Protein Phosphorylation And Regulation Of Cytoskeleton I

细胞骨架蛋白磷酸化的拓扑调控 正常神经元和神经变性期间。 主要的问题涉及调节神经元细胞骨架蛋白磷酸化的区室化的因素和负责神经退行性疾病的失调机制。为了解决这个问题，我们正在使用两种实验方法（1）研究局部外源性信号，可能激活轴突内的激酶和（2）从哺乳动物和鱿鱼系统中的细胞体和轴突中提取的大分子磷酸化复合物的性质分析。关于第一个，我们已经表明，脯氨酸定向激酶（MAP激酶和cdk 5）是主要负责广泛的轴突磷酸化的多个KSP重复位点在NF-M和NF-H尾域。我们还证明了这些激酶在细胞培养中被信号转导级联激活，并磷酸化NF-M和NF-H KSP重复序列。在轴突中，负责髓鞘形成的神经胶质是外源性信号的一个来源。我们的研究髓鞘相关糖蛋白（MAG）- null小鼠，其中神经胶质细胞不能髓鞘轴突，已经表明，Erk 1/2和cdk 5的活动，以及细胞骨架蛋白磷酸化的MAG null小鼠的DRG下调。为了进一步探讨这种信号转导的机制，我们建立了一个体外培养系统的PC 12或DRG细胞与MAG转染的COS细胞共培养。在这里，我们也观察到激活Erk 1/2和cdk 5激酶耦合到神经元细胞中的NF磷酸化。关于第二种方法，我们正在使用鱿鱼巨轴突系统来了解细胞骨架蛋白磷酸化的地形调节。我们的初步研究已经证明了神经元细胞体和轴突之间的细胞骨架蛋白磷酸化的差异模式。在鱿鱼巨纤维系统中，提取的轴突磷酸化复合物（细胞骨架蛋白，激酶，磷酸酶和调节剂的多聚体复合物）表现出内源性底物的活性磷酸化，而来自细胞体的类似复合物相对不活跃。为了研究这种差异的性质，我们正在评估各种激酶/磷酸酶，以确定它们的活性差异是否是导致这种隔室特异性磷酸化模式的原因。经过四个夏天的工作，我们已经证实，在鱿鱼巨大的轴突系统，酪氨酸蛋白磷酸酶活性显着升高的perikarya（细胞体）相比，轴突室。 CDK 5：表达、调节及其在神经细胞功能中的作用： 虽然细胞周期蛋白依赖性激酶5（cdk 5）属于细胞周期依赖性激酶（CDK）家族，但由于其与分子量为35和39 kDa的神经元特异性蛋白结合，其仅在有丝分裂后细胞中有活性。在脑中，cdk 5被证明在存活和凋亡中起重要作用。自本实验室首次从大鼠脑cDNA文库中鉴定并克隆了该基因以来，我们对其在神经系统发育中的调控和作用进行了广泛的体内和体外研究。在cdk 5-/-小鼠中，我们观察到肿胀的脑干和脊髓神经元胞体中存在过度磷酸化的细胞骨架蛋白。这使我们开始寻找其他受cdk 5缺失影响的激酶。由于cdk 5在p35-/-小鼠中下调，我们使用这些小鼠的脑提取物，发现MAPK（Erk 1/2）被过度激活。事实上，我们已经表明，MEK 1是一个在体外和体内的cdk 5/p35磷酸化的目标; MEK 1的催化活性被抑制磷酸化在一个特定的网站，Thr 286。这些结果表明cdk 5参与了与其他信号转导途径的串扰。此外，我们发现与野生型相比，cdk 5（-/-）衍生的神经元凋亡增加。目前我们正在研究cdk 5在这一过程中的调控因素。我们的初步研究表明，cdk 5通过负调节c-jun N-末端激酶来防止神经元凋亡。我们计划研究细胞凋亡中cdk 5/p35表达的信号通路。p25是p35的截短形式，过度刺激cdk 5活性，促进神经元中细胞骨架蛋白的异常过度磷酸化，并且与细胞凋亡和神经退行性疾病如阿尔茨海默病相关。应激相关因子通过促进p35至p25的蛋白水解裂解来促进这种失调。一方面，重要的是调和cdk 5/p35通过其下调JNK途径抑制细胞凋亡和其与p25偶联时明显上调细胞凋亡。p35是体外和体内最有效的CDK 5激活剂。为了更好地理解激活机制，我们采用动力学方法研究p35或其各种截短形式与cdk 5之间的相互作用。与截短形式p25和p21形成的cdk 5复合物产生类似的最大激酶速率，而与全长p35或与跨越138至291个氨基酸残基的进一步截短形式（p16）复合的cdk 5产生比p25稍低的活化（80%）。缺乏必需的N-和C-末端结构域的另一截短肽比保留这些结构域中的任一个的肽对cdk 5表现出明显更高的亲和力，并且在体外表现为最具活性的抑制剂。我们将检查其特异性抑制cdk 5活性的能力，在体外以及在体内相对于其他成员的细胞周期蛋白依赖性激酶。

Topographic regulation of cytoskeletal protein phosphorylation in normal neurons and during neurodegeneration. The major questions relate to the factors that regulate the compartmentalization of cytoskeletal protein phosphorylation in neurons and the mechanisms responsible for deregulation in neurodegenerative disorders. To address this question we are using two experimental approaches (1) a study of local exogenous signals that may activate kinases within axons and (2) an analysis of the nature of macromolecular phosphorylation complexes extracted from cell bodies and axons in mammalian and squid systems. With respect to the first, we have shown that proline directed kinases (MAP kinase and cdk5) are primarily responsible for the extensive axonal phosphorylation of multiple KSP repeat sites in NF-M and NF-H tail domains. We have also demonstrated that these kinases are activated by signal transduction cascades in cell culture and phosphorylate NF-M and NF-H KSP repeats. In the axon one source of exogenous signals are the glia responsible for myelination. Our studies of myelin associated glycoprotein (MAG)- null mice in which glial cells fail to myelinate axons, have shown that the activities of Erk1/2 and cdk5, as well as cytoskeleton protein phosphorylation were down regulated in DRGs of MAG null mice. To further explore the mechanism of this signaling we have established an in vitro culture system of PC12 or DRG cells co-cultured with MAG-transfected COS cells. Here, too, we observed activation of Erk1/2 and cdk5 kinases coupled to NF phosphorylation in neuronal cells. With respect to the second approach, we are using the squid giant axon system to understand the topographic regulation of cytoskeletal protein phosphorylation. Our initial studies have demonstrated differential patterns of cytoskeletal protein phosphorylation between neuronal cell bodies and