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.     

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.     

Myosin light chain kinase expression during smooth muscle development

The expression of smooth muscle myosin light chain kinase (MLCK) was investigated during chicken gizzard development. The molecular weight and the antigenic properties of MLCK did not change during development. The use of anion exchange high performance liquid chromatography (HPLC) enabled us to distinguish between MLCKs from post-hatched and adult chickens. A partial amino acid sequence determination of 4-day-old gizzard MLCK failed to disclose differences in the primary sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all stages of gizzard development, although charge variants due to post-translational modifications may exist.     

d-尼古丁对血管平滑肌细胞迁移的影响

为了在分子水平上揭示吸烟导致动脉粥样硬化的机制,探讨了烟草致病的主要成分d-尼古丁对豚鼠大脑基底动脉血管平滑肌细胞GbaSM-4迁移作用的影响。应用Boyden小室实验发现,d-尼古丁具有促进GbaSM-4细胞迁移的作用。免疫荧光染色显示,在d-尼古丁作用下有GbaSM-4细胞伪足内肌动蛋白表达和分布增加的现象。为了进一步阐明d-尼古丁促进平滑肌细胞迁移作用的分子机制,应用RT-PCR方法检测到在GbaSM-4细胞内有α7型烟碱乙酰胆碱受体的表达。应用烟碱乙酰胆碱受体的特异性抑制剂甲基牛扁碱和肌肉收缩的关键酶——肌球蛋白轻链激酶（myosin light chain kinase,MLCK）抑制剂ML-9作用GbaSM-4细胞后,发现d-尼古丁对GbaSM-4细胞的诱导迁移作用被明显的抑制。采用RNA干扰技术,成功地使GbaSM-4细胞内MLCK的表达水平下调,观察到d-尼古丁对GbaSM-4细胞的诱导迁移作用也被明显的抑制。上述研究结果表明,d-尼古丁以趋化因子的作用促进血管平滑肌细胞迁移,其分子机制可能与α7型烟碱乙酰胆碱受体和MLCK等因素有关,这一发现为揭示吸烟导致动脉粥样硬化提供了实验依据。     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

Effect of phosphorylation on the actin-activated atpase activity of myosin

The purpose of this study was to test the hypothesis that the phosphorylation of myosin is solely responsible for the activation of the Mg²⁺-ATPase activity of gizzard actomyosin. Using a washed natural actomyosin and a reconstituted actomyosin it was shown that phosphorylation alone caused only a slight activation of ATPase activity. Full activity was obtained only when proteins in addition to the myosin light chain kinase were added. It is evident from these results that: 1) there is no simple relationship between the extent of myosin phosphorylation and the specific Mg²⁺-ATPase activity of actomyosin and 2) in order for full activation by actin of the Mg²⁺-ATPase activity of phosphorylated myosin additional factors are required.     

Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.     

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。     

Calcium,cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca²⁺ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca²⁺ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.     

Process Formation in Astrocytes: Modulation of Cytoskeletal Proteins

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.