Increased Protein Phosphatase 2A Activity Research Articles

Loss Of Calreticulin Function Decreases NFKB Activity By Stabilizing IKB Protein

Background: Transcription factor NFKB is activated by several processes including inflammation, endoplasmic-reticulum (ER) stress, increased Akt signaling and enhanced proteasomal degradation. Calreticulin is an ER Ca2+ binding chaperone, which regulates many cellular processes. Previously, we have shown that loss of calreticulin function results in the activation of ER stress that is accompanied by a significant increase in the proteasome activity. These changes increase the resistance of calreticulin deficient cells to apoptosis. A role for calreticulin has also been described in the regulation of immune response. Objectives: To examine the role of calreticulin in the activation of NFKB signaling leading to enhanced resistance to apoptosis of these cells. Methods:: Wild type and calreticulin deficient cells were used for measurement of transcriptional activity of NFKB. Cells were co-transfected with of NFKB reporter and-gal reporter plasmids followed by reporter gene assays. Western blot analysis was utilized to examine changes in protein expression. Results: Our data illustrate a significant decrease in the basal transcriptional activity of NFKB upon loss of calreticulin function. Furthermore, treatment with lipopolysaccharide increased the transcriptional activity of NFKB in both the wild type and calreticulin deficient cells. However, the transcriptional activity of NFKB was still significantly lower in the calreticulin deficient cells as compared to the wild type cells. Our data also showed that the reduced NFKB activity in calreticulin deficient cells is not due to decreased p65 or p50 protein levels. To determine the mechanism of decreased NFKB activity we examined changes in IKB protein stability. Our data showed a significant increase in the IKB protein level due to decreased level of phosphorylated IKB protein. Furthermore, we illustrated that loss of calreticulin function resulted in increased protein phosphatase2A activity that was abolished by Okadaic acid treatment. Inhibition of IKB de-phosphorylation decreased its ubiquitination and proteasomal degradation. Conclusion: Our data suggests that the reduced transcriptional activity of NFKB upon loss of calreticulin function is mediated via stabilization of IKB protein. To our knowledge, this is the first report on the role of calreticulin in the regulation of NFKB function.

Bortezomib Sensitizes HCC Cells to CS-1008, an Antihuman Death Receptor 5 Antibody, through the Inhibition of CIP2A

Previously, we have shown that bortezomib overcame TRAIL resistance in hepatocellular carcinoma (HCC) cells via the inhibition of Akt. Here, we report that bortezomib sensitizes these TRAIL-resistant cells, including Huh-7, Hep3B, and Sk-Hep1, to CS-1008, a humanized agonistic antihuman death receptor 5 antibody. Cancerous inhibitor of protein phosphatase 2A (CIP2A) mediated the sensitizing effect of bortezomib to CS-1008 through inhibiting protein phosphatase 2A (PP2A) activity. Combination treatment of bortezomib and CS-1008 downregulated CIP2A in a concentration- and time-dependent manner, and increased PP2A activity in HCC cells. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells. Moreover, silencing CIP2A by short interfering RNA enhanced CS-1008-induced apoptosis in HCC cells and ectopic expression of CIP2A in HCC cells abolished CS-1008-induced apoptosis, indicating that CIP2A plays an important role in the sensitizing effect of bortezomib to CS-1008. Finally, our in vivo data showed that CS-1008 and bortezomib combination treatment decreased tumor growth significantly. In conclusion, bortezomib sensitized HCC cells to CS-1008 through the inhibition of CIP2A.

Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKCδ) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKCδ negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKCδ–PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3–10 μM) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKCδ specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1–1 μM Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKCδ kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKCδ inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKCδ and PP2A activity.

