平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.     

平滑肌细胞迁移的肌球蛋白轻链非磷酸化途径

为了阐明平滑肌细胞迁移存在肌球蛋白轻链非磷酸化调节途径,研究花生四烯酸(arachidonicacid,AA)对肌球蛋白轻链非磷酸化状态下平滑肌细胞迁移的影响及其相关的信号传导途径.经Boyden小室跨膜迁移实验发现,AA对培养的兔血管平滑肌SM3细胞具有明显的诱导迁移作用.然而,当预先用10μmolL肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)特异性抑制剂ML7作用SM3细胞后,发现AA对SM3细胞仍然具有明显的诱导迁移作用,并呈剂量依赖性,这种诱导作用可被细胞外信号调节激酶12(ERK12)的特异性抑制剂PD98059或磷脂酶C(PLC)的特异性抑制剂U73122所拮抗.此外,Ⅱ型肌球蛋白抑制剂blebbistatin(BLB)可部分抑制“非磷酸化”状态下AA的诱导迁移作用.经Western印迹检测显示,10μmolLML7可完全抑制SM3细胞中20kD肌球蛋白轻链(MLC20)磷酸化,并且加入AA后MLC20仍为非磷酸化状态.应用免疫荧光染色法观察肌动蛋白在SM3细胞中分布的变化,发现在AA作用下肌动蛋白呈细胞边缘聚集现象,有伪足形成,细胞形态表现为迁移状态.预先用ML7作用后再加入AA,肌动蛋白的分布与上述结果相同.研究结果初步表明,在平滑肌细胞迁移的作用途径中,在MLC磷酸化调节途径受到抑制时,AA可诱导MLC非磷酸化的平滑肌细胞发生迁移,其分子机理可能与ERK12和PLC信号传导途径有关,非磷酸化的肌球蛋白直接参与了该迁移过程.     

肌球蛋白轻链激酶活性片段对肌球蛋白轻链的非钙依赖性磷酸化作用特征

肌球蛋白轻链激酶 (MLCK)的活性片段 (MLCKF)能比完整的MLCK更有效地、以非钙依赖性的方式磷酸化肌球蛋白轻链 (MLC2 0 )。该片段是用胰蛋白酶水解MLCK ,再经DEAE 5 2柱层析分离而获得的 ,分子量约为 6 1kD。Western印迹已证实该MLCKF与完整的MLCK同源。MLCKF对肌球蛋白轻链的磷酸化作用及其作用特征通过甘油电泳及ScoinImage扫描软件检测 ,肌球蛋白ATP酶活性通过分光光度法检测。实验结果证实 ,MLCKF催化的MLC2 0 非钙依赖性磷酸化 (CIPM)比MLCK催化的CIPM效力高、耗能多 ,但比MLCK催化的MLC2 0 钙依赖性磷酸化 (CDPM)效力低、耗能少 ;MLCKF催化的CIPM与MLCK催化的CIPM均较MLCK催化的CDPM稳定 ,不易受温育温度、温育时间及离子浓度等变化的影响 ,且对MLCK抑制剂ML 9敏感性低。     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

平滑肌肌球蛋白轻链激酶及ATP结合位点突变体对ATP酶活性的调节作用

目的：探寻MLCK的非激酶活性区域对MLCK活性的影响,进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制。方法：利用编码MLCK全长的pColdI表达载体对其ATP结合位点进行定点突变,获得无激酶活性的MLCK突变体;应用Glycerol—PAGE鉴定肌球蛋白磷酸化水平;应用孔雀绿方法检测重组MLCK对肌球蛋白ATP酶活性的影响。结果：MLCK／△ATP（突变型）失去磷酸化肌球蛋白轻链的激酶活性;重组MLCK（野生型）和MLCK／AATP（突变型）均可以在非钙条件下激活非磷酸化肌球蛋白Mg2＋-ATP酶活性,抑制磷酸化肌球蛋白的Mg2＋．ATP酶活性,而且激活与抑制作用均随着MLCK浓度的增加而增大,但二者对肌球蛋白的ATP酶活性的作用没有显著差异（P〉0．05）。结论：平滑肌肌球蛋白轻链激酶及ATP结合位点突变体具有激活非磷酸化肌球蛋白ATP酶活性的作用。     

