min ischemia followed by 120 min of reperfusion (control). Some mice were assigned to C‐K, or PI3K inhibitor, wortmannin. C‐K preconditioned significantly reduced infarct size compared to control group (22.4 ± 2.5% [n = 7] vs. 43.2 ± 2.0% [n = 12] respectively, p<0.05) and wortmannin eliminated the protection produced by C‐K (43.3 ± 2.4% [n = 7]). Additionally, the hearts treated with C‐K had a 2‐fold increase in basal phosphorylation of Akt and pretreated with wortmannin completely abolished Akt phosphorylation in response to C‐K treatment.These results reveal that C‐K produces cardiac protection in mice in association with PI3K/Akt signaling.

Abstract 412: Cardiac-Specific Overexpression of Caveolin-3 Induces Endogenous Cardiac Protection

Caveolae are lipid-rich microdomains of the plasma membrane that localize and enrich cardiac protective signaling molecules. Caveolin-3 (Cav3), the dominant caveolin isoform found in cardiac myocytes, is a determinant of caveolae formation. We hypothesized that cardiac-directed overexpression of Cav3 would enhance the formation of caveolae and augment protective signaling of the heart in vivo . Full-length cDNA for mouse Cav3 was cloned into a vector containing the α-myosin heavy chain promoter to allow for cardiac myocyte-specific expression of Cav3. A transgenic (Tg) mouse line was derived with a 5.5-fold increase in Cav3 mRNA and a 2.4-fold increase in Cav3 protein that localized to the sarcolemma. No significant increase in Cav3 expression was observed in lung, liver, brain, or skeletal muscle from Tg-positive mice. Electron microscopy revealed enhanced formation of caveolae on the sarcolemmal membranes of Tg-positive mice compared to Tg-negative littermate controls and no apparent structural defects in the heart. Echocardiography of 7–9 month old Tg-positive and Tg-negative mice showed no significant differences in cardiac function. Tg-positive mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to Tg-negative mice (23.9 ± 3.3 vs. 44.4 ± 5.9 % risk area, p<0.01). The endogenous protection induced in Cav-3 Tg-positive mice was similar to that produced in wild-type mice undergoing ischemic preconditioning (27.2 ± 2.5 % risk area). Hearts excised from Tg-positive mice had a 1.5-fold increase in basal phosphorylation of Akt (p<0.05, n = 6 Tg positive, 4 Tg-negative). Cardiac protection in Cav3 Tg-positive mice was blocked by treatment prior to ischemia reperfusion with 5-hydroxydecanoate, a mitochondrial KATP channel blocker. These results reveal that cardiac-specific overexpression of Cav3 produces an endogenous protected phenotype in mice in association with greater basal Akt activation and a phenotype that is sensitive to antagonism via mitochondrial KATP channel inhibition. The findings suggest that approaches to increase Cav3 expression may provide a novel means to augment cardiac protection independent of ischemic preconditioning.

Cardiac‐Specific Overexpression of Caveolin‐3 Enhances Akt Phosphorylation

Caveolin-3 (Cav3), a membrane protein that helps define lipid raft/caveolar domains, is a potent activator of PI3-K/Akt signaling, an important pathway in cardiac protection. We hypothesized that cardiac-directed overexpression of Cav3 augments PI3-K/Akt cardiac protective signaling in vivo. Mouse Cav3 was cloned into a vector containing the α-myosin heavy chain promoter to allow for cardiac myocyte-specific expression of Cav3. A transgenic (Tg) mouse line was derived with a 5.5-fold increase in Cav3 mRNA and a 2.4-fold increase in Cav3 protein that localized specifically to the sarcolemma. No significant increase in Cav3 expression was observed in lung, liver, brain, or skeletal muscle from Tg-positive mice. Electron microscopy revealed enhanced formation of caveolae on the sarcolemmal membranes of Tg-positive mice compared to Tg-negative littermate controls and no apparent structural defects in the heart. Echocardiography of 7–9 month old Tg-positive and Tg-negative mice showed no significant differences in cardiac function. Hearts excised from Tg-positive mice had a 1.5-fold increase in basal phosphorylation of Akt (p<0.05, n=6 TG positive, 4 Tg-negative). Treatment with isoflurane (30 min, 1.4%), an agent that produces cardiac protection and activates Akt signaling, produced a 2.5-fold greater increase in phosphorylation of Akt in Tg-positive vs. Tg-negative animals (n=3/group). These results reveal that cardiac-specific overexpression of Cav3 produces a phenotype in mice with greater basal and stimulated PI3-K/Akt signaling, thus suggesting that approaches to increase Cav3 expression may provide a novel means to augment cardiac protection and perhaps other effects mediated by PI3-K/Akt.

