G蛋白对离子通道的调控

Ｇ蛋白对离子通道的调控鲍国斌濮璐裴钢（中国科学院上海细胞生物学研究所,上海２０００３１关键词Ｇ蛋白Ｇ蛋白偶联受体离子通道Ｇ蛋白（Ｇｐｒｏｔｅｉｎ）由α、β、γ三个亚基组成,位于细胞膜的胞液侧。Ｇα亚基具有ＧＴＰ酶活性,使Ｇ蛋白偶联受体和效应分子发生可...     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

受体及离子通道蛋白磷酸化对跨膜信号传递的调节

跨膜信号转导是细胞信息传递的起始环节,受体和离子通道在此环节上起重要作用,受体和通道蛋白易受多种因素的调节。蛋白南磷酸化是受体及离子通道调节的关键步骤,不仅使受体及离子通道的功能发生改变,而且地影响到其在细胞的分布状况。受体 子通道蛋白酸化过程及其调节机制对于分析细胞信号转导的过程及细胞功能有着重要的作用。     

蛋白酪氨酸磷酸酶SHP-2与疾病的相关性研究进展

蛋白酪氨酸磷酸酶家族由130多种蛋白酪氨酸磷酸酶组成,它们和蛋白质酪氨酸激酶家族一起调控蛋白质中酪氨酸残基的磷酸化以及去磷酸化的动态平衡,它们的活性直接决定细胞内蛋白质的磷酸化水平的高低。SHP-2是蛋白酪氨酸磷酸酶家族的一员,在各种细胞和组织中均有广泛的表达,参与多个信号传导通路,介导细胞的生长、分化、迁移、粘附及凋亡等。SHP-2的表达异常会导致多种疾病的产生,但是相关综述较少,同时未见文献报道其在胶质瘤中的作用,因此本文简要介绍SHP-2的结构、功能、信号传导,并阐述了SHP-2与常见疾病的关系。     

酪氨酸蛋白磷酸酶

本文介绍了酪氨酸蛋白磷酸酶的研究现状。对各种组织中纯化的多种酪氨酸蛋白磷酸酶的性质研究,发现在大多数组织和细胞中存在多种形式的该酶,它们可分成三大类,但各种形式的酶之间的相互关系尚不清楚。酪氨酸蛋白磷酸酶在细胞的生长、分化、转化及信号传递过程中可能起重要作用。     

蛋白磷酸酶2A对微管的调控作用研究进展

微管是细胞骨架的主要成分之一,几乎存在于所有真核生物细胞之中,参与细胞众多生理功能。PP2A是真核生物体内存在最广泛的蛋白磷酸酶之一,可以调控大部分细胞生命活动,其中,包括微管所介导的许多生命活动。该文从以下方面介绍了PP2A在微管功能行使中的重要作用,包括PP2A参与微管蛋白翻译后修饰、调控分子马达和微管相关蛋白的活性、维持细胞周期中微管的动态平衡以及PP2A异常与微管类疾病的相关性。     

溶酶体离子通道蛋白在神经退行性疾病发生发展中的作用及机制研究

溶酶体离子通道蛋白异常引起溶酶体功能障碍是导致阿尔茨海默病(Alzheimer’s disease,AD)和帕金森病(Parkinson’s disease,PD)等神经退行性疾病的重要因素.溶酶体离子通道蛋白调节溶酶体内离子稳态、溶酶体膜电压以及溶酶体的酸度.溶酶体离子通道蛋白的结构或功能缺陷会引起溶酶体降解功能障碍,导致神经退行性疾病的发生发展.在这篇综述中,我们总结了各种离子通道蛋白调节溶酶体功能的过程及机制,以及离子通道蛋白异常参与神经退行性疾病的过程和机制.调节离子通道蛋白改善溶酶体的功能、促进异常聚集蛋白的清除,是神经退行性疾病治疗的潜在途径.     

植物蛋白磷酸酶及其在植物抗逆中的作用

随着多种蛋白磷酸酶在植物中的发现,可逆磷酸化作用在植物各种生理活动中的研究有许多重要的进展。本文概述了蛋白磷酸酶的类型与特点,并着重介绍近年来植物中多种磷酸酶的活性、基因、蛋白等各个水平上的鉴定工作。讨论了几类主要蛋白磷酸酶参与植物抵抗逆境胁迫的有关研究成果。     

植物蛋白磷酸酶及其在植物抗逆中的作用

随着多种蛋白磷酸酶在植物中的发现,可逆磷酸化作用在植物各种生理活动中的研究有许多重要的进展。本文概述了蛋白磷酸酶的类型与特点,并着重介绍近年来植物中多种磷酸酶的活性、基因、蛋白等各个水平上的鉴定工作。讨论了几类主要蛋白磷酸酶参与植物抵抗逆境胁迫的有关研究成果。     