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

鸡肌球蛋白轻链激酶表达载体的构建

鸡肌球蛋白轻链激酶（ＭＬＣＫ）在调节平滑肌细胞收缩中具有重要作用．用ＰＣＲ产生构建所需的限制性内切酶ＳａｌⅠ位点,将鸡ＭＬＣＫｃＤＮＡ插入质粒ｐＢＫｒｓｖ中,构建成ｐＢＫｒｓｖ－ＭＬＣＫ,并通过酶切图谱、ＳａｌⅠ位点序列分析得到证实．重组质粒在大肠杆菌中获得表达．     

花生四烯酸诱导肌球蛋白磷酸化被抑制的平滑肌细胞 迁移的信号传导途径

在应用肌球蛋白轻链激酶特异抑制剂ML-7抑制了肌球蛋白轻链磷酸化后,花生四烯酸(arachidonic acid,AA)仍可诱导兔血管平滑肌细胞（SM3）发生迁移.为了进一步阐明其信号传导途径,应用多种信号抑制剂,采用免疫印迹、Boyden小室和提取细胞膜蛋白等实验方法,对上述迁移作用的信号传导途径进行了深入的研究.结果显示,PTX（Gi蛋白抑制剂）、U73122（PLC抑制剂）、staurosporine (PKC抑制剂)、PD98059（ERK1/2抑制剂）和SB203580（p38抑制剂）分别可拮抗上述AA诱导的SM3细胞迁移作用,而SP600125（JNK抑制剂）的作用较弱.免疫印迹结果显示,AA可提高SM3细胞中PKC(ε)、ERK1/2、p38和JNK信号的磷酸化水平,呈时间依赖性, PTX或U73122可抑制上述作用;staurosporine可抑制由AA 引起的ERK1/2和JNK的磷酸化水平增强,但对p38的磷酸化水平无影响.还发现AA可促进PLCβ2的细胞膜移位, PTX可抑制其作用.上述结果表明,当肌球蛋白轻链的磷酸化被抑制后, AA可通过Gi蛋白的活化促进PLCβ2向细胞膜移位,进而通过激活PKC(ε)、ERK1/2、p38和JNK等信号转导途径而诱导SM3细胞发生迁移     

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

The contraction of smooth muscle is regulated primarily by intracellular Ca²⁺ signal. It is well established that the elevation of the cytosolic Ca²⁺ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca²⁺ concentration and force development revealed that the alteration of the Ca²⁺-sensitivity of the contractile apparatus as well as the Ca²⁺ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca²⁺ concentration is considered to contribute to the major mechanisms regulating the Ca²⁺-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca²⁺ elevation is increased either by Ca²⁺-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca²⁺-dependent manner but also by multiple kinases in a Ca²⁺-independent manner, thus adding a novel mechanism to the regulation of the Ca²⁺-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.     

动物肌球蛋白碱性轻链研究进展

肌球蛋白碱性轻链是肌球蛋白的组成成分之一,作为结构和调节蛋白在肌纤维的发生分化、肌肉运动和代谢等过程中发挥重要生理功能。简要综述肌球蛋白碱性轻链亚型的分析、碱性轻链基因的数量和分布、碱性轻链发育性表达等,为肌肉发育生物学研究提供参考。     

平滑肌肌球蛋白B的超沉淀与肌球蛋白轻链磷酸化

本文报导了牛胃肌球蛋白B(天然肌动球蛋白)的超沉淀性质。当钙离子、钙调蛋白和ATP存在时,肌球蛋白B出现超沉淀,在pH6.8和7.5处,有两个峰值。Ca~(2+)(PCa值8-4)对超沉淀影响的浓度-反应曲线呈典型的S形,表明当Ca~(2+)浓度处于微摩尔水平时产生超沉淀。伴随超沉淀发生了肌球蛋白调节轻链磷酸化。这说明肌球蛋白轻链的Ca~(2+)-CaM依赖性磷酸化可能包含在脊椎动物平滑肌收缩活动的调节机制中。     

鸡平滑肌肌球蛋白轻链激酶在NIH 3T3细胞中的表达

肌球蛋白轻链激酶（ＭＬＣＫ）在调节平骨肌细胞收缩过程中具有十分重要的作用。本言语通过将ＭＬＣＫｃＤＮＡ插到质粒ｐＢＫｒｓｖ中构建ｐＢＫｒｓｖ－ＭＬＣＫ,并转染至ＮＩＨ３Ｔ３细胞中,ＤＮＡ－ＰＣＲ、ＲＴ－ＰＣＲ和Ｗｅｓｔｅｒｎ ｂｌｏｔ分析表达转染细胞可表达ＭＬＣＫ。活生分析表明所表达的ＭＬＣＫ具有生物学活性。为进一步研究ＭＬＣＫ在信号传导,调节平骨肌收缩等作用奠定了基础。     

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK

Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2α stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide. The same decoy peptide also significantly inhibits PGF-2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par-4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par-4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par-4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.     