axons. In the squid giant fiber system, extracted axonal phosphorylation complexes (multimeric complexes of cytoskeletal proteins, kinases, phosphatases and regulators) exhibit active phosphorylation of endogenous substrates whereas similar complexes derived from cell bodies are relatively inactive. To study the nature of this difference, we are evaluating various kinases/phosphatases to determine whether differences in their activities are responsible for this compartment-specific pattern of phosphorylation. After four summers of work we have confirmed that in the squid giant axon system, tyrosine protein phosphatase activity is significantly elevated in the perikarya (cell body) compared to the axonal compartment. CDK5: Expression, regulation and its role in nerve cell function: While cyclin-dependent kinase5(cdk5) belongs to the family of cell cycle-dependent kinase (CDKs), it is active only in post mitotic cells due to its binding with neuron specific proteins of molecular weight 35 and 39 kDa. In brain, cdk5 has been shown to play an important role including survival and apotosis. Since its initial identification and cloning from the rat brain cDNA library in our laboratory, we have studied its regulation and role in nervous system development both in vivo and in vitro extensively. In cdk5-/- mice, we observed the presence of hyperphosphorylated cytoskeletal proteins in swollen brain stem and spinal cord neuronal perikarya. This led us to look for other kinases affected by the absence of cdk5. Since cdk5 is down regulated in p35-/-mice, we used brain extracts from these mice and found that MAPK (Erk1/2) was hyperactivated. Indeed, we have shown that MEK1 is an in vitro and in vivo target for cdk5/p35 phosphorylation; MEK1 catalytic activity was inhibited by phosphorylation at a specific site, Thr 286. These results suggest that cdk5 is involved in cross talk with other signal transduction pathways. In addition we found elevated apoptosis of neurons derived from cdk5(-/-) compared to wild type. Currently we are studying the factors regulated by cdk5 in this process. Our preliminary studies show that cdk5 prevents neuronal apoptosis by negative regulation of c-jun N-terminal kinase. We plan to study the signaling pathways that regulate the expression of cdk5/p35 in apoptosis. p25, a truncated form of p35, overstimulates cdk5 activity, promotes abnormal hyperphosphorylation of cytoskeletal proteins in neuron, and correlates with apoptosis and neurodegenerative disorders such as Alzheimers disease. Stress related factors promote this deregulation by favoring proteolytic cleavage of p35 to p25. It is important to reconcile, on the one hand, cdk5/p35 inhibition of apoptosis via its down regulation of the JNK pathway and its apparent upregulation of apoptosis when coupled to p25. p35 is the most effective activator of cdk5, both in vitro and in vivo. To better understand the mechanism of activation we have taken a kinetic approach to study the interaction between p35, or its various truncated forms, with cdk5. The cdk5 complexes formed with the truncated forms p25 and p21 produced similar maximum kinase rates, while cdk5 complexed with full length p35 or with a further truncated form spanning amino acid residues from 138 to 291, (p16) produced slightly less activation ( 80 %) compared to p25. A further truncation peptide that lacks both the essential N- and C-terminal domains exhibits markedly higher affinity for cdk5 than peptides retaining either one of these domains and behaves, in vitro, as a most active inhibitor. We will examine its ability to inhibit specifically the cdk5 activity in vitro as well as in vivo with respect to other members of cyclin-dependent kinases.