Cdk5/p35激酶与肌动蛋白细胞骨架结合关系的鉴定

Cdk5,一种多功能的丝氨酸/苏氨酸蛋白激酶,其活性只有通过结合其神经特异性调节亚基才能被激活.p35是Cdk5的两个主要调节亚基之一.尽管Cdk5/p35激酶可以调控神经细胞中肌动蛋白细胞骨架的动态变化,但直到目前为止Cdk5/p35激酶与肌动蛋白细胞骨架的结合关系仍不是很清楚.现利用几种不同的方法对两者的结合关系进行了初步鉴定.目前的试验结果表明在鼠脑组织中肌动蛋白细胞骨架是Cdk5/p35超大蛋白复合体的一个组分,p35可以直接结合纤维状肌动蛋白,这说明在鼠脑组织或神经细胞中Cdk5很有可能是通过p35结合到肌动蛋白细胞骨架上并进一步调控肌动蛋白细胞骨架的动态活动的.     

Peptides derived from Cdk5 activator p35, specifically inhibit deregulated activity of Cdk5

Normal Cdk5 activity, conferred mainly by association with its primary activator p35, is critical for normal function of the cell and must be tightly regulated. During neurotoxicity, p35 is cleaved to form p25, which becomes a potent and mislocalized hyperactivator of Cdk5, resulting in a deregulation of Cdk5 activity. p25 levels have been found to be elevated in Alzheimer's disease (AD) brain and overexpression of p25 in a transgenic mouse results in the formation of phosphorylated tau, neurofibrillary tangles and cognitive deficits that are pathological hallmarks of AD. p25/Cdk5 also hyperphosphorylates neurofilament proteins that constitute pathological hallmarks found in Parkinson's disease and amyotrophic lateral sclerosis. The selective targeting of p25/Cdk5 activity without affecting p35/Cdk5 activity has been unsuccessful. In this review we detail our recent studies of selective p25/Cdk5 inhibition without affecting p35/Cdk5 or mitotic Cdk activities. We found that a further truncation of p25 to yield a Cdk5 inhibitory peptide (CIP) can specifically inhibit p25/Cdk5 activity in transfected HEK cells and primary cortical neurons. CIP was able to reduce tau hyperphosphorylation and neuronal death induced caused by p25/Cdk5 and further studies with CIP may develop a specific Cdk5 inhibition strategy in the treatment of neurodegeneration.     

Cdk5--p39 is a labile complex with the similar substrate specificity to Cdk5--p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.     

Expression and Activity of Cyclin-Dependent Kinase 5/p35 in Adult Rat Peripheral Nervous System

Abstract: In spite of the clarification in the temporal and spatial expression pattern of a cyclin-dependent kinase (Cdk) 5 and its neuron-specific activator, p35, in the CNS, it remains to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, we have examined their expression and activity in the PNS by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicated the expression of Cdk5 and p35 proteins in dorsal root ganglion (DRG) and sciatic nerve alike in the CNS. By immunohistochemistry, both proteins were shown to be present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicated the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity was found only in DRG, but not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains.     

Structural Basis for the Different Stability and Activity between the Cdk5 Complexes with p35 and p39 Activators

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.     

Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cyclin-dependent kinase 5 complexes

Cdk5 is a member of the cyclin-dependent kinases (Cdks), activated by the neuron-specific activator p39 or p35. The activators also determine the cytoplasmic distribution of active Cdk5, but the mechanism is not yet known. In particular, little is known for p39. p39 and p35 contain localization motifs, such as a second Gly for myristoylation and Lys clusters in the N-terminal p10 region. Using mutant constructs, we investigated the cellular distribution mechanism. We observed that p39 localizes the active Cdk5 complex in the perinuclear region and at the plasma membrane as does p35. We demonstrated the myristoylation of both p39 and p35, and found that it is a major determinant of their membrane association. Plasma membrane targeting depends on the amino acid sequence containing the Lys-cluster in the N-terminal p10 region. In contrast, a non-myristoylated Ala mutant (p39G2A or p35G2A) showed nuclear localization with stronger accumulation of p39G2A than p35G2A. These results indicate that myristoylation regulates the membrane association of p39 as well as p35 and that the Lys cluster controls their trafficking to the plasma membrane. The differential nuclear accumulation of p39 and p35 suggests their segregated functions, p35–Cdk5 in the cytoplasm and p39–Cdk5 in the nucleus.     

p39, the Primary Activator for Cyclin-dependent Kinase 5 (Cdk5) in Oligodendroglia,Is Essential for Oligodendroglia Differentiation and Myelin Repair

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39^−/− mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair.     