Gene regulation mediating fiber-type transformation in skeletal muscle cells is partly glucose- and ChREBP-dependent

Adaptations in the oxidative capacity of skeletal muscle cells can occur under several physiological or pathological conditions. We investigated the effect of increasing extracellular glucose concentration on the expression of markers of energy metabolism in primary skeletal muscle cells and the C2C12 muscle cell line. Growth of myotubes in 25 mM glucose (high glucose, HG) compared with 5.55 mM led to increases in the expression and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a marker of glycolytic energy metabolism, while oxidative markers peroxisome proliferator-activated receptor γ coactivator 1α and citrate synthase decreased. HG induced metabolic adaptations as are seen during a slow-to-fast fiber transformation. Furthermore, HG increased fast myosin heavy chain (MHC) IId/x but did not change slow MHCI/β expression. Protein phosphatase 2A (PP2A) was shown to mediate the effects of HG on GAPDH and MHCIId/x. Carbohydrate response element-binding protein (ChREBP), a glucose-dependent transcription factor downstream of PP2A, partially mediated the effects of glucose on metabolic markers. The glucose-induced increase in PP2A activity was associated with an increase in p38 mitogen-activated protein kinase activity, which presumably mediates the increase in MHCIId/x promoter activity. Liver X receptor, another possible mediator of glucose effects, induced only an incomplete metabolic shift, mainly increasing the expression of the glycolytic marker. Taken together, HG induces a partial slow-to-fast transformation comprising metabolic enzymes together with an increased expression of MHCIId/x. This work demonstrates a functional role for ChREBP in determining the metabolic type of muscle fibers and highlights the importance of glucose as a signaling molecule in muscle.

CIP2A mediates effects of bortezomib on phospho-Akt and apoptosis in hepatocellular carcinoma cells

Previously, we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. In this study, we report that cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediates the apoptotic effect of bortezomib in HCC. Silencing PP2A by small interference RNA (siRNA) abolishes bortezomib-induced down-regulation of phospho-Akt and apoptosis. Bortezomib increases PP2A activity in sensitive HCC cells, including Sk-Hep1, Hep3B and Huh-7, but not in resistant PLC5 cells. Bortezomib down-regulates CIP2A in a dose- and time-dependent manner in all sensitive HCC cells, whereas no alterations in CIP2A were found in resistant PLC5 cells. Knockdown of CIP2A by siRNA restored bortezomib's effects on apoptosis and PP2A activity in PLC5 cells. Moreover, over-expression of CIP2A up-regulated phospho-Akt and protected Sk-Hep1 cells from bortezomib-induced apoptosis. It is significant that, ectopic expression of CIP2A decreased Akt-related PP2A activity, whereas silencing CIP2A increased this activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells, furthermore, our in vivo data showed that bortezomib down-regulates CIP2A and up-regulates PP2A activity in Huh-7 tumors, but not in PLC5 tumors. In conclusion, inhibition of CIP2A determines the effects of bortezomib on apoptosis and PP2A-dependent Akt inactivation in HCC.

Abstract 355: CIP2A mediates effects of bortezomib on phospho-Akt and apoptosis in hepatocellular carcinoma cells

Abstract Previously, we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib (Chen et al, Cancer Res 2008). Here, we report that cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediates the apoptotic effect of bortezomib in HCC. Silencing PP2A by small interference RNA (siRNA) abolishes bortezomib-induced down-regulation of phospho-Akt and apoptosis. Bortezomib increases PP2A activity in sensitive HCC cells, including Sk-Hep1, Hep3B and Huh-7 but not in resistant PLC5 cells. Bortezomib down-regulates CIP2A in a dose- and time-dependent manner in all sensitive HCC cells whereas no alterations in CIP2A were found in resistant PLC5 cells. Knockdown of CIP2A by siRNA restored bortezomib's effects on apoptosis and PP2A activity in PLC5 cells. Moreover, over-expression of CIP2A up-regulated phospho-Akt and protected Sk-Hep1 cells from bortezomib-induced apoptosis. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity whereas silencing CIP2A increased this activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells, Furthermore, our in vivo data showed that bortezomib down-regulates CIP2A and up-regulates PP2A activity in Huh-7 tumors but not in PLC5 tumors. In conclusion, inhibition of CIP2A determines effects of bortezomib on apoptosis and PP2A-dependent Akt inactivation in HCC. CIP2A may be a biomarker for predicting clinical response of bortezomib in HCC treatment. Supported by NTUH98P03, NTUH98FTN21, NSC97-2314-B-002-182, NSC98-2314-B-002-067-MY3, and NSC98-3112-B-002-037. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 355.

