Protein Phosphorylation And Regulation Of Cytoskeleton I

蛋白质磷酸化和细胞骨架调控 I

• 批准号: 6533324
• 负责人: HARISH C PANT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6533324
• 关键词: astrocytes; axon; axoplasm; biological signal transduction; cyclin dependent kinase; cytoskeletal proteins; cytoskeleton; developmental neurobiology; enzyme activity; gene expression; hippocampus; laboratory mouse; laboratory rat; mitogen activated protein kinase; neural degeneration; neurofilament proteins; neuronal transport; neurons; nucleic acid repetitive sequence; phosphomonoesterases; phosphorylation; protein structure function; secretion; squid; tau proteins; tissue /cell culture

Phosphorylation of neurofilament proteins (NFPs) in vertebrate neurons regulates neurofilament assembly, transport and stability of large axonal structures. Phosphorylation of KSP (lys-ser-pro) repeat motifs in carboxy tail domains of high molecular weight neurofilament sub-units (NF-M and NF-H) contributes to increased axonal caliber, and, thereby, the conducting velocity of impulses in nerve fibers. In a normal neuron, phosphorylation of these KSP repeats seems to be topographically localized within the axonal compartment of neurons. Although all components of the cytoskeletal system of axons are synthesized in cell bodies, including the kinases involved in their phosphorylation, the phosphorylation of some elements such as neurofilaments occurs primarily in axons. The aberrant phosphorylation of the KSP repeats in perikarya has been proposed as a mechanism for pathological NF-accumulation in the cell bodies and proximal axons in several neurodegenerative diseases. These include Amyotrophic Lateral Sclerosis (ALS), Parkinson’s Disease, Dementia with Lewy bodies, diabetic neuropathy and Alzheimer's Disease with concomitant Lewy body pathology. Thus, phosphorylation of the NFs, and its regulation, seem to be involved in several physiological functions and pathological processes. However, the mechanisms responsible for the regulation of this phosphorylation are not well understood. The primary objective of this laboratory has been to understand the mechanism(s) of phosphorylation of NFs as well as other cytoskeletal proteins in neurons. This may provide insight into the processes underlying neurodegenerative disorders. Our previous studies have demonstrated that most ser/thr residues in the KSP repeats in human and rat NF-H are phosphorylated in vivo, suggesting that proline-directed kinases are responsible for their phosphorylation. In our recent studies we have shown that a family of mitogen activated kinases (Erk1/2, SAPK) and neuronal cyclin dependent kinase 5 (cdk5) can phosphorylate KSP repeats of NF-M and NF-H in their C-terminal tail domains in vitro and in vivo in neuronal and in transfected cells. For example, non-neuronal cell lines co-transfected with NF-M or NF-H, and kinases, that are either constituitively active or activated by a signal transduction pathway (MAP kinases), will induce robust phosphorylation of KSP repeats in neurofilament tail domains. These studies suggest that the activation of proline-directed kinase