Apoptosis-associated tyrosine kinase is a Cdk5 activator p35 binding protein

A 3(')-terminal fragment of a splice variant of KIAA0641, a human homologue of apoptosis-associated tyrosine kinase (AATYK), was screened from human brain cDNA libraries by a yeast two-hybrid system using a Cdk5 activator p35 as a bait. The cloned cDNA encoded 477 amino acids, composed of internal 458 amino acids of KIAA0641 and 19 amino acids unique to this variant after splicing, then referred to this clone as hAATYKs-p35BP (human AATYK short isoform-p35 binding polypeptide). Using GST-fusion protein, hAATYKs-p35BP was shown to bind to Cdk5/p35 in a rat brain extract. hAATYKs made by fusing the kinase domain of KIAA0641 to the N-terminus of hAATYKs-p35BP was used for binding to Cdk5/p35 in HEK293 cells. Both hAATYKs and KIAA0641 bound to and were phosphorylated by Cdk5/p35. These results suggest that both isoforms of hAATYK are novel Cdk5/p35-binding and substrate proteins.     

MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK

Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.     

Twice switched at birth: cell cycle-independent roles of the neuron-specific cyclin-dependent kinase 5 (Cdk5) in non-neuronal cells

Cdk5 (cyclin-dependent kinase 5 or initially NCLK for neuronal CDC2-like kinase) was switched twice at its birth nearly twenty years ago: first it was thought to be cyclin-dependent, second it was assumed to be primarily of importance in neuronal cells—both turned out not to be the case. In this review we want to discuss issues of pharmacological inhibition, to highlight the versatile roles, and to summarize the growing evidence for the functional importance of Cdk5 in non-neuronal tissues, such as blood cells, tumor cells, epithelial cells, the vascular endothelium, testis, adipose and endocrine tissues. The organizing principles we follow are apoptosis/cell death, migration/motility, aspects of inflammation, and, finally, secretion/metabolism.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.     

A Cdk5–p35 Stable Complex Is Involved in the β-Amyloid-Induced Deregulation of Cdk5 Activity in Hippocampal Neurons

The cdk5 and its activator p35 constitute one of the main tau-phosphorylating systems in neuronal cells. Under normal conditions for neurons, its activity is required for modulating tau involvement in neuronal polarity and in development of the mammalian central nervous system. Recently, we reported that the treatment of rat hippocampal cells in culture with fibrillary β-amyloid (Aβ) results in deregulation of the protein kinase cdk5. The neurotoxic effects of Aβ fibrils were prevented by inhibition of cdk5 activity by butyrolactone I or by using antisense oligonucleotides that control the expression of this kinase. Here, we show that the Aβ-promoted increase of cdk5 activity is associated with changes in tau phosphorylation patterns and in the intraneuronal distribution of tau. In addition to hippocampal cells, deregulation of cdk5 was observed in other cell types. However, butyrolactone I prevented Aβ-induced cell death only in neuronal cells in which cdk5 activation was sensitive to Aβ fibrils. This lost of cdk5 regulation in hippocampal cells exposed to Aβ fibrils appears to be associated with an increase in the cdk5–p35 complex stability. Complex stabilization was sensitive to phosphorylation of cdk5. However, no changes in cdk5 and p35 mRNAs were observed, suggesting that the main effects on cdk5 occur at the posttranslational level. These studies indicate that cdk5 phosphorylation and the formation of an abnormally active cdk5–p35 complex are directly involved in the molecular paths leading to the neurodegenerative process of rat hippocampal neurons triggered by Aβ fibrils.     