PP2A attenuates cardiac response to LPS‐induced sepsis

Lipopolysaccharide (LPS), a cell membrane component from Gram‐negative bacteria, is involved in sepsis‐associated cardiac dysfunction. We wanted to know if protein phosphatase 2A (PP2A) influences LPS‐signaling in the heart. Therefore, we used transgenic (TG) mice with heart‐specific overexpression of PP2A. TG and littermate wild type (WT) mice were intraperitoneally injected with 30 μg LPS per g body weight or with NaCl as control (n = 14 – 18 per group). After 3 days, successful induction of LPS‐induced sepsis was monitored by enhanced plasma levels of interleukin‐6. Contractility of WT and TG hearts was analyzed using the isolated work performing heart preparation. Left ventricular systolic pressure (LVP) and the first derivative of developed pressure (+dP/dt and −dP/dt) were decreased in hearts of LPS‐treated WT mice, whereas hearts of TG mice showed no decrease of contractility after LPS‐treatment (Table). Contractile parameters Control LPS WT TG WT TG LVP (mmHg) 74±2.2 66±4.3 50±4.3* 61±1.9# +dP/dt (mmHg/s) 2481±186 2145±254 1306±246* 1901±171# −dP/dt (mmHg/s) −1371±114 −1073±139 −720±135* −1006±115 p<0.05 vs. control, p<0.05 vs. WT Quantitative PCR revealed that LPS increased IL‐6 mRNA expression in TG and WT hearts but TNFα mRNA levels only were enhanced in WT. In summary, LPS‐induced myocardial dysfunction was attenuated in TG mice compared to WT. It is tempting to speculate that increased PP2A activity in TG hearts is protective against LPS‐induced sepsis probably via diminished TNFα expression. (Supported by the DFG)

CAMP-stimulated Protein Phosphatase 2A Activity Associated with Muscle A Kinase-anchoring Protein (mAKAP) Signaling Complexes Inhibits the Phosphorylation and Activity of the cAMP-specific Phosphodiesterase PDE4D3

The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085-2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56delta. Like PDE4D3, B56delta is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56delta enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56delta mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56delta phosphorylation. In general, PKA.PP2A.mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling.

Progesterone decreases tyrosine hydroxylase phosphorylation state and increases protein phosphatase 2A activity in the stalk-median eminence on proestrous afternoon

The progesterone (P(4)) rise on proestrous afternoon is associated with dephosphorylation of tyrosine hydroxylase (TH) and reduced TH activity in the stalk-median eminence (SME), which contributes to the proestrous prolactin surge in rats. In the present study, we investigated the time course for P(4) effect on TH activity and phosphorylation state, as well as cAMP levels and protein phosphatase 2A (PP2A) activity and quantity, in the SME on proestrous morning and afternoon. P(4) (7.5 mg/kg, s.c.) treatment on proestrous afternoon decreased TH activity and TH phosphorylation state at Ser-31 and Ser-40 within 1 h, whereas morning administration of P(4) had no 1 h effect on TH. PP2A activity in the SME was enhanced after P(4) treatment for 1 h on proestrous afternoon without a change in PP2A catalytic subunit quantity, whereas P(4) treatment had no effect on PP2A activity or quantity on proestrous morning. cAMP levels in the SME were unchanged with 1 h P(4) treatment. At 5 h after P(4) treatment, TH activity and phosphorylation state declined coincident with an increase in plasma prolactin in both P(4)-treated morning and afternoon groups. PP2A activity in the SME was unchanged in 5 h P(4)-treated rat. Our data suggest that P(4) action on tuberoinfundibular dopaminergic (TIDA) neurons involves at least two components. A more rapid (1 h) P(4) effect engaged only on proestrous afternoon likely involves the activation of PP2A. The longer P(4) action on TIDA neurons is evident on both the morning and afternoon of proestrus and may involve a common, as yet unidentified, mechanism.

