Synthesis of a substrate of protein kinase C and its corresponding phosphopeptide

The syntheses of a protein kinase C (PKC) peptide substrate, H-Lys-Arg-Thr-Leu-Arg-OH, and a phosphopeptide analog of the synthetic substrate, H-Lys-Arg-Thr(P)-Leu-Arg-OH, are reported. PKC phosphorylates the peptide with an apparent K_M of 0.30 ± 0.04 mM and an apparent V_max equal to one-tenth that of histone III-S. The synthesis of the phosphopeptide features a recently developed convenient phosphorylation procedure for serine and threonine using N,N-diethylamino-dibenzylphosphoramidite. A complete characterization of the PKC substrate and its corresponding phosphopeptide by C-H COSY 2D n.m.r. is included.     

蛋白激酶C在肿瘤中的作用及其抑制剂研究进展

蛋白激酶C(protein kinase C,PKC)在肿瘤的发生、发展和转移中均发挥着重要作用,因此,PKC是开发治疗肿瘤疾病药物的潜在分子靶点。目前,几种PKC抑制剂已经进入临床研究阶段,但是同时也为靶向PKC抗肿瘤药物的研究开发带来了新的挑战。该文拟对近年来蛋白激酶C在肿瘤中的作用及其抑制剂研究进展作一综述。     

Naturally appearing N-feruloylserotonin isomers suppress oxidative burst of human neutrophils at the protein kinase C level

N-feruloylserotonin (N-f-5HT) isomers, isolated from seeds of Leuzea carthamoides (Wild) DC, inhibited dose-dependent oxidative burst in human whole blood and isolated neutrophils in vitro, which were measured by luminol- and/or isoluminol-enhanced chemiluminescence in the following rank order of stimuli: PMA > OpZ > calcium ionophore A23187. In isolated neutrophils that were stimulated with PMA, N-f-5HT isomers were effective against extracellular and intracellular reactive oxygen species. Liberation of ATP, analysis of apoptosis, and recombinant caspase-3 activity revealed that N-f-5HT isomers, used in concentrations up to 100 μM, did not alter the viability and integrity of isolated neutrophils. Western blot analysis documented that in concentrations of 10 and 100 μM, N-f-5HT isomers significantly decreased PMA-induced phosphorylation of PKC α/β II. The results suggest that N-f-5HT isomers are an effective, naturally occurring substance with a potent pharmacological effect on the oxidative burst of human neutrophils. It should be further investigated for its pharmacological activity against oxidative stress in ischemia-reperfusion, inflammation and other pathological conditions.     

糖尿病合并症与蛋白激酶C

糖尿病 (DM )可引起体内各系统的合并症。实验研究表明 ,二酰基甘油 (DAG) 蛋白激酶C(PKC)通路的活动增加与DM合并症的产生密切相关 ,可能是DM时多种生化指标引起合并症产生的最后的细胞内共同通路。在已知的 11中PKC亚型中 ,PKC β和 ε与DM合并症的产生最为密切     

从天然产物中寻找蛋白激酶C抑制剂的研究进展

蛋白激酶C(PKC)抑制剂对抗肿瘤、抗病毒、治疗心血管疾病等多种药物的合理设计有着非常重要的意义。该文对文献报道的天然产物中的PKC抑制剂依据化学结构进行分类,并对其生物来源、生物活性和作用位点进行综述。     

蛋白激酶C在心肌预适应中的作用机制

心肌预适应后 ,内源性物质释放 ,分别作用于心肌中与G蛋白耦联的相应受体 ,蛋白激酶C激活并转位至细胞膜及细胞骨架 ,并可通过线粒体KATP通道、MAPK等通路引发心肌保护作用。在不同的动物中 ,分别发现不同的蛋白激酶C亚型激活转位。目前公认 :在参与心肌预适应过程的多种因素中 ,PKC起到了关键性作用 ,是多种因素作用的一条共同通路     

Effect of barbiturates on polyphosphoinositide biosynthesis and protein kinase C activity in synaptosomes