Deletion of synapsins I and II genes alters the size of vesicular pools and rabphilin phosphorylation

Previous studies established that genetic deletion of synapsins, synaptic vesicle-associated phosphoproteins that regulate neurotransmitter release, decreases the number of synaptic vesicles in nerve terminals. To investigate whether these changes affect the release properties of the remaining synaptic vesicles, we used a radioactive labeling technique to measure release independently of the total number of synaptic vesicles. 3H-glutamate and 14C-γ-amino-butyric-acid (GABA) release from isolated nerve terminals prepared from the neocortex of synapsins I and II double knock-out mice (DKO) was assayed and compared to wild-type preparations. Hyperosmotic shock-evoked 3H-glutamate was reduced by 20 ± 3% from DKO nerve terminals and potassium depolarization-evoked glutamate release was also decreased by 28 ± 2%. Surprisingly, sucrose or potassium depolarization-evoked release of 14C-GABA was increased by 32 ± 4% and 29 ± 5%, respectively. The basal efflux of both 3H-glutamate and 14C-GABA increased by 17 ± 2% and 12 ± 2% from DKO nerve terminals. As lack of synapsins I and II, major phosphoproteins of synaptic vesicles, may lead to deregulation of phosphorylation events, we compared phosphorylation state of another synaptic vesicle protein, rabphilin. In DKO nerve terminals, membrane-associated rabphilin level was reduced by ∼0.28-fold, its phosphorylation at 234serine was increased by ∼1.61-fold whereas cytosolic rabphilin levels showed both more dramatic reduction in abundance, ∼16.5-fold, and increase in phosphorylation, ∼4.8-fold. Collectively, these data suggest that deletion of major synapsin isoforms leads to (1) deregulation of basal neurotransmission causing “leaky” basal release, (2) changes in either the size or mobilization of releasable or reserve pools, and (3) a decrease in rabphilin abundance accompanied by an increase in basal phosphorylation of the remaining rabphilin.

The role of STAT-3 in the mediation of smooth muscle cell response to cyclic strain

Hemodynamic forces, including shear stress and cyclic strain, have been recognised as important modulators of vascular cell morphology and function. However, the mechanism by which vascular cells sense and transduce the extracellular mechanical signals into the cell nucleus has not yet been clarified. The purpose of our study was to assess the involvement of the signal transducer and activator of transcription-3 (STAT-3) in the signaling pathway mediating the response of vascular smooth muscle cells (SMC) to cyclic strain. Embryonic A7r5 SMC derived from thoracic aortas of DB1X rats were seeded on flexible collagen I-coated plates. Cells were subjected to 10% average strain at 60 cycles/min for various time periods. Activation of STAT-3, p38, extracellular signal-regulated kinase (ERK) 1/2 and Src was assessed by immunoblotting using phosphospecific antibodies. The interactions between STAT-3 phosphorylation and p38, ERK1/2, phosphatidylinositol-3 (PI3K), mammalian target of rapamycin (mTOR), Janus kinase (JAK) 2 and Src were evaluated by pretreating the cells with specific inhibitors including SB202190, PD98059, LY294002, wortmannin, rapamycin, AG490 and PP1. Serine phosphorylation of STAT-3 was increased by 2-fold after 15 min of cyclic strain, while tyrosine phosphorylation was increased by 2.3-fold after 60 min. Inhibition of ERK1/2 by PD98059 prevented serine phosphorylation of STAT-3, whereas inhibition of Src by PP1 prevented STAT-3 tyrosine phosphorylation. Pretreating the cells with SB202190, a specific inhibitor of p38, resulted in an increase in basal phosphorylation of ERK1/2 and a subsequent increase in basal serine phosphorylation of STAT-3. In conclusion, both serine and tyrosine phosphorylation of STAT-3 are involved in the signaling pathway mediating the effects of cyclic strain on vascular SMC. Serine phosphorylation of STAT-3 is mediated by ERK1/2, while tyrosine phosphorylation is mediated by Src. A negative feedback loop was also found between p38 and ERK1/2.

Reconstitution of Regulated Phosphorylation of FcϵRI by a Lipid Raft-excluded Protein-tyrosine Phosphatase

To examine the exquisite regulation of IgE-FcepsilonRI tyrosine phosphorylation by Lyn kinase that is stimulated by antigen-mediated cross-linking, we utilized co-expression of FcepsilonRI and Lyn in Chinese hamster ovary cells, which results in high basal levels of Lyn kinase activity and spontaneous phosphorylation of FcepsilonRI. We found that co-expression of a lipid raft-excluded transmembrane tyrosine phosphatase, PTPalpha, suppresses Lyn kinase activity and markedly reduces the level of spontaneous phosphorylation of FcepsilonRI, while facilitating its antigen-stimulated phosphorylation. Other tyrosine phosphatases, including SHP-1, CD45, and a lipid raft-preferring chimeric version of PTPalpha fail to reconstitute antigen-dependent FcepsilonRI phosphorylation. We concluded that both substrate specificity and submembrane location are critical to phosphatase-mediated regulation of Lyn kinase activity that supports activation of FcepsilonRI.