Glutamate treatment and p25 transfection increase Cdk5 mediated tau phosphorylation in SH-SY5Y cells

Neurofibrillary tangles (NFT) of hyperphosphorylated tau protein are a major pathological hallmark of Alzheimer's disease (AD). One of the tau phosphorylating kinases with pathological relevance in AD has been suggested to be the cyclin-dependent kinase 5 (Cdk5). The proposed mechanism leading to pathological Cdk5 activity is through induced cleavage of p35 to a proteolytic product, p25. To further study activation of Cdk5 and its role in tau phosphorylation in vitro, we used differentiated SH-SY5Y cells treated with neurotoxic stimuli or transfected with p25. We show that glutamate increased tau phosphorylation, concomitant with an increased Cdk5 activity achieved by upregulation of Cdk5 and p35 protein levels. Treatment with the calcium ionophore A23187 generated the calpain cleaved p25 fragment but only in toxic conditions that caused dephosphorylation and loss of tau. When p25 was transfected to the cells, increased tau phosphorylation was achieved. However, application of the Cdk5 inhibitor Roscovitine did not result in inhibition of tau phosphorylation possibly due to activation of extracellular regulated kinase 1/2 (Erk1/2), which also is capable of phosphorylating tau. Cdk5 and Erk1/2 kinases share some common substrates but impact of their cross talk on tau phosphorylation has not previously been demonstrated. We also show that p25 is degraded via the proteasome in Roscovitine treated cells.     

Hyperactive Innate Immunity Causes Degeneration of Dopamine Neurons upon Altering Activity of Cdk5

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Cdk5与学习和记忆的研究进展

越来越多的证据表明,Cdk5通过与大量蛋白相互作用而在学习和记忆过程中发挥重要作用。近来,关于Cdk5的研究结果不仅证实其参与药物成瘾过程中细胞间的通路改变,且其活性与一些神经退行性疾病的发生有关。本文就Cdk5对学习和记忆的影响及其相关机制的研究进展予以综述。     

CDK5 and Its Activator P35 in Normal Pituitary and in Pituitary Adenomas: Relationship to VEGF Expression

Pituitary tumors are monoclonal adenomas that account for about 10-15% of intracranial tumors. Cyclin-dependent kinase 5 (CDK5) regulates the activities of various proteins and cellular processes in the nervous system, but its potential roles in pituitary adenomas are poorly understood. The kinase activity of CDK5 requires association with an activating protein, p35 (also known as CDK5 activator 1, p35). Here, we show that functional CDK5, associated with p35, is present in normal human pituitary and in pituitary tumors. Furthermore, p35 mRNA and protein levels were higher in pituitary adenomas than in the normal glands, suggesting that CDK5 activity might be upregulated in pituitary tumors. Inhibition of CDK5 activity in rat pituitary cells, reduced the expression of vascular endothelial growth factor (VEGF), a protein that regulates vasculogenesis and angiogenesis. Our results suggest that increased CDK5-mediated VEGF expression might play a crucial role in the development of pituitary adenomas, and that roscovitine and other CDK5 inhibitors could be useful as anticancer agents.     

Reelin signaling and Cdk5 in the control of neuronal positioning

Neuronal positioning is important for higher brain function because it is the architectural basis of the formation of precise synaptic circuits. Analysis of neurological mutant mice has led to dramatic progress in the identification and characterization of molecules important for neuronal positioning in the developing mammalian brain. Among these molecules, identification of signal pathways mediated by Reelin and Cdk5 kinase has provided a conceptual framework for exploring the molecular mechanisms underlying proper neuronal positioning in the developing mammalian brain. Recent evidence has implicated synergism between Reelin signaling and Cdk5 in contributing to the proper positioning of selective neuronal populations.     

p35/cdk5复合物的功能及在阿尔茨海默病病理中的作用

阿尔茨海默病（Alzheimer's disease,AD)是老年痴呆症中最常见的一种,以严重的记忆减退和认知障碍为主要临床表现,目前有关AD的发病机制尚不甚明了,神经原纤维缠结由高度磷酸化的tau蛋白聚集而成,是AD的特征性病变。细胞周期蛋白依赖的蛋白激酶5（cyclin-dependent ki-nase5,cdk5)与其调节蛋白p35,p25等参与了AD病人脑内tau蛋白的异常磷酸化,在AD的发病过程中可能发挥重要作用,本文简要介绍p35及cdk5的结构特征,在中枢神经系统内的分布,对p35/cdk5参与AD发病机制进行了初步的探讨,为临床防治AD提供一些新的思路。