Protein Phosphorylation And Regulation Of Cytoskeleton I

在神经系统中，细胞骨架蛋白在神经系统中被广泛磷酸化。这种磷酸化大部分发生在细胞骨架蛋白的脯氨酸导向丝氨酸/苏氨酸（S/TP）残基上，如神经丝高分子量和中等分子量（NF-H和NF-M）以及微管相关蛋白MAP-2和tau。正常情况下，这些蛋白在神经元的轴突室中被选择性磷酸化。虽然所有的激酶、磷酸酶及其底物和调节因子都是在细胞体中合成的，但在细胞体中很少或根本没有检测到S/T残基上的细胞骨架蛋白磷酸化。这种磷酸化的区隔化受到严格调控。然而，在一些神经病理条件下，如肌萎缩性侧索硬化症（ALS）、阿尔茨海默病(Alzheimer？阿尔茨海默病（AD）和匹克病（pick disease）就会被解除管制。这些细胞骨架蛋白在S/T残基上有异常磷酸化，在细胞体中它们以聚集形式积累。这会导致这些受影响区域的大量神经元细胞死亡。主要的焦点是确定磷酸化这些S/T残基的激酶。在寻找这些激酶的过程中，我们发现除了有丝分裂原活化蛋白激酶（MAPKs），例如ERK1/2， Cdk5是另一个主要的激酶，可以磷酸化NF-M、NF-H和tau蛋白羧基末端的多个赖氨酸/丝氨酸/脯氨酸（KSP）重复序列。Cdk5从脑组织中纯化，并从我们实验室的大鼠cDNA文库中克隆。我们已经证明，在原代神经元培养和转染的非神经元细胞中，各种信号转导级联可以激活cdk5。髓鞘胶质细胞是外源性激活信号的主要来源。使用髓鞘相关糖蛋白（MAG）敲除小鼠，没有髓鞘形成，我们发现与野生型小鼠相比，脑和坐骨神经提取物中的ERK1/2和Cdk5活性下调。这些研究还表明，神经胶质-轴突相互作用诱导的ERK1/2和Cdk5活性升高刺激了NF-M、NF-H、MAP-2B和tau的轴突磷酸化。我们已经得出结论，MAG可能作为配体激活适当的激酶级联，导致轴突细胞骨架蛋白磷酸化。此外，整合素等基质元素也可以激活这些激酶。最近，我们的实验室专注于研究cdk5在神经系统功能和发育中的作用。尽管Cdk5在所有细胞中普遍表达，并且与周期蛋白依赖性激酶家族（Cdks）的其他成员具有高度的同源性，但其活性仅在有丝分裂后神经元中发现，因为其激活因子p35和p39主要在神经元中表达。包括我们在内的不同实验室的研究表明，Cdk5是一种多功能S/T蛋白激酶，参与从神经突生长和神经元迁移到突触活性和细胞存活的广泛神经元功能。例如，我们已经证明，cdk5 KO小鼠（-/-）是致命的，在E16和P0之间死亡之前表现出异常的皮质生成和其他神经元异常。最近，我们也证明了cdk5在cdk5 KO小鼠的神经元中的实验性重新表达完全恢复了野生型，清楚地表明神经元而非胶质细胞的cdk5活性是正常发育和生存所必需的。cdk5的多种作用部分是基于它在神经细胞存活、生长和分化的信号转导网络中起关键作用的证据。我们观察到cdk5-/-小鼠肿胀的脑干和脊髓核周存在过度磷酸化的细胞骨架蛋白。这导致我们寻找受cdk5缺失影响的其他激酶。由于cdk5活性在p35-/-小鼠中下调，我们发现MAPK （Erk1/2）在脑提取物中被过度激活，这进一步表明cdk5调节了MAP激酶级联中的一个位点。这些结果表明，Cdk5参与了与其他信号转导和生存途径的“串扰”，可能调节了对特定信号的反应强度。Cdk5活性在神经系统中受到严格调控，它可能促进神经元细胞存活或诱导程序性细胞死亡（PCD）。这种反应取决于细胞类型、其增殖或分化状态、外源信号的性质以及所涉及的特定信号通路。cdk5（-/-）致死性表型表明，cdk5可能参与存活途径，因为在E16-18胚胎的大脑皮层区域发现了凋亡水平升高。为了进一步探讨这一点，我们研究了cdk5对jnk3介导的凋亡途径的影响。我们可以证明活化的c-Jun n -末端激酶3 （JNk3）的凋亡作用可以被cdk5/p35抑制，这是cdk5与信号转导途径串扰的另一个例子，在这种情况下，其中一个参与PCD。我们证明cdk5/p35可以磷酸化Thr131上的JNk-3，并下调其活性和c-jun的磷酸化。此外，体内研究表明，与野生型相比，cdk5 KO小鼠脑提取物中Jnk3活性升高，这可以解释皮质细胞凋亡增加的原因。紫外线诱导的细胞凋亡在培养的cdk5 -/-皮质神经元中也增加，显示出caspase表达水平升高，这与cdk5/p35在防止Jnk-3激活的凋亡级联中的作用一致。关于cdk5在细胞凋亡中的作用的另一个线索来自cdk5 KO小鼠。我们观察到，在Cdk5基因敲除的大脑中，磷酸化磷脂的水平显著降低，这表明磷脂肌醇-3激酶（pi3激酶）活性受到损害。PI3K是一个关键的激酶，在存活信号级联中磷酸化Akt激酶下游，表明cdk5在发育过程中神经元存活中起作用。有人提出，通过异常产生p25 （p35的一个截断的、更活跃的片段）来解除大脑中cdk5活性的调节，可导致人类AD大脑中特有的tau过度磷酸化病理。我们对cdk5与其p35调节因子的截断形式之间的位点特异性相互作用的研究揭示了p35中心片段，125个氨基酸残基（CIP）对cdk5 /p25复合物具有高亲和力并抑制其体外活性。我们已经证明，CIP在转染细胞中特异性抑制Cdk5/p25活性，并降低共转染tau蛋白的磷酸化。值得注意的是，CIP不影响cdc2激酶的活性。然而，问题是CIP是否会抑制cdk5/p25在初级神经元中的活性。如果CIP特异性抑制原代神经元中cdk5的过度活性，那么它是否可以抑制p25转基因小鼠和ALS转基因模型诱导的AD模型表型中tau和NF蛋白的过度磷酸化？

Protein Phosphorylation And regulation of Cytoskeleton in Nervous system In the nervous system: The cytoskeletal proteins are extensively phosphorylated in the nervous system. Most of this phosphorylation occurs on the proline-directed serine/threonine (S/TP) residues of cytoskeletal proteins, such as neurofilament high and medium molecular weight (NF-H and NF-M) and the microtubule-associated proteins MAP-2 and tau. Normally, these proteins are phosphorylated selectively in the axonal compartment of the neuron. Although all kinases, phosphatases, their substrates and their regulators are synthesized in the cell body, little or no cytoskeletal protein phosphorylation on S/T residues is detected in the cell body. This compartmentalization of phosphorylation is tightly regulated. However, in a