Synergistic interactions between sorafenib and bortezomib in hepatocellular carcinoma involve PP2A-dependent Akt inactivation

Previously we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. Here we report that combined treatment with sorafenib and bortezomib shows synergistic effects in HCC. HCC cell lines (PLC/PRF/5, Huh-7, and Hep3B) were treated with sorafenib and/or bortezomib and analyzed in terms of apoptosis signal transduction. In vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. Pretreatment with sorafenib enhanced bortezomib-induced apoptotic cell death by restoring bortezomib's ability to inactivate Akt in PLC/PRF/5 cells. Knocking down Akt1 by RNA-interference overcame apoptotic resistance to bortezomib in PLC/PRF/5 cells and ectopic expression of active Akt in HCC cells abolished the bortezomib sensitizing effect of sorafenib, indicating Akt inactivation plays a key role in mediating the combinational effects. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of phospho-Akt (P-Akt) expression induced by co-treatment with sorafenib and bortezomib, and 1, 9 di-deoxy-forskolin, a PP2A agonist, restored bortezomib's effect on P-Akt and apoptosis. Importantly, silencing of PP2A by RNA-interference reduced the apoptotic effect induced by sorafenib-bortezomib co-treatment, indicating that PP2A is indispensable for mediating the effects of these drugs. Notably, sorafenib with bortezomib increased PP2A activity in PLC/PRF/5 cells without altering protein levels of PP2A complex or the interaction between PP2A and Akt proteins. Finally, sorafenib plus bortezomib significantly suppressed PLC/PRF/5 xenograft tumor growth, down-regulated P-Akt expression, and up-regulated PP2A activity. The combination of sorafenib and bortezomib shows synergy in HCC through PP2A-dependent Akt inactivation.

Protein phosphatase 2A contributes to the cardiac dysfunction induced by endotoxemia

AimsSepsis-associated cardiac dysfunction represents an intrinsic impairment of cardiomyocyte function due in part to a decrease in myofilament Ca2+ sensitivity associated with a sustained increase in cardiac troponin I (cTnI) phosphorylation at Ser23/24. Dephosphorylation of cTnI is under regulatory control. Thus, muscarinic and adenosine A1-receptor agonists antagonize β-adrenergic stimulation via activation of protein phosphatase 2A (PP2A). The aim of this study was to determine whether modulation of PP2A and thus cTnI phosphorylation could improve sepsis-induced contractile dysfunction.Methods and resultsCardiomyocytes were isolated from control or septic mice 16–18 h after an injection of vehicle or lipopolysaccharide (LPS; 9 mg/kg ip) respectively. Protein expression and phosphatase activity were determined in homogenates of control and septic hearts. Our data showed that LPS significantly increased cTnI phosphorylation at Ser23/24 in cardiomyocytes and reduced contraction amplitude without affecting Ca2+-transients. Treatment of cardiomyocytes with the A1 agonist cyclopentyladenosine (CPA) or the protein kinase A inhibitor H89 significantly attenuated the LPS-induced contractile dysfunction without effect on Ca2+-transients. Co-treatment with CPA and H89 completely reversed the contractile dysfunction. Increased cTnI phosphorylation in septic hearts was associated with a significant reduction in the protein expression of both the catalytic and regulatory subunits (B56α) of PP2A and a decrease in PP2A activity. CPA treatment of septic hearts increased PP2A activity. An increase in the protein expression of demethylated PP2A and a decrease in the PP2A-methyltransferase (PPMT; the methyltransferase that catalyses this reaction) were also observed.ConclusionThese data support the hypothesis that sustained cTnI phosphorylation underlies the contractile dysfunction seen in sepsis.

