Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase in the secretory cascade of the pancreatic beta-cell

Protein phosphorylation by myosin light-chain kinase (MLCK), protein kinase A, and protein kinase C (PKC) plays a positive role in insulin secretion from the pancreatic beta-cell. To investigate the underlying mechanisms, we examined intracellular distribution of the insulin granules and MLCK by immunofluorescence and immunoelectron microscopies and also investigated intracellular traffic of the granules in cultured beta-cells (MIN6) by video microscopy. Considerable parts of MLCK immunoreactivity were colocalized with the insulin granules. Subcellular fractionation of MIN6 cell extracts revealed that myosin light chain (MLC) may be distributed with the insulin-rich fractions, and immunofluorescence staining using specific antibodies against mono- and diphosphorylated MLCs depicted presence of phosphorylated MLCs in the cytoplasm, in part, with colocalization with the insulin granules. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) caused a shift of both insulin granules and MLCK to the cell periphery, which was not reproduced by the adenylate cyclase activator, forskolin. In contrast, forskolin, but not TPA, increased the granule movement. Costimulation of the beta-cell by TPA and forskolin induced drastic translocation of insulin granules and MLCK to the cell periphery, resulting in enormous potentiation of insulin release. These findings suggest that these protein kinases increase insulin granules in the ready-releasable pool by acting on different steps in the secretory cascade.     

Receptor tyrosine kinase and phosphoinositide-3 kinase signaling in malignant mesothelioma

OBJECTIVE: The phosphoinositide-3 kinase signaling pathway is implicated in the development of malignancy and promotes cell-cycle progression and resistance to apoptosis. Malignant mesothelioma tumor specimens demonstrate high levels of the phosphoinositide-3 kinase downstream mediator phosphorylated Akt. Exposure of mesothelioma cell lines to LY294002, a phosphoinositide-3 kinase inhibitor, results in apoptotic cell death and decreased phosphorylated Akt in vitro and tumor burden reduction in vivo. Phosphoinositide-3 kinase is activated by cell-surface receptor tyrosine kinases. We sought to determine which receptors are present in mesothelioma and their role in cellular survival and phosphoinositide-3 kinase signaling. METHODS: Western blot analysis was performed to determine the relative expression of epidermal growth factor receptor, insulin-like growth factor receptor, and platelet-derived growth factor receptor in the mesothelioma cell lines I-45 and REN and the mesothelial line Met5a. After exposure of mesothelioma lines to kinase inhibitors, a cell viability assay was performed, cell-cycle analysis was performed to determine the percentage of apoptosis, and Western blot analysis was performed for phosphorylated Akt. RESULTS: Inhibition of epidermal growth factor receptor resulted in apoptotic cell death and Akt hypophosphorylation in mesothelioma cell lines. Insulin-like growth factor receptor inhibition led to apoptotic cell death without affecting Akt phosphorylation. Platelet-derived growth factor receptor inhibition did not affect cellular survival or phosphoinositide-3 kinase signaling. CONCLUSION: In malignant mesothelioma constitutive activation of phosphoinositide-3 kinase/Akt results in cellular survival and contributes to the malignant phenotype. We have demonstrated that epidermal growth factor receptor inhibition leads to apoptotic cell death through downregulation of phosphoinositide-3 kinase signaling in mesothelioma cell lines, whereas insulin-like growth factor receptor inhibition leads to apoptosis independent of phosphoinositide-3 kinase. Epidermal growth factor receptor, insulin-like growth factor receptor, and phosphoinositide-3 kinase inhibition might be clinically relevant in malignant mesothelioma.     

The map kinase ERK regulates renal activity of cyclin-dependent kinase 2 in experimental glomerulonephritis.

BACKGROUND: In vitro, the extracellular signal-regulated kinase (ERK) is an intracellular convergence point of multiple stimuli, which affect the cell cycle. However, the role of ERK in cell cycle regulation in vivo is unknown. METHODS: To address this issue, ERK activity was blocked both in vitro in mesangial cells (MC) and in vivo in experimental glomerulonephritis (GN) by a pharmacological inhibitor (U0126) of the ERK-activating kinase. RESULTS: In stimulated MC, inhibition of ERK reduced cyclin-dependent kinase 2 (CDK2) phosphorylation, CDK2 activity and cyclin E/A expression, whereas downregulation of CDK inhibitor p27(Kip1) expression was inhibited. In vivo, U0126 was given to rats in the acute phase of anti-Thy 1.1 GN. We previously showed that glomerular cell proliferation was reduced by 67% upon treatment with the inhibitor compared to nephritic controls. Now, we detected a significant increase in renal CDK2-activity/phosphorylation in the nephritic controls, that was significantly and dose-dependently reduced by ERK inhibition. CDK2 activation was accompanied by an increase in renal expression of cyclins E/A and the enhanced binding of these cyclins to CDK2 in the nephritic controls. These changes were blunted by U0126 treatment. Finally, we noted an increased expression and CDK2-binding of p27(KIP1) protein in the nephritic controls which was decreased in U0126 treated rats. CONCLUSIONS: Our observations provide the first evidence that ERK is an intracellular regulator of renal CDK2 activity in vivo in a glomerulonephritis model.     

