Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.     

Phosphorylation of smooth muscle myosin: evidence for cooperativity between the myosin heads

The relationship between the actin-activated adenosinetriphosphatase activity of smooth muscle myosin and the extent of myosin light chain phosphorylation is nonlinear. It is suggested that the phosphorylation of the two heads of smooth muscle myosin is an ordered process and that the two heads are influenced by cooperative interactions.     

Inhibition of myosin light chain kinase by p21-activated kinase

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.     

蛋白质磷酸化调控羊肉肌原纤维蛋白的功能

【目的】通过激活蛋白激酶的活性提高羊肉肌原纤维蛋白的磷酸化水平,分析磷酸化水平的提高对羊肉肌原纤维蛋白降解程度、收缩功能的影响,进一步揭示蛋白质磷酸化对羊肉肌肉嫩化的作用机理。【方法】通过添加蛋白激酶A激活剂Forskolin、蛋白激酶C激活剂佛波酯（PMA）,提高蛋白激酶的活性,改变羊肉肌原纤维蛋白的磷酸化水平。比较激活剂处理组与空白对照组肌原纤维小片化指数（MFI）、条带降解程度、肌节长度等指标的差异,确定蛋白质磷酸化水平对羊肉肌肉收缩和肌肉降解的影响。【结果】将羊肉样品在蛋白激酶溶液中培养24 h,在培养结束后1、2和4 h,PMA处理组（PKC激活组）的蛋白激酶活性显著高于空白对照组（P＜0.05）,而在培养后1和4 h,Forskolin处理组（PKA激活组）的蛋白激酶活性显著高于空白对照组（P＜0.05）。PMA处理组和Forskolin处理组的最高蛋白激酶活性出现在培养后1 h。Forskolin和PMA通过提高激酶活性显著提高肌联蛋白（Titin）、肌球蛋白结合蛋白C（Myosin binding protein C）、原肌球蛋白（Tropomyosin）、肌球蛋白轻链2（Myosin light chain 2）等蛋白质的磷酸化水平,肌球蛋白重链、肌动蛋白质的磷酸化水平没有显著变化,Forskolin组和PMA组的蛋白质降解程度及肌节长度均低于对照组。【结论】肌原纤维蛋白磷酸化水平提高不利于羊肉肌原纤维蛋白的降解。另一方面,磷酸化水平可能通过对肌球蛋白轻链2的作用增强羊肉肌肉的收缩作用力,通过促进相邻原肌球蛋白的相互作用促进肌细丝收缩,影响肉的僵直进程和嫩化进程。     

蛋白质磷酸化对肌动球蛋白解离及其乙酰化水平的影响

[目的]研究肌球蛋白重链和肌动蛋白磷酸化对其乙酰化水平、肌动球蛋白解离及ATP酶活性的影响,为通过调控磷酸化水平改善肉品嫩度提供理论依据.[方法]以羊背最长肌为材料制备肌肉匀浆液,采用碱性磷酸酶抑制剂(抑制去磷酸化)和蛋白激酶抑制剂(抑制磷酸化)调控其磷酸化水平,在4℃分别孵育0、0.5、4、12、24、48和72 h...     

Myosin light chain phosphorylation does not modulate cross-bridge cycling rate in mouse skeletal muscle

An attempt was made to determine whether phosphorylation of the myosin light chain represents a thick filament-associated mechanism for modulating the rate of cross-bridge cycling in mouse skeletal muscle. When the degree of light chain phosphorylation was varied independently of tetanus duration, there was no correlation of phosphorylation with cross-bridge turnover rate, as measured by the shortening velocity of the muscle. It is concluded that in intact skeletal muscle phosphorylation of the myosin light chain does not in itself modulate cross-bridge cycling rate and that previously reported changes in cycling rate were due to other factors that may vary with tetanus duration.     

Protein kinase injection reduces voltage-dependent potassium currents

Intracellular iontophoretic injection of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase increased input resistance and decreased a delayed voltage-dependent K+ current of the type B photoreceptor in the nudibranch Hermissenda crassicornis to a greater extent than an early, rapidly inactivating K+ current (IA). This injection also enhanced the long-lasting depolarization of type B cells after a light step. These findings suggest the involvement of cyclic adenosine monophosphate-dependent phosphorylation in the differential regulation of photoreceptor K+ currents particularly during illumination. On the other hand, conditioning-induced changes in IA may also be regulated by a different type of phosphorylation (for example, Ca2+-dependent).     