Protein Phosphatase 2A and Rapamycin Regulate the Nuclear Localization and Activity of the Transcription Factor GLI3

Gain-of-function alterations to the sonic hedgehog (SHH) signaling cascade have been found in a wide range of tumors. Three SHH effectors, GLI1, GLI2, and GLI3, regulate transcription of diverse genes involved in cell growth and cell proliferation. Here, we show that protein phosphatase 2A (PP2A), its regulatory subunit, alpha4, and rapamycin, an inhibitor of the mammalian target of rapamycin kinase complex 1 (mTORC1), regulate the nuclear localization and transcriptional activity of GLI3. An increase in PP2A activity or treatment with rapamycin leads to cytosolic retention of GLI3 and, consequently, reduced transcription of the GLI3 target gene and cell cycle regulator, cyclin D1. Conversely, inhibition of PP2A results in increased expression of cyclin D1. In summary, our findings reveal the existence of a hitherto unrecognized molecular cross-talk between the oncogenic SHH pathway and the tumor suppressor PP2A and suggest a novel mechanism underlying the anticancerogenic effects of rapamycin.

Induced TRPC1 expression increases protein phosphatase 2A sensitizing intestinal epithelial cells to apoptosis through inhibition of NF-κB activation

Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs), and induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-kappaB activation. However, the exact mechanism by which increased TRPC1 results in NF-kappaB inactivation remains elusive. Protein phosphatase 2A (PP2A) is a widely conserved protein serine/threonine phosphatase that is implicated in the regulation of a wide array of cellular functions including apoptosis. The present study tests the hypothesis that induced TRPC1 expression inhibits NF-kappaB activation by increasing PP2A activity through Ca2+ influx in IECs. The expression of TRPC1 induced by stable transfection with the wild-type TRPC1 gene increased PP2A activity as indicated by increases in levels of PP2A proteins and their phosphatase activity. Increased levels of PP2A activity in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1) were associated with decreased nuclear levels of NF-kappaB proteins and a reduction in NF-kappaB-dependent transcriptional activity, although there were no changes in total NF-kappaB protein levels. Inhibition of PP2A activity by treatment with okadaic acid or PP2A silencing with small interfering RNA not only enhanced NF-kappaB transactivation but also prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis induced by treatment with tumor necrosis factor-alpha (TNF-alpha)/cycloheximide (CHX). Decreasing Ca2+ influx by exposure to the Ca2+-free medium reduced PP2A mRNA levels, destabilized PP2A proteins, and induced NF-kappaB activation, thus blocking the increased sensitivity of IEC-TRPC1 cells to TNF-alpha/CHX-induced apoptosis. These results indicate that induced TRPC1 expression increases PP2A activity through Ca2+ influx and that increased PP2A sensitizes IECs to apoptosis as a result of NF-kappaB inactivation.

PP2A activity is controlled by methylation and regulates oncoprotein expression in melanoma cells: A mechanism which participates in growth inhibition induced by chloroethylnitrosourea treatment

Protein phosphatase 2A (PP2A), an Akt pathway inhibitor, is considered to be activated by methylation of its catalytic subunit. Also PP2A downregulation was proposed to take part in carcinogenesis. Recently, PP2A activation was shown to be activated in response to DNA damage. To obtain further information on the role of PP2A in tumors and response to DNA damage, we investigated the relationship between PP2A methylation and activity, cell proliferation, Akt activation, c-Myc expression and PTEN activity in B16 melanoma cells untreated and after chloroethylnitrosourea (CENU) treatment. In untreated cells, okadaic acid, an antagonist of PP2A methylation, inhibited PP2A activity, stimulated cell proliferation, increased Akt activation and c-Myc expression. Xylulose-5-phosphate, an agonist of PP2A methylation, increased PP2A activity, decreased cell proliferation, Akt activation and c-Myc expression. However, both PP2A methylation modulators increased PTEN activity. During the response to CENU treatment, PP2A methylation and activity were strongly increased, Akt activation and c-Myc expression were decreased. However PTEN activity was increased. After tumor cell growth recovery, these modifications were moderately decreased. PP2A methylation was quantified and correlated positively with PP2A activity, and negatively with criteria for cell aggressiveness (cell proliferation, Akt activation, c-Myc expression). Based on these data, PP2A methylation status controls PP2A activity and oncoproteins expression and PP2A is strongly activated after CENU treatment thus partly explaining the growth inhibition in response to this agent. It follows that PP2A promethylating agents are potential candidates for anticancer drugs.