Negative regulation by p70 S6 kinase of FGF-2-stimulated VEGF release through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts.

To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2-stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. INTRODUCTION: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. MATERIALS AND METHODS: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. RESULTS: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2-stimulated VEGF release and VEGF mRNA expression. The FGF-2-induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2-stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2-stimulated VEGF release and phosphorylation of SAPK/JNK. CONCLUSIONS: These results strongly suggest that p70 S6 kinase limits FGF-2-stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts.     

Roles of protein kinase A and protein kinase G in synaptic plasticity in the visual cortex

Monocular deprivation leads to clear physiological and anatomical changes in the visual cortex known as ocular dominance plasticity. Protein kinase A (PKA) is necessary for ocular dominance plasticity, while protein kinase G (PKG) is not. We have now tested the role of PKA and PKG in long-term potentiation (LTP) and long-term depression (LTD). We have shown that PKA inhibitors have a major effect on both LTP and LTD in the visual cortical slices, whereas a PKG inhibitor affects LTP but not LTD. The PKA activator, 8-chloroadenosine-3',5'-monophosphorothioate, Sp-isomer (Sp-8-Cl-cAMPS), by itself induces a slowly rising form of LTP, which is occluded by theta-burst stimulation (TBS)-induced LTP. These results support the point that the PKA signaling pathway is crucial for neuronal plasticity in visual cortex, and the dissociation of the role of PKA and PKG in long-term synaptic plasticity in the visual cortex suggests that LTP alone is not sufficient to support ocular dominance plasticity, or LTD plays a more fundamental role than LTP in ocular dominance plasticity.     

ERK/MAPK通路参与肝癌产生多药耐药的胞内信号传导

目的探讨微环境诱导肝癌产生多药耐药的胞内信号传导途径。方法分别使HepG2细胞在缺氧、低糖环境下生长或稳定整合HBX基因,运用Western蛋白印迹法检测这些细胞内ERK／MAPK的活性。用ERK／MAPK特异性阻断剂U0126处理这些细胞后,用Western蛋白印迹法检测缺氧诱导因子-1α（HIF—1α）和多药耐药相关蛋白的表达变化,逆转录聚合酶链反应和免疫细胞化学技术分别检测HIF-1α在mRNA水平表达量和部位的改变。结果不同环境下生长的HepG2细胞中,磷酸肜非磷酸化ERK／MAPK比例均有不同程度的增高。用U0126处理12h后,这些细胞中HIF-1α和多药耐药相关蛋白的表达下降,且HIF-1α表达由胞核向胞质转位,其mRNA水平无显著变化。结论ERK／MAPK信号通路是微环境诱导肝癌产生多药耐药的重要胞内信号传导途径。     

丝裂酶原活化蛋白激酶激酶1/2抑制对家兔关节软骨保护作用的研究

目的评价丝裂酶原活化蛋白激酶（mitogen—activated protein kinase，MAPK）信号转导通路中MAPK激酶1／2（MEKI／2）在骨关节炎发病过程中的作用。方法新西兰家兔30只，制作OA模型，随机分成3组，每组10只。从造模术后第4天开始，第1组和第2组分别注射100μmol／L和40μmol／L的PD98059，第3组注射PBS液体作为安慰剂对照组，每周2次。另取10只家兔，正常关节内注射PBS液体作为正常对照组。8周后处死动物，进行股骨髁关节软骨退变的大体评分。用Western Blot印记杂交法测定软骨组织中ERK1／2以及磷酸化ERK1／2的表达。用实时定量PCR方法测定基质金属蛋白酶-1／13（MMp-1／13）的mRNA表达水平。结果与使用PBS的安慰剂对照组相比，使用PD98059的关节软骨破坏明显减轻。磷酸化的ERK1／2在100μmol／LPD98059干预组明显低于40μmol／L干预组和安慰剂对照组。而MMP-1／13的mRNA表达在不同浓度的干预组均低于安慰剂对照组。结论MAPK信号转导通路参与了骨关节炎关节软骨破坏的病理过程。MEK1／2选择性阻滞剂PD98059可以在关节炎动物活体内有效抑制关节炎软骨破坏的程度，该作用可能与其有效抑制ERK1／2的磷酸化激活有关。     