Ribosomal p70S6K basal activity increases upon induction of differentiation of myelomonocytic leukemic cell lines HL60, AML14 and MPD

The role of ribosomal p70S6K in the cell cycle has been studied extensively, and it is known that this enzyme is crucial for cell advancement through G 1. Conversely, the participation of p70S6K in cell differentiation is not well understood. We have studied the response of p70S6K to the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in three differentiation-capable leukemic cell lines (MPD, AML-14 and HL-60) and in normal mature neutrophils. Immature leukemic cells starved for 16 h showed a robust (∼3.5-fold over controls) p70S6K phosphorylation on T 421/S 424 residues in response to an acute (5 min) 10 nM GM-CSF stimulation. On the other hand, cells that had been induced to differentiate and express granulocytic phenotypes, showed an increased (∼6-fold) basal level of p70S6K T 421/S 424 phosphorylation over immature cells, as well as an increased baseline tyrosyl phosphorylation of the GM-CSF receptor β subunit (GM-CSF·Rβ). However, the differentiated cells displayed a weak (∼1.4-fold over controls) response to GM-CSF even at prolonged incubation times (20 min). In vitro p70S6K enzymatic activity paralleled p70S6K T 421/S 424 phosphorylation in both high basal, unstimulated, levels in immature cells and a low degree of response to GM-CSF. Lastly, peripheral blood mature neutrophils had low basal GM-CSF·Rβ and p70S6K activity, with both parameters being robustly stimulated following addition of GM-CSF, a situation in contrast with the cell lines, indicative perhaps of their incomplete terminal differentiation. In summary, these findings show the increase in basal phosphorylation of p70S6K upon granulocytic differentiation of myeloid leukemic cells and their responses to GM-CSF that are closely paralleled with tyrosyl phosphorylation of its receptor, and help in pointing to specific cell signaling molecules that are different in leukemic blasts from normal leukocytes.

Effect of Activating and Inactivating Mutations on the Phosphorylation and Trafficking of the Human Lutropin/Choriogonadotropin Receptor

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.

Agonist-induced Phosphorylation of the Angiotensin II (AT1A) Receptor Requires Generation of a Conformation That Is Distinct from the Inositol Phosphate-signaling State

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT(1A)) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar(1),Ile(4),Ile(8)]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT(1A) receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT(1A) receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT(1) receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar(1),Ile(4),Ile(8)]AngII. Paradoxically, [Sar(1),Ile(4), Ile(8)]AngII produced a robust ( approximately 85% of AngII), dose-dependent phosphorylation of the wild type AT(1A) receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar(1),Ile(4),Ile(8)]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT(1A) receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.

Phosphorylation of the inositol trisphosphate receptor in isolated rat hepatocytes.

The inositol trisphosphate receptor (IP3R) in brain has been shown to be a substrate for several different protein kinases in vitro. We have studied the phosphorylation of the IP3R in intact cells by using isolated hepatocytes and an antibody to immunoprecipitate the receptor protein from detergent extracts. Stimulation of 32P-labeled hepatocytes with glucagon or N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP) markedly increased phosphorylation of the IP3R. However, no increase was observed in response to angiotensin II, vasopressin, 12-O-tetradecanoyl-phorbol-13-acetate, or epidermal growth factor. The kinetics of phosphorylation in response to glucagon was both rapid and transient. In agreement with previous studies, physiological concentrations of Ca2+ stimulated D-myo-inositol 1,4,5-trisphosphate (IP3) binding to permeabilized hepatocytes (Pietri, F., Hilly, M., and Mauger, J.-P. (1990) J. Biol. Chem. 265, 17478-17485). Pretreatment of cells with db-cAMP had no effect on binding in the absence of added Ca2+ but enhanced binding measured in the presence of basal low concentrations (0.16-0.25 microM) of Ca2+ and decreased the concentration of Ca2+ required for half-maximal stimulation. The effect of db-cAMP was associated with an increase in affinity of the IP3 binding site without a change in maximum number of binding sites. Preincubation of intact hepatocytes with okadaic acid alone produced an increase in basal phosphorylation of the IP3R, and maximal phosphorylation of the receptor was observed in the presence of both okadaic acid and db-cAMP. However, okadaic acid blocked the effect of db-cAMP and inhibited the effect of Ca2+ on IP3 binding. Detergent-solubilized binding sites were already fully activated and insensitive to modulation by Ca2+ or cAMP-dependent protein kinase. It is proposed that the receptor in native membranes is inhibited and that Ca2+ and cAMP-dependent protein kinase may act to relieve this inhibition.