Previously, it has been found that phenobarbital inhibited protein kinase C (PKC) and the enzymes of the metabolism of polyphosphoinositide, especially phosphatidylinositol 4-phosphate (PIP) kinase (PIP-kinase). As a continuation of these studies, a number of barbiturates (barbituric acid, barbital, butabarbital, pentobarbital, amobarbital, phenobarbital, secobarbital and hexobarbital) were tested for inhibition of these enzymes and also of phosphatidylinositol (PI) kinase (PI-kinase), in a synaptosomal preparation at pH 7.8 from the brain of rat. All compounds, except barbituric acid (and Na-barbital for PI-kinase) inhibited the three kinases. However, PKC was approximately 3–5 fold more sensitive to inhibition by the drugs (measured by Ki values) than PIP-kinase, which was 2- to 4-fold more sensitive than PI-kinase. The inhibitory potency of the drugs increased with their lipophilicity, although to a lesser degree than expected from the differences in partition coefficients; the largest deviation from a positive correlation (i.e. hexobarbital) may be the result of the blockade of an imide (-NH) group at one position of the barbituric ring. Concentrations of drugs (after correction for the greater than normal ionization (pH 7.8) of the drugs in the assays) necessary for half-maximal inhibition were well within, or smaller than, those reported necessary for in vitro blocking of nerves. The possibility, therefore, exists that the physiological effects of the barbiturates are, in part, the result of an inhibition of protein kinase C and PIP-kinase.     

溶血性磷脂酰胆碱诱导血小板活化及蛋白质酪氨酸激酶和蛋白激酶C的作用

应用双道血小板聚集仪和荧光分光光度计分别测定溶血性磷脂酰胆碱(LysoPC)诱导的兔洗涤血小板聚集,细胞内钙离子浓度(［Ca²⁺］_i),细胞内pH值(pH_i)的变化及5-羟色胺(5-HT)的释放,并观察蛋白质酪氨酸激酶(PTK)抑制剂金雀异黄素(Gen)和蛋白激酶C(PKC)抑制剂星形孢菌素(Sta)对其作用的影响. 结果表明：LysoPC(30-300 μmol·L^-1)诱导血小板聚集,5- HT释放和［Ca²⁺］_i 升高,均呈现良好的浓度依赖性,100 μmol·L^-1以上时伴有pH_i的增加;Gen使LysoPC 诱导的聚集浓度效应曲线右移,半效聚集浓度值从(100±23) μmol·L^-1增加到(200±42) μmol·L^-1,明显抑制 ［Ca²⁺］_i 升高和胞浆碱化,对5-HT释放无明显影响;Sta对较低浓度LysoPC 诱导的血小板聚集,5-HT释放, ［Ca²⁺］_i 和pH_i的增加均有明显抑制作用,但对高浓度LysoPC 诱导的血小板活化无明显影响. 提示：LysoPC通过内Ca²⁺调节和Na^＋/H^＋交换介导血小板聚集和致密颗粒释放;PTK的激活参与了LysoPC诱导的血小板聚集,内Ca²⁺调节和Na^＋/H^＋交换,而在5-HT释放的机理中意义不大;PKC的激活在较低浓度LysoPC 诱导的血小板活化中起重要作用,高浓度LysoPC通过不依赖于PKC的途径激活血小板.     

A phosphorylation assay using [γ‐32P]ATP: A highly sensitive detection of protein kinase C

The involvement of protein kinase C (PKC) in many biological processes such as development, memory, cell differentiation and proliferation, and carcinogenesis has been demonstrated. Using the mep45 gene encoding the 45‐kDa major envelope protein (Mep45) of Selenomonas ruminantium, a protein‐fused substrate (neurogranin‐Mep45, MFS‐PKC) was cloned, which is a highly selective substrate for PKC. The recombinant protein‐fused substrate can be constantly produced in reasonable quantities with a small outlay. In this study, a suitable strategy for the detection of the phosphorylation of a peptide‐type substrate and a Mep45‐fused substrate catalyzed by PKC by using a sensitive radiodetection is described. This strategy can be applicable to the development of protein microarray, which can be a useful tool for high‐throughput screening in biological and medical research. Copyright © 2010 John Wiley & Sons, Ltd.     