宰后肌肉中肌球蛋白磷酸化调控肌动球蛋白解离作用机制

【目的】研究宰后肌肉中肌球蛋白磷酸化与肌动球蛋白解离之间的关系,分析其磷酸化水平的变化对肌动球蛋白解离的影响,探究肌球蛋白磷酸化对宰后肌肉肌节长度与嫩度的作用。【方法】取宰后30 min内的羊背最长肌,在4℃条件下分别成熟6、24、48和72 h,通过SDS-PAGE电泳、Pro-Q染色和蛋白质免疫印迹测定肌球蛋白的磷酸化水平和肌动球蛋白解离程度随宰后时间的变化;测定肌动球蛋白ATP酶的活性,分析宰后不同时间点肌球蛋白与肌动蛋白结合作用力的强弱;采用透射电镜分析宰后肌节长度随时间的变化。【结果】研究发现宰后肌肉中肌球蛋白轻链2的磷酸化水平在0.5—48 h快速降低(P0.05),并在48 h达到最低点,在48—72 h有所升高(P0.05),但其最终磷酸化水平明显低于初始值。肌动球蛋白的解离程度在宰后初期(0.5—6 h)显著降低(P0.05),在6—48 h显著升高(P0.05),并于48—72 h维持稳定,其最终解离程度显著高于宰后0.5 h的初始值。肌动球蛋白ATPase活性在宰后初期(0.5—6 h)略有升高,6—24 h快速上升(P0.05),并在24 h达到最高点,24—72 h逐渐降低;而肌节长度的变化则与之相反,呈先下降后上升的趋势,并在24 h达到肌节最短点。【结论】羊宰后肌肉中的肌球蛋白轻链2磷酸化水平的变化对肌球蛋白与肌动蛋白的相互作用有较大的影响,且肌节收缩(肌球蛋白与肌动蛋白的相互作用力)与肌动球蛋白的解离(肌球蛋白与肌动蛋白的相互作用量)并不是一个同步的进程。肌球蛋白轻链2的磷酸化修饰负向调控肌动球蛋白解离和肌动球蛋白ATPase活性,导致肌节的收缩与舒张,进而调控肉品最终的嫩度。     

Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum

The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.     

Myosin rod phosphorylation and the catch state of molluscan muscles

"Catch" is a prolonged state of tension in molluscan smooth muscles shown by mechanical measurements to be associated with the level of protein phosphorylation. Myosin isolated from these muscles is unusual in being phosphorylated in the rod portion by an endogenous kinase, like certain nonmuscle myosins. These findings suggest that the myosin rod is a target for phosphorylation and that this reaction may control the transition from catch to relaxation.     

Catecholamine and dibutyryl cyclic AMP effects on myosin adenosine triphosphatase in cultured rat heart cells

Catecholamines and dibutyryl adenosine 3', 5'-monophosphate (dibutyryl cyclic AMP) increase the activity of myosin adenosine triphosphatase in cultured rat heart cells. Dichloroisoproterenol, an inhibitor of the beta receptor of the catecholamines, inhibits the action of the catecholamines but not of cyclic AMP.     

Muscle contraction and free energy transduction in biological systems

Muscle contraction occurs when the actin and myosin filaments in muscle are driven past each other by a cyclic interaction of adenosine triphosphate (ATP) and actin with cross-bridges that extend from myosin. Current biochemical studies suggest that, during each adenosine triphosphatase cycle, the myosin cross-bridge alternates between two main conformations, which differ markedly in their strength of binding to actin and in their overall structure. Binding of ATP to the cross-bridge induces the weak-binding conformation, whereas inorganic phosphate release returns the cross-bridge to the strong-binding conformation. This cross-bridge cycle is similar to the kinetic cycle that drives active transport and illustrates the general principles of free energy transduction by adenosine triphosphatase systems.     