number of neuropathological conditions, such as amyotrophic lateral sclerosis (ALS), Alzheimer?s disease (AD) and Picks Disease it gets deregulated. There is an aberrant phosphorylation of these cytoskeletal proteins on S/T residues, in the cell bodies where they accumulate in aggregated form. This leads to massive neuronal cell death in these affected areas. The major focus has been to identify the kinases that phosphorylate these S/T residues. In search of these kinases, we discovered that in addition to the mitogen activated protein kinases (MAPKs) e.g. ERK1/2, Cdk5 is another major kinase that phosphorylates multiple lysine/serine/proline (KSP) repeats in the carboxy-terminal domains of NF-M, NF-H, and tau. Cdk5 was purified from brain tissue and also was cloned from a rat cDNA library in our laboratory. We have demonstrated that various signal transduction cascades in primary neuronal cultures and transfected non-neuronal cells activate cdk5. Myelinating glial cells are a major source of exogenous activating signals. Using myelin associated glycoprotein (MAG) knockout mice, which exhibit no myelination, we have shown that ERK1/2 and Cdk5 activities in brain and sciatic nerve extracts are down-regulated as compared to wild-type mice. These studies also indicated that increased ERK1/2 and Cdk5 activity induced by glial-axon interactions stimulated axonal phosphorylation of NF-M, NF-H, MAP-2B and tau. We have concluded that MAG may act as a ligand to activate the appropriate kinase cascade causing axonal cytoskeletal protein phosphorylation. In addition, matrix elements e.g. integrins may also activate these kinases . More recently, our laboratory has focused to study the role of cdk5 in nervous system function and development. Although Cdk5 is ubiquitously expressed in all cells and shares a high degree of homology with other members of the cyclin-dependent kinase family (Cdks), its activity is found specifically in post-mitotic neurons because its activators, p35 and p39 are expressed primarily in neurons. Studies from different laboratories, including ours, have shown that Cdk5 is a multi-functional S/T protein kinase that is involved in a wide range of neuronal functions from neurite outgrowth and neuronal migration to synaptic activity and cell survival. We have shown, for example, that cdk5 KO mice (-/-) are lethal, exhibiting abnormal corticogenesis and other neuronal abnormalities before dying between E16 and P0. Recently, we have also demonstrated that experimental re-expression of cdk5 in neurons of cdk5 KO mice in vivo completely restored the wild type, clearly demonstrating that neuronal and not glial cdk5 activity is necessary for normal development and survival . The diverse roles of cdk5 are based, in part, on evidence that it is a key player in signal transduction networks underlying neuronal cell survival, growth and differentiation. We observed the presence of hyperphosphorylated cytoskeletal proteins in swollen brain stem and spinal cord perikarya in cdk5-/- mice. This led us to look for other kinases affected by the absence of cdk5. Since cdk5 activity is down regulated in