Cyclin G-associated kinase regulates protein phosphatase 2A by phosphorylation of its B'γ subunit

Protein phosphatase 2A (PP2A) bearing the B’γ (= B’α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B’γ/cyclin G complex, directly phosphorylates the B’γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B’γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B’γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B’γ subunit.     

7,7′-Diazaindirubin—A small molecule inhibitor of casein kinase 2 in vitro and in cells

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7′-diazaindigo (10) and 7,7′-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 μM, 7-azaindirubin (12) and 7′-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7′-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 μM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.     

Tau phosphorylation and neuronal apoptosis induced by the blockade of PP2A preferentially involve GSK3β

Overactivation of GSK3β (glycogen synthase kinase-3β) and downregulation of PP2A (protein phosphatase-2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation in Alzheimer’s disease (AD). GSK3β and PP2A signaling pathways were reported to be interconnected. Targeting tau kinases was suggested to represent a therapeutic strategy for AD. Here, tau phosphorylation and neuronal apoptosis were induced in cortical cultured neurons by the inhibition of PP2A by okadaic acid (OKA). In this in vitro model of ‘tau pathology’ and neurodegeneration, we tested whether GSK3β and other tau kinases including DYRK1A and CDK5 were implicated. Our results show that the inhibitors of GSK3β, lithium and 6-BIO (6-bromoindirubin-3′-oxime), prevented OKA-induced tau phosphorylation and neuronal apoptosis. The implication of GSK3β in these OKA-induced effects was confirmed by its silencing by hairpin siRNA. By contrast, inhibition of DYRK1A (dual-specificity tyrosine-phosphorylation regulated kinase-1A) and CDK5 (cyclin-dependent kinase-5) reversed OKA-induced tau phosphorylation at certain sites but failed to prevent neuronal apoptosis. These results indicate that OKA-induced effects, especially neuronal apoptosis, are preferentially mediated by GSK3β. Furthermore, since chronic exposure to lithium and 6-BIO might be deleterious for neurons, we tested the effect of a new 6-BIO derivative, 6-BIBEO (6-bromoindirubin-3′-(2-bromoethyl)-oxime), which is much less cytotoxic and more selectively inhibits GSK3β compared to lithium and 6-BIO. We show that 6-BIBEO efficiently reversed OKA-induced tau phosphorylation and neuronal apoptosis. It will be interesting to test neuroprotection by 6-BIBEO in an in vivo model of tau pathology and neurodegeneration.     

Role of protein kinase C in oxidant — mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells

The present study was undertaken to test the hypothesis that activation of cell membrane associated protein kinase C (PKC) plays a role in stimulating cell membrane associated phospholipase A₂ (PLA₂) activity, and subsequent liberation of arachidonic acid (AA) under exposure of rabbit pulmonary arterial smooth muscle cells to the oxidant hydrogen peroxide (H₂O₂). Exposure of the smooth muscle cells to H₂O₂ dose-dependently stimulates [¹⁴C] AA release, and enhances the cell membrane associated PLA₂ activity. Pretreatment of the cells with protein kinase C (PKC) inhibitors H₇ and sphingosine prevent the cell membrane associated PLA₂ activity, and AA release caused by H₂O₂. Treatment of the smooth muscle cells with H₂O₂ stimulates the cell membrane associated PKC activity. Pretreatment of the cells with an antioxidant vitamin E prevents H₂O₂ caused stimulation of the cell membrane associated PKC activity. The cell membrane associated PLA₂ and PKC activities correlate linearly. These results suggest that H₂O₂ caused stimulation of the smooth muscle cell membrane associated PLA₂ activity, and subsequent liberation of AA can occur through an increase in the activity of the cell membrane associated PKC. (Mol Cell Biochem122: 9–15, 1993)Abbreviations AA Arachidonic Acid - PLA₂ Phospholipase A₂ - PKC Protein Kinase C - PBS Phosphate Buffered Saline - HBPS Hank's Buffered Physiological Saline - HEPES 4-(2-Hydroxyethyl)-1-Piperazine N-2-Ethanesulfonate - FCS Fetal Calf Serum - ATP Adenosine Triphosphate - H₇ 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine - DMEM Dulbecco's Modified Eagles Medium - TCA Trichloroacetic Acid     

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by α2β1 integrin low clustering through focal adhesion kinase

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.     

Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.It is now clear that chronic inflammation is a hallmark of type I and type II diabetes, affecting both β-cell mass and insulin secretion.¹ Type I diabetes is characterized by drastic decreases in β-cell mass and insulin secretion, in part mediated by proinflammatory cytokines produced following autoimmune activation.¹ Proinflammatory cytokines, particularly interleukin-1β (IL-1β), in combination with interferon-γ (IFN-γ) and/or tumor necrosis factor-α (TNF-α), promote death by apoptosis and decrease function of differentiated β-cells, leading to β-cell destruction.¹ Pancreatic islet transplantation is a promising alternative therapy for some type I diabetic patients.² However, clinical outcome is not always achieved because of significant loss of islet mass during and after transplantation.³ Up to 80% of transplanted islets can die during the post-transplantation period as a result of apoptosis because of several mechanisms, notably the instant blood-mediated inflammatory response (IBMIR) and the release of a mix of cytokines including IL-1β, TNF-α and IFN-γ.⁴Immune-modulatory strategies for type I diabetes therapy and improvement of islet transplantation outcomes have emerged, targeting a single specific cytokine, such as IL-1β or TNF-α.^{2, 5} However, these strategies may only target inflammation partially.² Indeed, multiple cytokines, originating from surrounding immune cells and/or β-cells themselves, are more likely to be present simultaneously^{4, 6} and act synergistically to induce β-cell death and dysfunction.^{7, 8, 9} Preclinical and clinical studies demonstrated that glucagon-like peptide-1 (GLP-1) analogs, in addition to regulating glucose homeostasis in vivo, contribute to the restoration of normoglycemia after islet transplantation.^{10, 11, 12, 13} GLP-1 receptor (GLP-1R) analogs protect β-cell survival and function from proinflammatory cytokine attack.^{12, 14, 15} However, some studies have shown only modest and short-term anti-inflammatory effects of GLP-1 analogs when used alone.^{11, 13, 16}Mitogen-activated protein kinases (MAPKs) (i.e., extracellular-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK) play important roles in cytokine-induced β-cell dysfunction and death.¹ Conversely, ERK1/2 are involved in the beneficial effects of glucose and GLP-1 analogs.^{17, 18, 19} In this context, upstream protein kinases that specifically control the activation of MAPK in response to a combination of inflammatory cytokines (IL-1β, TNF-α and IFN-γ), rather than a single cytokine, may be useful targets for therapeutic interventions against pancreatic β-cell failure.The serine/threonine kinase tumor progression locus 2 (Tpl2) (also known as COT (Cancer Osaka Thyroid) in humans) is a member of the MAP3K family (the MAP3K8) whose activation stimulates primarily the ERK1/2 pathway, but also JNK and/or p38 MAPK in some cell types, specifically in response to various inflammatory stimuli.^{20, 21, 22} Dysregulation of Tpl2 expression and signaling is associated with acute and chronic inflammatory diseases,^{20, 21, 22} and several studies highlight a critical function of Tpl2 in the control of inflammatory responses and survival in adipocytes, fibroblasts and immune and epithelial cells.^{21, 22, 23, 24}However, there is currently nothing known about the effects of Tpl2 in β-cells. The aim of this study was to determine whether Tpl2 may be a new key inflammatory regulator in β-cells or islets. We demonstrate that Tpl2 contributes to cytokine-induced β-cell apoptosis and dysfunction, and suggest that Tpl2 inhibition, either alone or combined with a GLP-1 receptor agonist, represents a potential new therapeutic strategy for the treatment of diabetes.     

B56α subunit of protein phosphatase 2A mediates retinoic acid-induced decreases in phosphorylation of endothelial nitric oxide synthase at serine 1179 and nitric oxide production in bovine aortic endothelial cells

We previously showed that all-trans retinoic acid (atRA) decreased nitric oxide (NO) production through Akt-mediated decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser¹¹⁷⁹) in bovine aortic endothelial cells (BAEC). Since protein phosphatase 2A (PP2A) was also reported to decrease eNOS-Ser¹¹⁷⁹ phosphorylation, we investigated using BAEC whether PP2A mediates atRA-induced eNOS-Ser¹¹⁷⁹ dephosphorylation and subsequent decreased NO production. Treatment with okadaic acid (5 nM), a selective PP2A inhibitor, or ectopic expression of small interference RNA (siRNA) of PP2A catalytic subunit α (PP2A Cα) significantly increased eNOS-Ser¹¹⁷⁹ phosphorylation and NO production. Each treatment also significantly reversed atRA-induced observed effects, suggesting a role for PP2A. We also found that atRA significantly increased cellular PP2A activity. However, Western blot analysis revealed that atRA did not increase the expression of PP2A Cα, although it significantly increased the level of B56α of PP2A regulatory B subunit (PP2A B56α), but not PP2A B55α and PP2A B56δ. Real-time PCR assay confirmed a significant increase in PP2A B56α mRNA expression in atRA-treated cells. Ectopic expression of siRNA of PP2A B56α significantly reversed atRA-induced inhibitory effects on eNOS-Ser¹¹⁷⁹ phosphorylation and NO production, suggesting a role for PP2A B56α. Our study demonstrates for the first time that atRA decreases eNOS-Ser¹¹⁷⁹ phosphorylation and NO release at least in part by increasing PP2A B56α-mediated PP2A activity in BAEC.