Role of mitochondrial KATP channels and protein kinase C in ischaemic preconditioning

1. Activation of mitochondrial KATP (mitoKATP) channels and protein kinase C (PKC) has been implicated in cardioprotective mechanisms of ischaemic preconditioning (IPC). However, the exact role of these events in early IPC remains unclear. 2. Isolated and perfused rat hearts underwent IPC with three cycles of 5 min ischaemia and 5 min reperfusion. The heart was subjected to 30 min global ischaemia followed by 120 min reperfusion. Flavoprotein oxidation was monitored to assess mitoKATP channel activity. Cardioprotection was evaluated by recovery of isovolumic left ventricular (LV) function and infarct size. 3. Diazoxide (50 mgr;mol/L) increased flavoprotein oxidation and conferred cardioprotection in a manner sensitive to the selective mitoKATP channel blocker 5-hydroxydecanoate (5-HD; 0.5 mmol/L). 4. Pretreatment with 0.5 mmol/L 5-HD abrogated IPC-induced flavoprotein oxidation and cardioprotection, whereas late treatment with 5-HD after IPC required a higher dose (2 mmol/L) to abolish flavoprotein oxidation and cardioprotection afforded by IPC. 5. Pretreatment with the PKC inhibitors Ro318425 (1 micro mol/L) and chelerythrine (5 micro mol/L) abolished IPC-induced flavoprotein oxidation and cardioprotection, whereas late treatment with Ro318425 required a higher dose (4 micro mol/L) to abolish flavoprotein oxidation and cardioprotection. 6. In conclusion, these results suggest that activation of mitoKATP channels is the trigger and the mediator of IPC and that PKC plays a crucial role in both phases of mitoKATP channel activation, although mitoKATP channels and PKC may be more activated during the mediator phase.     

蛋白激酶C 抑制剂的研究新进展

摘要： 蛋白激酶 C （PKC） 是一组磷脂依赖性的丝氨酸/苏氨酸蛋白激酶, 与蛋白激酶 A （PKA） 和蛋白激酶 G （PKG） 共同构成丝氨酸/苏氨酸蛋白激酶 AGC 超家族。PKC 包括传统型 PKC、 新型 PKC、 非典型 PKC 和与 PKC 相关的一些激酶 （PRK） 成员。PKC 广泛分布于哺乳动物的组织和细胞中, 对细胞的生长代谢、 增殖分化等起着重要的生物学作用。研究表明, 多种细胞的变异和疾病的发生发展都与 PKC 的异常表达有关。因此, 设计和寻找高效的 PKC 抑制剂对于多种有效药物的合成和临床上包括肿瘤、 心血管疾病、 高血压等多种疾病的治疗都具有非常重要的意义。近年来, 关于 PKC 抑制剂的研究已经成为国内外研究的焦点。大量文献报道了多种有效的 PKC 抑制剂, 并对其作用位点、 作用机制以及临床试验数据等进行了分析。这些 PKC 抑制剂的发现对于 PKC 的结构分析和疾病的治疗具有重要的意义。因此, 本文对这些高效的PKC 抑制剂进行综述。     

灯盏花素对慢性低氧大鼠PKC的影响

目的　探讨灯盏花素对慢性低氧大鼠PKC信号途径的影响。方法　将SD大鼠分为 :对照组 (A) ,低氧组 (B) ,低氧 +灯盏花素组 (C) ,低氧时间为 4wk。采用透射电镜、放射活性测定法、免疫组化等方法研究灯盏花素对慢性低氧大鼠肺动脉平均压 (mPAP)、左右心室重量比 (RV/LV +S)、肺细小动脉管壁面积 /管总面积 (WA/TA)、中膜平滑肌细胞核密度 (SMC)、肺细小动脉超微结构、肺组织PKC活性、肺细小动脉管壁PKC的影响。结果　 (1)B组mPAP、RV/LV +S明显高于A组 (P <0 0 1) ,C组mPAP、RV/LV +S明显低于B组 (P <0 0 1) ;(2 )光镜下B组WA/TA、SMC明显高于A组 (P <0 0 1) ,C组WA/TA、SMC明显低于B组 (P <0 0 1) ;电镜显示B组肺动脉中膜平滑肌细胞增生 ,胶原纤维丰富 ;C组肺动脉中膜平滑肌细胞、胶原纤维较B组明显减少 ;(3)B组肺组织PKC总活性 (PKCt)、胞膜PKC活性(PKCm)、胞质PKC活性 (PKCc)及胞膜PKC活性 (PKCm)占PKC总活性 (PKCt)的百分比明显高于A组 (P <0 0 1) ,C组PKCt、PKCm、PKCc及PKCm占PKCt的百分比明显低于B组 (P <0 0 1) ;(4 )免疫组化显示B组肺细小动脉 (直径约10 0～ 2 0 0 μm)PKC含量明显高于A组 (P <0 0 1) ,C组较B组明显为低 (P <0 0 1)。结论　灯盏花素抑制PKC信号途径可能是其抑制慢性低氧     