Regulation of the Actin-Activated Mg-ATPase of Brain Myosin via Phosphorylation by the Brain Ca2+, Calmodulin-Dependent Protein Kinases

We have previously isolated two Ca2+, calmodulin-dependent protein kinases with molecular weights of 120,000 (120K enzyme) and 640,000 (640K enzyme), respectively, by gel filtration analysis from rat brain. Chicken gizzard myosin light-chain kinase and the 120K enzyme phosphorylated two light chains of brain myosin, whereas the 640K enzyme phosphorylated both the two light chains and the heavy chain. The phosphopeptides of the light chains digested by Staphylococcus aureus V8 protease were similar among chicken gizzard myosin light-chain kinase, the 120K enzyme, and the 640K enzyme. Only the seryl residue in the light chains and the heavy chain was phosphorylated by the enzymes. The phosphorylation of brain myosin by any of these enzymes led to an increase in actin-activated Mg-ATPase activity. The results suggest that brain myosin is regulated by brain Ca2+, calmodulin-dependent protein kinases in a similar but distinct mechanism in comparison with that of smooth muscle myosin.     

肌球蛋白轻链激酶非激酶活性调节磷酸化肌球蛋白ATP酶活性及肌丝运动

肌球蛋白轻链激酶(myosin light chain kinase,MLCK)具有激酶和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为进一步阐明MLCK非激酶活性在平滑肌收缩过程中的调节作用,利用已删除部分激酶区域的MLCK重组体(pGEXF6.5)在大肠杆菌中进行表达,采用亲和层析技术纯化表达的MLCK片段,应用EnzChek磷分析试剂盒检测MLCK片段对磷酸化肌球蛋白、水解重酶解肌球蛋白(heavymeromyosin,HMM)及肌球蛋白亚片段1(subfragmentl,S1)ATP酶活性的影响,体外检测MLCK片段对肌动蛋白肌丝运动的调节.研究结果显示,pGEX-F6.5重组表达载体在大肠杆菌中以可溶性GST融合蛋白的形式表达.该融合蛋白经Glutathione-Sepharose4B纯化、SDS-PAGE鉴定得到较纯的单一表达条带.纯化的MLCK片段对磷酸化肌球蛋白、HMM和S1的ATP酶活性均有明显激活作用.MLCK片段激活磷酸化肌球蛋白ATP酶活性为:Vmax=(19.426±1.669)倍;Km=(0.486±0.106)μmol/L,MLCK片段对磷酸化HMM和S1的ATP酶活性也有相似的刺激作用.体外肌丝运动研究表明,随着MLCK片段浓度的增加,磷酸化肌球蛋白与肌动蛋白结合的数量不断增加,肌丝运动的速度也随之增加.上述结果表明,MLCK的C端非激酶活性具有调节磷酸化的肌球蛋白ATP酶活性及肌丝运动的作用.     

Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes

The kinetics of myosin regulatory light chain (MLC) phosphorylation by recombinant AMP-activated protein kinase (AMPK) were compared with commercial AMPK from rat liver and smooth muscle myosin light chain kinase (smMLCK). With identical amounts of activity units, initial rates of phosphorylation of MLC were at least 100-fold less with recombinant AMPK compared to smMLCK, whereas with rat liver AMPK significant phosphorylation was seen. In Madin-Darby Canine Kidney cells, AMPK activation led to an increase in MLC phosphorylation, which was decreased by a Rho kinase inhibitor without affecting AMPK activation. Therefore, MLC phosphorylation during energy deprivation does not result from direct phosphorylation by AMPK.

Structured summary

MINT-6800264: smMLCK (uniprotkb:P11799) phosphorylates (MI:0217) MLC (uniprotkb:P08590) by protein kinase assay (MI:0424)

MINT-6800252: AMPK (uniprotkb:Q13131) phosphorylates (MI:0217) ACC2 (uniprotkb:000763) by protein kinase assay (MI:0424)