Oxidative stress and adenosine A1 receptor activation differentially modulate subcellular cardiomyocyte MAPKs

The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 500 nM) or H(2)O(2) (100 microM) for 15 min. Nuclear/myofilament, cytosolic, Triton-soluble membrane, and Triton-insoluble membrane fractions were generated. CCPA and H(2)O(2) activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In Triton-soluble membrane fractions, H(2)O(2) activated p38 MAPK and p42 ERK, whereas CCPA had no effect on MAPK activation in this fraction. The greatest difference between H(2)O(2) and CCPA was in the Triton-insoluble membrane fraction, where H(2)O(2) increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the Triton-insoluble membrane fraction, suggesting that the activation of this phosphatase may mediate CCPA effects in this fraction. The Triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3, and >85% of p38 MAPK and p42 ERK was bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 microM) reduced H(2)O(2)-mediated MAPK activation but failed to attenuate CCPA-induced MAPK activation. H(2)O(2) but not CCPA increased reactive oxygen species (ROS). Thus the adenosine A(1) receptor and oxidative stress differentially modulate subcellular MAPKs, with the main site of divergence being the Triton-insoluble membrane fraction. However, the adenosine A(1) receptor-mediated MAPK activation does not involve ROS formation.

Protein Kinase C-mediated Down-regulation of Cyclin D1 Involves Activation of the Translational Repressor 4E-BP1 via a Phosphoinositide 3-Kinase/Akt-independent, Protein Phosphatase 2A-dependent Mechanism in Intestinal Epithelial Cells

We reported previously that protein kinase Calpha (PKCalpha), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKCalpha-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr(45) and Ser(64) on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKCalpha-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an approximately 2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H(2)O(2) and ceramide, two naturally occurring PKCalpha agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKCalpha-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/PKCalpha-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells.

Activation of glycogen synthase kinase-3 inhibits protein phosphatase-2A and the underlying mechanisms

The activity of protein phosphatase-2A (PP-2A) is significantly suppressed in the brain of Alzheimer's disease (AD) patients, but the mechanism is not understood. Here, we found an in vivo association of glycogen synthase kinase 3β (GSK-3β) with inhibitor-2 of PP-2A (I2PP-2A). The activation of GSK-3 resulted in accumulation of I2PP-2A with concomitant suppression of PP-2A activity and increases of tau phosphorylation in HEK293, N2a and PC12 cells, while inhibition of GSK-3 caused decreases of I2PP-2A with increased PP-2A activity and decreased tau phosphorylation. A positive correlation between GSK-3β and I2PP-2A (R=0.9158) and a negative correlation between GSK-3β and PP-2A (R=−0.9166) were detected. GSK-3 activation did not affect I2PP-2A mRNA level, while it increased the mRNA level of a heterogeneous ribonucleoprotein A18 (hnRNP A18). The activation of GSK-3 increased the expression and the activity of proteasome system. It suggests that activation of GSK-3 inhibits PP-2A through up-regulation of I2PP-2A with hnRNP A18-involved mechanism.