蛋白酪氨酸磷酸酶SHP-2在乳腺癌细胞移动及粘附中的作用

探讨蛋白酪氨酸磷酸酶SHP 2在乳腺癌细胞MCF 7的移动及粘附中的作用 .利用基因重组技术分别将野生型SHP 2与突变型SHP 2与绿色荧光蛋白GFP的基因片段构成重组质粒 (SHP 2 GFP、SHP 2C >S GFP) .脂质体转染法分别转入MCF 7中 ,表达成功后筛选并建立SHP 2 GFP和SHP 2C >S GFP细胞株 .荧光显微镜观察细胞移动情况 ,免疫印迹法检测粘附分子E 钙粘蛋白和金属蛋白酶MMP 1及MMP 9的表达 .实验后建立SHP 2 GFP及SHP 2C >S GFP细胞株 ,同时观察到SHP 2C >S GFP细胞的形态发生明显改变 :从梭形状态变成圆形状态 .荧光显微镜发现 ,MCF 7细胞和SHP 2 GFP、SHP 2C >S GFP转染的细胞在 3h、6h、9h的移动情况分别是MCF 7为 10 %、2 3%、5 4% ,SHP 2 GFP为 15 %、4 9%、98% ,SHP 2C >S GFP为 4 %、11%、30 % .免疫印迹结果表明 ,SHP 2C >S GFP细胞的E 钙粘蛋白表达比SHP 2 GFP细胞明显升高 (P <0 0 5 ) .MMP 1及MMP 9的表达量在SHP 2 GFP细胞中有所增强 (P <0 0 5 ) .实验表明 ,SHP 2可能通过调节粘附分子和基质金属磷酸酶而在细胞移动、粘附中发挥重要作用     

miXED messages in ion channel modulation

Ion channels are modulated by multiple molecular mechanisms. In this issue of Neuron, Pietrzykowski et al. expand the mechanistic repertoire by demonstrating that ethanol-induced microRNA can modulate the pattern of mRNA splice variants from which BK potassium channels are constructed. Because BK channels are important targets of ethanol, this finding has implications for mechanisms of ethanol sensitivity and tolerance.     

Reversible protein phosphorylation regulates the activity of the slow-vacuolar ion channel

Protein storage vacuoles (PSVs) within barley ( Hordeum vulgare ) aleurone cells contain abundant K, Ca, Mg and P reserves. These minerals are transported from the PSV and are used to support growth of the embryo. In this study, the regulation of transport through slow-vacuolar (SV) ion channels in the tonoplast of barley aleurone PSVs was examined using the patch—clamp technique. Okadaic acid (OA), an inhibitor of protein phosphatase types 1 and 2A, reduced whole-vacuole SV currents by 60%. This inhibition by OA was overcome by exogenous calcineurin. Adding ATP (200 µM) to the bath solution as a substrate for kinase activity decreased SV channel activity by 70%. This reduction in activity was prevented by the kinase inhibitor H-7. From these data, it is concluded that protein phosphorylation can inhibit SV channel activity, and that both the protein kinase and protein phosphatase involved in this regulation are present at the PSV tonoplast. Whole-vacuolar SV currents were significantly higher when 2 mM ATP was used to bathe PSVs than with 200 µM ATP. Calmodulin-like domain protein kinase (CDPK) at either ATP concentration increased SV channel activity by ∼ 150%, implying that protein phosphorylation can also stimulate SV channel activity. When PSVs were treated with the ATP analog AMP-PNP, SV channel activity was not reduced. Hence, ATP hydrolysis is not essential for sustained SV channel activity. A model in which SV channel activity is regulated by protein phosphorylation at two sites is presented.     

Influenza virus M2 protein has ion channel activity.

The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0°C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0°C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens. Received: 4 December 2001 / Accepted: 7 January 2002     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Glycosaminoglycans and a newly purified aortic chondroitin proteoglycan block polycationic modulation of protein phosphatase activity

Recently, we described a bovine aortic phosphatase which we called PCM-phosphatase (polycation modulable) because its activity in vitro can be modulated by polycations such as polylysine and histone-H1 (Di Salvo J, Gifford D, Kokkinakis A. Modulation of aortic protein phosphatase activity by polylysine. Proc Soc Exp Biol Med 177:24-32, 1984). We We suspected that polycationic modulation might be inhibited by polyanionic glycosaminoglycans. Accordingly, an aortic anionic substance was purified by sequential steps including (a) heating aortic extracts at 90 degrees C, (b) precipitation of protein with (NH4)2 SO4, and (c) anionic-exchange chromatography on a Mono Q HR 5/5 column using the Pharmacia fast protein liquid chromatography system. Electrophoresis (polyacrylamide-agarose) of the purified substance revealed one band which stained metachromatically with toluidine blue; however, no staining occurred with Coomassie blue. Electrophoretic mobility increased following proteolytic digestion of the substance with papain. The substance produced concentration-dependent reversal of polylysine-mediated inhibition of myosin light chain dephosphorylation, and it also reversed polylysine-mediated stimulation of phosphorylase phosphatase activity expressed by PCM-phosphatase. Its ability to inhibit or reverse polycationic modulation was abolished after incubation with either chondroitinase AC or chondroitinase ABC. Based on these properties the substance was identified as a chondroitin proteoglycan. Commercially available glycosaminoglycans (heparin and chondroitin sulfates) also reversed polycationic modulation. The results show that modulation of phosphatase activity may be significantly modified by naturally occurring glycosaminoglycans. These studies may also have an important bearing on the purported roles of phosphatase(s) and glycosaminoglycans in calcification of soft tissues.     

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl⁻ current (I_Cl.PKA) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I_Cl.PKA and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I_Cl.PKA in a concentration- and voltage-dependent fashion (IC₅₀ at +100 mV = 0.3 ± 1.5 μM and at −100 mV = 5.1 ± 1.3 μM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I_Cl.PKA (30 μM) was tested on other cardiac ion currents. Inward current at −120 mV, comprised predominantly of the inward-rectifier background K⁺ current, I_K1, was reduced by ∼43% (n = 5). Under selective recording conditions, the Na⁺ current (I_Na) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at −40 mV of ∼82%; n = 8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca²⁺ channel current (I_Ca,L); fractional block at +10 mV of ∼49% and of ∼28% at −10 mV; n = 11, with a ∼−3 mV shift in the voltage-dependence of I_Ca,L activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I_Cl.PKA channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.