p35-/-mice, we found that MAPK (Erk1/2) was hyper-activated in brain extracts which further suggested that cdk5 modulated a site in the MAP kinase cascade.These results suggest that Cdk5 is involved in "cross-talk" with other signal transduction and survival pathways, perhaps acting to modulate the intensity of the response to specific signals. . Cdk5 activity is tightly regulated in the nervous system and it may promote neuronal cell survival or induce programmed cell death (PCD). The response depends on cell type, its state of proliferation or differentiation, the nature of exogenous signals, and the specific signaling pathways involved. The cdk5 (-/-) lethal phenotype suggests that cdk5 may be involved in the survival pathway since elevated levels of apoptosis were seen in cortical brain regions of E16-18 embryos. To explore this further, we studied the effect of cdk5 on the well-studied JNk3-mediated apoptotic pathway. We could show that the apoptotic effects of the activated c-Jun N-terminal kinase 3 (JNk3) can be inhibited by cdk5/p35, another illustration of cdk5 cross talk with signal transduction pathways, in this case, with one involved in PCD. We demonstrated that cdk5/p35 could phosphorylate JNk-3 on Thr131 and down regulate its activity and phosphorylation of c-jun. Moreover, in vivo studies showed that Jnk3 activity was elevated in brain extracts of cdk5 KO mice compared to wild type, which could explain the increased cortical apoptosis. UV-induced apoptosis was also increased in cdk5 -/- cortical neurons in culture which exhibited elevated levels of caspase expression, consistent with a role for cdk5/p35 in protecting against a Jnk-3 activated apoptotic cascade. An additional clue about cdk5's role in apoptosis came from the cdk5 KO mice. We observed that the level of phosphorylated phospholipid is considerably reduced in Cdk5 knockout brain, suggesting that phospholipid inositol-3 kinase (PI3-kinase) activity is compromised. PI3K is a key kinase, which phosphorylates Akt kinase downstream in a survival-signaling cascade suggesting a role for cdk5 in neuronal survival during development. It has been proposed that deregulation of cdk5 activity in the brain, by abnormal production of p25, a truncated, more active fragment of p35, can lead to hyperphosphorylated tau pathologies characteristic of AD brains in humans . Our study of site specific interactions between cdk5 and truncated forms of its p35 regulator have revealed a central p35 fragment, 125 amino acids residues (CIP) that has high affinity for and inhibits the in vitro activity of the Cdk5/p25 complex. We have shown that CIP specifically inhibits Cdk5/p25 activity in transfected cells and also reduces phosphorylation of co-transfected tau. It is important to note that CIP does not affect the activity of cdc2 kinase. The question, however, arises as to whether CIP will inhibit cdk5/p25 activity in primary neurons. If CIP specifically inhibits cdk5 hyperactivity in primary neurons, can it inhibit the hyperphosphorylation of tau and NF proteins in AD model phenotypes induced in p25 transgenic mice and in an ALS transgenic model?