CaM Kinase Inhibition

See related article, pages 1092–1099 Calcium/calmodulin-dependent protein kinases (CaM kinases) encompass a family of serine/threonine kinases (CaMKI-CaMKIV) that are regulated by calcium bound to calmodulin.1,2 Four separate genes encode the CaMKII subunits (α, β, γ, δ), and 6 to 12 subunits homo- or heteromultimerize to form the active enzyme. Each subunit contains an N-terminal catalytic domain which binds ATP and substrate, a regulatory domain which includes an autoinhibitory domain and a Ca/Calmodulin binding domain, and a C-terminal association (multimerization) domain. In the heart, a number of splice variants of CaMKIIδ are expressed, including CaMKIIδ2 (CaMKIIδC) and CaMKIIδ3 (CaMKIIδB) which differ from each other by the presence of a nuclear localization signal between the regulatory domain and the association domain in CaMKIIδ3.3 CaMKII functions as a local calcium sensor in the heart. At baseline, the autoinhibitory domain prevents substrate binding to the enzyme.1,2 In cardiac myocytes, intracellular Ca2+ [Ca2+]i rises because of transmembrane influx through L-type Ca2+ channels ( I Ca) or the Na/Ca exchanger and release from internal stores such as the sarcoplasmic reticulum (SR). When intracellular Ca2+ rises, it binds to the EF-hand motifs at the N- and C-terminals calmodulin, an ≈150 amino acid ubiquitous intracellular protein. The Ca/CaM complex then binds to the regulatory domain of CaMKII and removes …

Protein phosphatase 2A and Ca 2+-activated K + channels contribute to 11,12-epoxyeicosatrienoic acid analog mediated mesenteric arterial relaxation

Epoxyeicosatrienoic acids (EETs) are considered to be endothelium-derived hyperpolarizing factors, and are potent activators of the large-conductance, Ca 2+-activated K + (BK Ca) channel in vascular smooth muscle. Here, we investigate the signal transduction pathway involved in the activation of BK Ca channels by 11,12-EET and 11,12-EET stable analogs in rat mesenteric vascular smooth muscle cells. 11,12-EET and the 11,12-EET analogs, 11-nonyloxy-undec-8( Z)-enoic acid (11,12-ether-EET-8-ZE), 11-(9-hydroxy-nonyloxy)-undec-8( Z)-enoic acid (11,12-ether-EET-8-ZE-OH) and 11,12- trans-oxidoeicosa-8( Z)-enoic acid (11,12-tetra-EET-8-ZE), caused vasorelaxation of mesenteric resistance arteries. Mesenteric myocyte whole-cell (perforated-patch) currents were substantially (∼150%) increased by 11,12-EET and 11,12-EET analogs. Single-channel recordings were conducted to identify the target for 11,12-EET. 11,12-EET and 11,12-EET analogs also increased mesenteric myocyte BK Ca channel activity in cell-attached patches. Similar results were obtained in cell-free patches. Baseline mesenteric myocyte BK Ca channel activity (NPo) in cell-free patches averaged less than 0.001 at +50 mV and 11,12-EET (1 μmol/L) increased NPo to 0.03 ± 0.02 and 11,12-EET analogs (1 μmol/L) increased NPo to 0.09 ± 0.006. Inhibition of protein phosphatase 2A (PP2A) activity with okadaic acid (10 nmol/L) completely reversed 11,12-EET stimulated BK Ca channel activity and greatly attenuated 11,12-ether-EET-8-ZE mesenteric resistance artery vasorelaxation. 11,12-EET and 11,12-EET analogs increased mesenteric myocyte PP2A activity by 3.5-fold. Okadaic acid and the EET inhibitor, 14,15-epoxyeicosa-5( Z)-enoic acid (14,15-EEZE) inhibited the 11,12-EET mediated increase in PP2A activity. These findings provide initial evidence that PP2A activity contributes to 11,12-EET and 11,12-EET analog activation of mesenteric resistant artery BK Ca channels and vasorelaxation.

D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension

A defect in the coupling of the D(1) receptor (D(1)R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D(1)R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D(1)R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3',5' monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D(1)Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0+/-1.5 density units, DU) than in normotensive rats (7.4+/-2.9 DU). The increased basal serine phosphorylation of D(1)Rs in SHRs was accompanied by decreased expression of D(1)R at the cell surface, and decreased ability of a D(1)-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3',5' monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17+/-4%) and alter D(1)R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D(1)R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26+/-6%) in SHRs. Uncoupling of the D(1)R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D(1)R serine phosphorylation.