pathways in vivo are responsible for axonal NF-phosphorylation. We found that the selectivity of these kinases depends upon the nature of the KSP motifs. For example, in rat NF-H, where the majority of KSP repeats (repeating 41 times) are present as KSPXXXK motifs, are primarily phosphorylated by the activation of ERK1/2 kinases, while integrin a1/b1-mediated activation of cdk5 phosphorylation of human NF-H in a human cell line, has higher efficacy compared to activated ERK1/2. This is due to the fact that human NF-H has more KSPXK repeat motifs,, the consensus sequence for cdk5, repeating 32 times, than KSPXXXK (repeating 11 times). These studies suggest that different kinase cascades may mutually interact in the sequential phosphorylation of multiple sites in NFs, depending upon the species and environmental cues. A similar mechanism may be operative for other cytoskeletal proteins such as human tau; the microtubule associated protein found in axons. Numerous proline and non-proline-directed kinases have been shown to phosphorylate human tau in vivo and in vitro, producing hyperphosphorylated tau in the abnormal aggregates of paired helical filaments found in perikarya of Alzheimer neurons. Some clues as to the topographic regulation of phosphorylation in neurons have come from our summer studies on the squid giant axon at the Marine Biological Laboratory in Woods Hole. In axoplasm of the giant axon we have demonstrated active multimeric complexes of cytoskeletal proteins (tubulin and neurofilaments) associated with proline directed kinases such as cdc2-like kinases and Erk1/2 together with non-proline directed kinases such as CKI, PKA and CAMK. Such complexes are absent in the cell bodies of those axons; instead we found different, relatively inactive, complexes of cytoskeletal proteins and kinases. This suggests that the compartmentalization of cytoskeletal phosphorylation and its regulation depends on the nature of the “phosphorylating machines” that are segregated into the respective compartments. Significantly, we have followed up these studies by demonstrating that similar multimeric phosphorylating complexes can be extracted from rat brain lysates by identical techniques. We have continued our studies on neuronal cyclin dependent kinase 5 (cdk5) to further understand its role in nerve cell function and regulation. Our previous studies have shown that cdk5 null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering due to abnormal neuronal migration, lack of cerebellar foliation, and degeneration of neurons in the brain stem and spinal cord. Recently, in an attempt to rescue the cdk5 knockout phenotype, we have reconstituted cdk5 overexpression in a tissue specific manner in the brain in cdk5 null mice and examined its effect on the lethal phenotype. Unlike the cdk5 null mice, cdk5 that overexpress the cdk5 transgene with a p35 (the cyclin like cdk5 activator) promoter (TgKO mice) were viable and fertile. Since p35 is expressed primarily in the nervous system, in these mice cdk5 overexpression was limited to neurons. In contrast, wild type mice express cdk5 in neurons as well as