Cardioprotection by St Thomas' solution is mediated by protein kinase C and tyrosine kinase

BACKGROUND: Intracellular signaling pathways, specifically the activation of protein kinase C and tyrosine kinase, are essential to the cardioprotection of ischemic preconditioning. We proposed that activation of PKC and TK contribute to the myocardial protection of St. Thomas' No. 2 cardioplegia solution (STC). MATERIALS AND METHODS: Isolated rat hearts were exposed to 40 min of global ischemia followed by 120 min of reperfusion. Before ischemia, hearts received no treatment (control; n = 7), STC (n = 7), phorbol 12-myristate 13-acetate (PMA; n = 6), PMA + chelerythrine (n = 6), anisomycin (n = 6), anisomycin + genistein (n = 7), STC + chelerythrine (n = 7), STC + genistein (n = 7), PMA + genistein (n = 7) or anisomycin + chelerythrine (n = 7). Left ventricular developed pressure (LVDP) recovery, myocardial infarct size, and lactate dehydrogenase release were measured. RESULTS: STC as well as PMA (protein kinase C activator) and anisomycin (tyrosine kinase activator) significantly reduced infarct size (6.9 +/- 2.9%, 9.6 +/- 2.1%, 14.0 +/- 4.4%) compared with controls (42.4 +/- 2.9%, P < 0.05). The infarct reduction of PMA and anisomycin were blocked by their inhibitors chelerythrine and genistein, respectively. Both chelerythrine (29.2 +/- 4.1%, P < 0.05) and genistein (40.4 +/- 4.3%, P < 0.05) attenuated the reduction of infarct size provided by STC. The recovery of LVDP improved with STC, PMA and anisomycin (72.6 +/- 1.4%, 60.4 +/- 4.7%, 57.2 +/- 4.6%) compared with control (33.8 +/- 3.6%, P < 0.05). Addition of chelerythrine or genistein to STC impaired recovery of LVDP (52.3 +/- 4.4%, 35.1 +/- 2.5%, P < 0.05) compared with STC treatment. CONCLUSION: Administration of the pharmacologic inhibitors chelerythrine and genistein blunts the cardioprotection caused by STC treatment.     

CaM kinase II: a protein kinase with extraordinary talents germane to insulin exocytosis

CaM kinase II, a multifunctional Ca2+/calmodulin-dependent protein kinase, is expressed in the pancreatic beta-cell and is activated by glucose and other secretagogues in a manner correlating with insulin secretion. It is proposed that the activation of CaM kinase II mediates some of the actions of Ca2+ on the exocytosis of insulin secretory granules. This suggestion is supported by the localization of CaM kinase II to the insulin secretory granule and by the identification of two secretory-relevant proteins, MAP-2 and synapsin I, as endogenous substrates in the beta-cell. Mechanistically, CaM kinase II appears to be involved in secretory steps proximal to granule fusion at the plasmalemma, and may facilitate protracted secretion through control of the interaction of granules with the cell cytoskeleton and their mobilization from intracellular synthesis sites. Through its unique regulatory properties, however, CaM kinase II is predicted to serve in more specialized aspects of the secretory process. In particular, the ability of CaM kinase II to remain active after cell stimulation is suggested to represent a mechanism by which releasable pools of granules are replenished between stimuli.     

Thymidine kinase in brain-tumor cysts

A recently developed method for deoxythymidine kinase (TK) determination was applied to brain-tumor cyst fluid and fluid from a non-neoplastic intracerebral cyst. The fluid from all tumors tested positive for TK whereas the non-neoplastic cyst lacked TK. Cyst fluid was also analyzed for TK before and after intracystic instillation of BCNU. It is suggested that TK activity in the fluid in cystic brain lesions could prove useful in deciding whether an intracerebral lesion is neoplastic. Also, TK activity can be used to evaluate the effect of topical therapy.     

蛋白激酶C活性及δ亚型在肾透明细胞癌的表达

目的　探讨蛋白激酶C(PKC)、PKC δ在肾透明细胞癌中的表达及其临床意义。方法　采用PKC比活性测定和WesternBlotting方法检测 38例肾透明细胞癌和 5例正常肾组织中PKC比活性和PKC δ的表达。结果　癌组织和正常组织胞浆PKC比活性值分别为 ( 6 .97± 2 .6 5 )10 - 2 pmol·ml- 1 ·min- 1 和 ( 14 .16± 5 .6 7) 10 - 2 pmol·ml- 1 ·min- 1 ;而胞膜中的比活性值分别为( 2 7.90± 7.5 8) 10 - 2 pmol·ml- 1 ·min- 1 和 ( 5 7.0 6± 18.13) 10 - 2 pmol·ml- 1 ·min- 1 ;PKC δ在癌组织细胞胞浆中的相对灰度值小于 1,在胞膜中则大于 1,各病理分期和细胞分级间差异均有显著性 (P<0 .0 5 )。结论　PKC活性在肾透明细胞癌中下降 ,PKC δ表达与病理分期、细胞分级相关 ,是判断肾细胞癌预后的指标。     

Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells.