蛋白激酶C在心肌纤维化中的作用

心肌纤维化是多种心脏疾病发展的共同结果,心肌成纤维细胞的增殖导致细胞外基质蛋白代谢紊乱,引起心肌重塑,最终导致恶性心律失常、心功能衰竭、甚至心脏性猝死的发生。有效地抑制心肌重塑可预防猝死的发生。明确蛋白激酶C(protein kinase C,PKC)对心肌纤维化的调控机制,可能为逆转心肌重塑提供新的治疗靶点。     

Effect of verapamil on cardiac protein kinase C activity in diabetic rats

We examined the effect of verapamil treatment on cardiac protein kinase C (PKC) activity in streptozocin-induced diabetic rats. Basal cardiac PKC activity in diabetes increased in both cytosolic (by 94%, P < 0.01) and membrane (by 41%, P < 0.05) fractions as compared with that in controls. Subcutaneous administration of 8 mg/kg verapamil twice a day for 8 weeks induced a significant decrease in both cytosolic (by 59%, P < 0.01) and membrane (by 50%, P < 0.01) PKC activity in diabetes as compared with the activity in the non-treated diabetic groups. In contrast, cardiac cytosolic PKC activity in control rats was significantly (P < 0.01) decreased by 41% as compared with that of the non-treated control group without there being any change in membrane PKC activity. Our data demonstrate that verapamil treatment may ameliorate the abnormal activation of cardiac PKC in diabetes.     

Inhibition of protein kinase C but not protein kinase A attenuates morphine withdrawal excitation of rat hypothalamus-pituitary-adrenal axis

Our previous studies have shown an enhanced activity of the hypothalamus-pituitary-adrenocortical axis response in rats withdrawn from morphine, which results from an increase in the hypothalamic paraventricular nucleus noradrenergic activity that is dependent on alpha-adrenoceptor activation. The first objective of this work was to examine the effect of protein kinase A (PKA) and protein kinase C (PKC) inhibitors on morphine withdrawal-induced changes in corticosterone release (an index of the hypothalamus-pituitary-adrenocortical axis activity) and in catecholaminergic turnover in the paraventricular nucleus. Plasma corticosterone levels as well as the concentration of noradrenaline, 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the paraventricular nucleus were determined. The second purpose of the study was to assess whether kinase inhibitors, administered continuously through s.c. osmotic minipumps, get into the brain. Chronic pretreatment for 7 days with the selective PKA inhibitor N-(2'guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004) concomitantly with morphine did not affect the increase in corticosterone release observed after naloxone-precipitated morphine withdrawal. However, pretreatment with the selective PKC inhibitor, calphostin-C significantly antagonized the corticosterone hypersecretion in morphine-withdrawn rats. Neither HA-1004 nor calphostin-C co-administered with morphine for 7 days did modify the morphine withdrawal-induced increase in noradrenaline turnover. Pretreatment with HA-1004 inhibits the increase in dopamine turnover during morphine withdrawal, whereas calphostin-C did not affect the DOPAC/dopamine ratio. Our results might indicate that expression of morphine dependence for hypothalamus-pituitary-adrenocortical axis hyperactivity involves PKC but not PKA signaling mechanisms. It is suggested that in rats PKC may be up-regulated during morphine dependence. High-performance liquid chromatography (HPLC) analysis of hypothalamic tissue from rats perfused with kinase inhibitors demonstrates that both calphostin-C and HA-1004 can cross the blood-brain barrier when administered peripherally.     

Screening of natural compounds and their derivatives as potential protein kinase C inhibitors

Protein kinase C (PKC) isozymes are important signal transducers in a number of cellular responses that makes PKC inhibition a topical target for drug discovery. In this study, a set of natural compounds and their derivatives (43 in total) were screened to establish their potential inhibitory effects on PKC, exploiting kinase activity and [³H]‐phorbol ester binding assays. Statistical evaluation of the assays yielded signal‐to‐background, signal‐to‐noise, and Z′ values of 16.1, 11.3, and 0.62, respectively, for the kinase activity assay and 47.4, 15.9, and 0.73 for the binding assay, demonstrating their suitability for screening. Of the compounds investigated, the most potent PKC inhibitor was (?)‐epigallocatechin gallate (EGCG), which had an IC₅₀ of 4.8 µM. In addition, (?)‐epicatechin gallate, dodecyl gallate, and the flavonoids myricetin, quercetin, rhamnetin, luteolin, isorhamnetin, and kaempferol were effective while naringin and scopoletin demonstrated negligible effects. None of the compounds was able to significantly inhibit the binding of phorbol ester to the regulatory domain of PKCα. The highest inhibition, 59%, was observed in the case of EGCG at a concentration of 150 µM. Taken together, nine PKC inhibitors were identified, none of which was able to compete with the binding of phorbol ester to PKCα, suggesting that the mechanism of PKC inhibition could be a result of binding to the catalytic domain of PKC. Drug Dev Res 63:76–87, 2004. © 2004 Wiley‐Liss, Inc.     