astrocytes. The cerebral cortical layering pattern and cerebellar foliation was normal, and no degenerating neurons were seen in the brain stem and the spinal cord. We were thus able to reverse the phenotype and associated lethality of cdk5 null mice. This investigation confirms that cdk5 expression in the nervous system is critical for embryonic development and survival. In a separate study we have provided the evidence for a novel role for cross talk between cdk5 and MAP kinase pathway. Cdk5 down regulates the activity of MAP kinase kinase 1 (MEK1), the upstream regulator of ERK1 by phosphorylating its Thr-286 residue both in vitro and in vivo. Using p35 null mice which lack appreciable cdk5 activity, we show that MEK1 activity is regulated by cdk5 in vivo. These observations provide evidence for a cross talk between cdk5 and the MAP kinase pathway in the brain. This may serve as another regulatory mechanism that modulates signal transduction pathways during growth, differentiation and apoptotic cell death.

脊椎动物神经元中神经丝蛋白 (NFP) 的磷酸化调节神经丝组装、运输和大轴突结构的稳定性。高分子量神经丝亚基（NF-M 和 NF-H）羧基尾域中 KSP（lys-ser-pro）重复基序的磷酸化有助于增加轴突口径，从而提高神经纤维中脉冲的传导速度。在正常神经元中，这些 KSP 重复序列的磷酸化似乎在神经元的轴突区室中局部定位。尽管轴突细胞骨架系统的所有成分都是在细胞体中合成的，包括参与其磷酸化的激酶，但某些元素（例如神经丝）的磷酸化主要发生在轴突中。核周体中 KSP 重复序列的异常磷酸化已被认为是几种神经退行性疾病中细胞体和近端轴突中病理性 NF 积累的机制。这些疾病包括肌萎缩侧索硬化症 (ALS)、帕金森病、路易体痴呆、糖尿病性神经病变和伴有路易体病理的阿尔茨海默病。因此，NF 的磷酸化及其调节似乎涉及多种生理功能和病理过程。然而，负责调节这种磷酸化的机制尚不清楚。该实验室的主要目标是了解神经元中 NF 以及其他细胞骨架蛋白的磷酸化机制。这可能有助于深入了解神经退行性疾病的潜在过程。我们之前的研究表明，人和大鼠 NF-H 的 KSP 重复序列中的大多数丝氨酸/苏氨酸残基在体内被磷酸化，表明脯氨酸定向激酶负责其磷酸化。在我们最近的研究中，我们发现丝裂原激活激酶家族（Erk1/2、SAPK）和神经元细胞周期蛋白依赖性激酶 5 (cdk5) 可以在体外和体内神经元和转染细胞中磷酸化其 C 端尾部结构域中 NF-M 和 NF-H 的 KSP 重复序列。例如，与 NF-M 或 NF-H 和激酶（组成型活性或由信号转导途径（MAP 激酶）激活）共转染的非神经元细胞系将诱导神经丝尾区中 KSP 重复序列的强烈磷酸化。这些研究表明体内脯氨酸定向激酶途径的激活是轴突 NF 磷酸化的原因。我们发现这些激酶的选择性取决于 KSP 基序的性质。例如，在大鼠 NF-H 中，大多数 KSP 重复序列（重复 41 次）以 KSPXXXK 基序存在，主要通过 ERK1/2 激酶的激活而磷酸化，而在人类细胞系中，整合素 a1/b1 介导的人 NF-H 的 cdk5 磷酸化激活与激活的 ERK1/2 相比具有更高的功效。这是因为人类 NF-H 比 KSPXXXK（重复 11 次）具有更多的 KSPXK 重复基序（cdk5 的共有序列），重复 32 次。这些研究表明，不同的激酶级联可能在 NF 中多个位点的顺序磷酸化中相互相互作用，具体取决于物种和环境因素。类似的机制可能适用于其他细胞骨架蛋白，例如人类 tau 蛋白；轴突中发现的微管相关蛋白。许多脯氨酸和非脯氨酸定向激酶已被证明可以在体内和体外磷酸化人 tau，在阿尔茨海默神经元核周中发现的成对螺旋丝异常聚集体中产生过度磷酸化的 tau。关于神经元磷酸化的地形调节的一些线索来自于我们在伍兹霍尔海洋生物实验室对鱿鱼巨轴突的夏季研究。在巨轴突的轴浆中，我们证明了细胞骨架蛋白（微管蛋白和神经丝）的活性多聚体复合物，与脯氨酸定向激酶（例如cdc2样激酶和Erk1/2）以及非脯氨酸定向激酶（例如CKI、PKA和CAMK）相关。这些轴突的细胞体中不存在这种复合物；相反，我们发现了不同的、相对不活跃的细胞骨架蛋白和激酶复合物。这表明细胞骨架磷酸化的区室化及其调节取决于被隔离到各个区室中的“磷酸化机器”的性质。值得注意的是，我们对这些研究进行了后续研究，证明可以通过相同的技术从大鼠脑裂解物中提取类似的多聚体磷酸化复合物。我们继续对神经细胞周期蛋白依赖性激酶5（cdk5）进行研究，以进一步了解其在神经细胞功能和调节中的作用。我们之前的研究表明，cdk5缺失小鼠表现出独特的表型，其特征是围产期死亡率、由于神经元迁移异常而导致的大脑皮层分层破坏、小脑叶状结构的缺乏以及脑干和脊髓神经元变性。最近，为了挽救 cdk5 敲除表型，我们在 cdk5 缺失小鼠的大脑中以组织特异性方式重建了 cdk5 过表达，并检查了其对致死表型的影响。与 cdk5 缺失小鼠不同，用 p35（细胞周期蛋白样 cdk5 激活剂）启动子过表达 cdk5 转基因的 cdk5（TgKO 小鼠）是有活力和可生育的。由于 p35 主要在神经系统中表达，因此在这些小鼠中 cdk5 过度表达仅限于神经元。相比之下，野生型小鼠在神经元和星形胶质细胞中表达cdk5。大脑皮质分层模式和小脑叶状结构正常，脑干和脊髓未见变性神经元。因此，我们能够逆转 cdk5 缺失小鼠的表型和相关致死率。这项研究证实，神经系统中的 cdk5 表达对于胚胎发育和存活至关重要。在另一项研究中，我们为 cdk5 和 MAP 激酶通路之间的串扰提供了新作用的证据。在体外和体内，Cdk5 通过磷酸化其 Thr-286 残基，下调 MAP 激酶激酶 1 (MEK1)（ERK1 的上游调节因子）的活性。使用缺乏明显 cdk5 活性的 p35 null 小鼠，我们表明 MEK1 活性在体内受到 cdk5 的调节。这些观察结果为 cdk5 和大脑中 MAP 激酶通路之间的串扰提供了证据。这可能作为另一种调节机制，在生长、分化和细胞凋亡过程中调节信号转导途径。