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low-(5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [gamma-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effect on autophosphorylation (30 +/- 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 +/- 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 microM to 80 microM) for ATP of IRK from high-glucose-treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells after acute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of the kinase domain in death-associated protein kinase attenuates tubular cell apoptosis in renal ischemia-reperfusion injury

Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase localized to renal tubular epithelial cells. To elucidate the contribution of DAPK activity to apoptosis in renal ischemia-reperfusion (IR) injury, wild-type (WT) mice and DAPK-mutant mice, which express a DAPK deletion mutant that lacks a portion of the kinase domain, were subjected to renal pedicle clamping and reperfusion. After IR, DAPK activity was elevated in WT kidneys but not in mutant kidneys (1785.7 +/- 54.1 pmol/min/mg versus 160.7 +/- 60.6 pmol/min/mg). Furthermore, there were more TUNEL-positive nuclei and activated caspase 3-positive cells in WT kidneys than in mutant kidneys after IR (24.0 +/- 5.9 nuclei or 9.4 +/- 0.6 cells per high-power field [HPF] versus 6.3 +/- 2.2 nuclei or 4.4 +/- 0.7 cells/HPF at 40 h after ischemia). In addition, the increase in p53-positive tubule cells after IR was greater in WT kidney than in mutant kidneys (9.9 +/- 1.4 cells/HPF versus 0.8 +/- 0.4 cells/HPF), which is consistent with the theory that DAPK activity stabilizes p53 protein. Finally, serum creatinine levels after IR were higher in WT mice than in mutant mice (2.54 +/- 0.34 mg/dl versus 0.87 +/- 0.24 mg/dl at 40 h after ischemia). Thus, these results indicate that deletion of the kinase domain from DAPK molecule can attenuate tubular cell apoptosis and renal dysfunction after IR injury.     

DAPK在肿瘤中的研究进展

死亡相关蛋白激酶(death-associated protein kinase,DAPK)是钙调蛋白调节的丝氨酸/苏氨酸蛋白激酶,在整个凋亡系统中发挥着重要作用,是凋亡正性调节因子之一.DAPK被认为是抑癌基因,与肿瘤的发生、发展、转移密切联系,在肿瘤早期诊断、评价预后及基因靶向治疗等方面有重要意义.     

Role of tyrosine kinase in desflurane-induced preconditioning

BACKGROUND: Short administration of volatile anesthetics preconditions myocardium and protects the heart against the consequences of subsequent ischemia. Activation of tyrosine kinase is implicated in ischemic preconditioning. The authors investigated whether desflurane-induced preconditioning depends on activation of tyrosine kinase. METHODS: Sixty-four rabbits were instrumented for measurement of left ventricular pressure, cardiac output, and myocardial infarct size (IS). All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Rabbits underwent a treatment period consisting of either no intervention for 35 min (control group, n = 12) or 15 min of 1 minimum alveolar concentration desflurane inhalation followed by a 10-min washout period (desflurane group, n = 12). Four additional groups received the tyrosine kinase inhibitor genistein (5 mg/kg) or lavendustin A (1.3 mg/kg) at the beginning of the treatment period with (desflurane-genistein group, n = 11; desflurane-lavendustin A group, n = 12) or without desflurane inhalation (genistein group, n = 9; lavendustin A group, n = 8). RESULTS: Hemodynamic values were similar in all groups during baseline (left ventricular pressure, 87 +/- 14 mmHg (mean +/- SD]; cardiac output, 198 +/- 47 ml/min), during coronary artery occlusion (left ventricular pressure, 78 +/- 12 mmHg; cardiac output, 173 +/- 39 ml/min), and after 2 h of reperfusion (left ventricular pressure, 59 +/- 17; cardiac output, 154 +/- 43 ml/min). IS in the control group was 55 +/- 10% of the area at risk. The tyrosine inhibitors had no effect on IS (genistein group, 56 +/- 13%; lavendustin A group, 49 +/- 13%; each P = 1.0 vs. control group). Desflurane preconditioning reduced IS to 40 +/- 15% (P = 0.04 vs. control group). Tyrosine kinase inhibitor administration had no effect on IS reduction (desflurane-genistein group, 44 +/- 13%; desflurane-lavendustin A group, 44 +/- 16%; each P = 1.0 vs. desflurane group). CONCLUSION: Desflurane-induced preconditioning does not depend on tyrosine kinase activation.