Protein kinase translocation as an early event in the hormonal control of uterine contraction

beta-Adrenergic stimulation with isoproterenol inhibits contractility, increases cyclic adenosine monophosphate (AMP) concentration, decreases the concentration of unsaturated cyclic AMP receptor sites, and increases cyclic AMP-independent kinase in the uterus of ovariectomized rats. The total soluble kinase activity is reduced. The protein kinase activity lost from the cytosol was translocated to the microsomal fraction mostly in a cyclic AMP-independent form, suggesting a particulate substrate for the activated enzyme.     

Oligodendrocyte adhesion activates protein kinase C-mediated phosphorylation of myelin basic protein

When isolated adult oligodendrocytes adhere to a substratum myelinogenesis occurs. Investigation of the mechanism by which this happens indicated that the oligodendrocyte-substratum interaction activated protein kinase C-dependent phosphorylation of myelin basic protein and promoted the synthesis of myelin basic protein. In addition, when agents that activate protein kinase C (second messenger diacylglycerol or a tumor-promoting phorbol ester) were added to nonattached oligodendrocytes, they mimicked the influence of the substratum by inducing phosphorylation of myelin basic protein; and reagents that increase cellular adenosine 3', 5'-monophosphate (cyclic AMP) inhibited phosphorylation of myelin basic protein. Thus, at least in vitro, the interaction between oligodendrocytes and the substratum may mediate myelinogenic events, and phosphorylation of myelin basic protein may be an early requirement in the sequence of steps that ultimately results in myelin formation.     

Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle

Stress development depended on calcium-stimulated myosin phosphorylation in an arterial smooth muscle preparation in which the concentration of calcium was controlled. However, developed stress was maintained at a concentration of calcium that did not support phosphorylation. These results, in conjunction with other evidence, suggest that the interaction of two regulatory mechanisms with different calcium sensitivities regulate both stress and the rate and energetics of contraction.     

Regulation of phosphodiesterase synthesis: requirement for cyclic adenosine monophosphate-dependent protein kinase

Endogenous cyclic adenosine monophosphate (AMP) and its dibutyryl derivative increase cyclic AMP phosphodiesterase activity in cultured lymphoma cells. This effect is prevented by cycloheximide. A variant population of cells deficient in cyclic AMP-dependent protein kinase contains lower basal phosphodiesterase activity, which cannot be induced by cyclic AMP.     

Inhibition of platelet aggregation by monoclonal antibody reactive with beta 2-microglobulin chain of HLA complex

A mouse monoclonal antibody that reacts with beta 2-microglobulin, the light chain of class I major histocompatibility antigens, inhibited the second wave of human platelet aggregation induced by adenosine diphosphate and epinephrine and blocked aggregation and platelet protein phosphorylation induced by sodium arachidonate. Thrombin-induced platelet aggregation was inhibited at threshold concentrations but not at higher concentrations. The antibody also inhibited aggregation and secretion in response to thromboxane A2 or the stable endoperoxide analog, U46619. These results suggest that beta 2-microglobulin in the histocompatibility complex is intimately associated with transmission of the endoperoxide-thromboxane signal at the platelet membrane.     

Cyclic adenosine monophosphate: potassium-dependent action on vascular smooth muscle membrane potential

Dibutyryl cyclic adenosine monophosphate and theophylline hyperpolarize smooth muscle of rabbit main pulmonary artery in low concentrations of potassium (1 millimole per liter) but do not have a significant effect on the membrane potential in the presence of high concentrations of potassium (10 millimoles per liter). The dependence of the hyperpolarizing effect on a low external concentration of potassium is similar to that observed with isoproterenol. Prior treatment with theophylline potentiated the hyperpolarizing action of isoproterenol. These findings are compatible with the assumption that potassium-dependent, beta-adrenergic hyperpolarization is mediated by cyclic adenosine monophosphate.     

Endogenous substrate for cyclic AMP-dependent protein kinase in adrenocortical polyadenylated messenger ribonucleoproteins

An endogenous polysomal cyclic AMP-dependent protein kinase specifically phosphorylates a 150,000-dalton peptide bound to an adrenocortical polyadenylated messenger ribonucleoprotein complex. There is a possibility that this protein is a physiological substrate of cyclic AMP-dependent protein kinase and that the phosphorylation and dephosphorylation of this substrate may be important in the translation control of adrenal polyadenylated messenger RNA.