Efficacy of the phosphorylation of synthetic peptides by purified catalytic subunit of PKA (PKAcat) from bovine lens depends on the amino acid sequence of the peptides

Abstract: Protein kinase (PK) A catalytic (PKAcat) subunit was purified to homogeneity from bovine lens using a 100‐kDa cut‐off membrane filtration followed by different chromatographic procedures. The molecular weight of PKAcat was found to be 41 kDa. The kinase phosphorylates histone IIIs and other synthetic modified peptides of VRKRTLRRL with different amino acid environment. The extent of phosphorylation depends not only on the presence of Ser or Thr (phosphorylating residues) but also on other surrounding amino acid residues. Although some peptides compete in phosphorylating histone, they are not very significant. The result suggests that the extent of phosphorylation depends on the amino acid residue(s) surrounding phosphorylable residue(s) on the peptide.     

Hyposmotic challenge modulates function of L-type calcium channel in rat ventricular myocytes through protein kinase C

Aim:

To study the effects and mechanisms by which hyposmotic challenge modulate function of L-type calcium current (I_Ca,L) in rat ventricular myocytes.

Methods:

The whole-cell patch-clamp techniques were used to record I_Ca,L in rat ventricular myocytes.

Results:

Hyposmotic challenge(∼220 mosmol/L) induced biphasic changes of I_Ca,L, a transient increase followed by a sustained decrease. I_Ca,L increased by 19.1%±6.1% after short exposure (within 3 min) to hyposmotic solution. On the contrary, long hyposmotic challenge (10 min) decreased I_Ca,L to 78.1%±11.0% of control, caused the inactivation of I_Ca,L, and shifted the steady-state inactivation curve of I_Ca,L to the right. The decreased I_Ca,L induced by hyposmotic swelling was reversed by isoproterenol or protein kinase A (PKA) activator foskolin. Hyposmotic swelling also reduced the stimulated I_Ca,L by isoproterenol or foskolin. PKA inhibitor H-89 abolished swelling-induced transient increase of I_Ca,L, but did not affect the swelling-induced sustained decrease of I_Ca,L. NO donor SNAP and protein kinase G (PKG) inhibitor Rp-8-Br-PET-cGMPS did not interfere with swelling-induced biphasic changes of I_Ca,L. Protein kinase C (PKC) activator PMA decreased I_Ca,L and hyposmotic solution with PMA reverted the decreased I_Ca,L by PMA. PKC inhibitor BIM prevented the swelling-induced biphasic changes of I_Ca,L.

Conclusion:

Hyposmotic challenge induced biphasic changes of I_Ca,L, a transient increase followed by a sustained decrease, in rat ventricular myocytes through PKC pathway, but not PKG pathway. PKA system could be responsible for the transient increase of I_Ca,L during short exposure to hyposmotic solution.     

Gene cloning and utility phophorylation assay of a protein‐fused substrate for a highly sensitive detection of cdc2 protein kinase using a radioisotope detection technique for the development of a protein biochip

The prototype of the cdc2 protein kinase in mammalian cells regulates its entry into mitosis by phosphorylating a group of key proteins in the major cell cycle transitions. In this study, using the mep45 gene encoding the 45 kDa major envelope protein (Mep45) of Selenomonas ruminantium, a rumen bacteria, a Mep45‐fused substrate (PKTPKKAKKL‐Mep45, MFS‐cdc2) was cloned to detect the activity of cdc2 protein kinase. We report here on a strategy for the detection of a phosphorylation of a substrate catalyzed by cdc2 protein kinase by using a radioisotope detection technique. It is possible to constantly obtain a reasonable quantity of MFS‐cdc2 for the cdc2 protein kinase assay and its cost can be as low as a synthesized peptide. Results of the study indicate that the Mep45‐fused protein can be used effectively as a substrate for detecting the activity of cdc2 protein kinase and it can be used in developing a protein biochip for a high‐throughput screening and also for studying protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.