Cdk5

Cell adhesion is a fundamental property of epithelial cells, required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.     

S-nitrosylation of Cdk5

Aberrant activation of Cdk5 has been implicated in the process of neurodegenerative diseases such as Alzheimer's disease (AD). We recently reported that S-nitrosylation of Cdk5 (forming SNO-Cdk5) at specific cysteine residues results in excessive activation of Cdk5, contributing to mitochondrial dysfunction, synaptic damage, and neuronal cell death in models of AD. Furthermore, SNO-Cdk5 acts as a nascent S-nitrosylase, transnitrosylating the mitochondrial fission protein Drp1 and enhancing excessive mitochondrial fission in dendritic spines. However, a molecular mechanism that leads to the formation of SNO-Cdk5 in neuronal cells remained obscure. Here, we demonstrate that neuronal nitric oxide synthase (NOS1) interacts with Cdk5 and that the close proximity of the two proteins facilitates the formation of SNO-Cdk5. Interestingly, as a negative feedback mechanism, Cdk5 phosphorylates and suppresses NOS1 activity. Thus, together with our previous report, these findings delineate an S-nitrosylation pathway wherein Cdk5/NOS1 interaction enhances SNO-Cdk5 formation, mediating mitochondrial dysfunction and synaptic loss during the etiology of AD.     

Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor

Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.     

Structural Insights into Cdk5 activation by a neuronal Cdk5 activator.

Although Cdk5 shows high sequence identity to Cdk1 and Cdk2, it can be fully activated by its neuronal activators p35/p25(nck5a) and p39(nck5ai) in a phosphorylation-independent manner. To understand structural basis of the Cdk5/p25(nck5a) activation, the complex is modelled to assume either an obstructed or an opened conformation based on X-ray structures of the unphosphorylated or the phosphorylated Cdk2/cyclin A complex, respectively. Comparison and analysis of the two models, along with mutagenesis studies of p25(nck5a), suggest that the opened form represents more closely the structure of active Cdk5/p25(nck5a). The results provide a rationale basis for understanding the phosphorylation-independent activation of Cdk5/p25(nck5a).     

Cdk1-dependent control of membrane-trafficking dynamics

Cyclin-dependent kinase 1 (Cdk1) is required for initiation and maintenance of polarized cell growth in budding yeast. Cdk1 activates Rho-family GTPases, which polarize the actin cytoskeleton for delivery of membrane to growth sites via the secretory pathway. Here we investigate whether Cdk1 plays additional roles in the initiation and maintenance of polarized cell growth. We find that inhibition of Cdk1 causes a cell surface growth defect that is as severe as that caused by actin depolymerization. However, unlike actin depolymerization, Cdk1 inhibition does not result in a massive accumulation of intracellular secretory vesicles or their cargoes. Analysis of post-Golgi vesicle dynamics after Cdk1 inhibition demonstrates that exocytic vesicles are rapidly mistargeted away from the growing bud, possibly to the endomembrane/vacuolar system. Inhibition of Cdk1 also causes defects in the organization of endocytic and exocytic zones at the site of growth. Cdk1 thus modulates membrane-trafficking dynamics, which is likely to play an important role in coordinating cell surface growth with cell cycle progression.     

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.     

Cdk5 regulates Rap1 activity

Rap1 signaling is important for migration, differentiation, axonal growth, and during neuronal polarity. Rap1 can be activated by external stimuli, which in turn regulates specific guanine nucleotide exchange factors such as C3G, among others. Cdk5 functions are also important to neuronal migration and differentiation. Since we found that pharmacological inhibition of Cdk5 by using roscovitine reduced Rap1 protein levels in COS-7 cells and also C3G contains three putative phosphorylation sites for Cdk5, we examined whether the Cdk5-dependent phosphorylation of C3G could affect Rap1 expression and activity. We co-transfected C3G and tet-OFF system for p35 over-expression, an activator of Cdk5 activity into COS-7 cells, and then we evaluated phosphorylation in serine residues in C3G by immunoprecipitation and Western blot. We found that p35 over-expression increased C3G-serine-phosphorylation while inhibition of p35 expression by tetracycline or inhibition of Cdk5 activity with roscovitine decreased it. Interestingly, we found that MG-132, a proteasome inhibitor, rescue Rap1 protein levels in the presence of roscovitine. Besides, C3G-serine-phosphorylation and Rap1 protein levels were reduced in brain from Cdk5^−/− as compared with the Cdk5^+/+ brain. Finally, we found that p35 over-expression increased Rap1 activity while inhibition of p35 expression by tetracycline or roscovitine decreased Rap1 activity. These results suggest that Cdk5-mediated serine-phosphorylation of C3G may control Rap1 stability and activity, and this may potentially impact various neuronal functions such as migration, differentiation, and polarity.     

Cdk5 on the brain.

Mammalian brains are highly compartmentalized into groups of functionally specialized neurons. Cell migration and neurite outgrowth must be tightly orchestrated to achieve this level of organization. A small serine/threonine kinase that shows homology to cyclin-dependent kinases (Cdks) has emerged as an important regulator of neuronal migration. Cdk5, unlike other Cdks, is not regulated by cyclins, and its activity is primarily detected in postmitotic neurons in developing and adult nervous systems. This review describes work indicating that Cdk5 links extracellular signaling pathways and cytoskeletal/membrane systems to direct neuronal migration, axon growth, and possibly neurosecretion. Despite its importance, unchecked Cdk5 activity is toxic to neurons, and may underlie some of the pathologies associated with neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis.     

ERM Proteins and Cdk5 in Cellular Senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-b-galactosidase (SA-b-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-b-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

Protein-protein interactions in Cdk5 regulation and function

Cdk5 is a unique member of the cyclin-dependent kinase (Cdk) family of small protein kinases. In association with its neuron-specific activator p35 or p39, Cdk5 displays many regulatory properties distinct from other Cdks. A growing body of evidence has suggested that Cdk5-p35 has important implications in a variety of neuronal activities occurring in the central nervous system. In brain, Cdk5-p35 appears to exist as large molecular complexes with other proteins, and protein-protein interactions appear to be a molecular principle for Cdk5-p35 to conduct its physiological functions. Over the past decade, a number of proteins have been identified to associate with Cdk5-p35. While the majority of these proteins mediate their interaction with Cdk5 through p35, implying that p35 may act not only as an activator of Cdk5 but also as an adaptor to associate Cdk5 with its regulators and physiological targets, a small group of other proteins are found to link directly with Cdk5. In addition, Cdk5 has been found to phosphorylate a diverse list of substrates, further implicating its regulatory roles in a wide range of cellular processes. In this review, we present an updated inventory of the interacting proteins of Cdk5-p35 kinase and its substrates as well as a discussion on the implicated effects of these interactions.     

ERM proteins and Cdk5 in cellular senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-beta-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

Cdk5: Links to DNA damage

Comment on: Huang E, et al. Nat Cell Biol 2010; 12:563-71.     

Distinct functions of Cdk5(Y15) phosphorylation and Cdk5 activity in stress fiber formation and organization

Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.     

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。     

Targeting Cdk5 Activity in Neuronal Degeneration and Regeneration

The major priming event in neurodegeneration is loss of neurons. Loss of neurons by apoptotic mechanisms is a theme for studies focused on determining therapeutic strategies. Neurons following an insult, activate a number of signal transduction pathways, of which, kinases are the leading members. Cyclin-dependent kinase 5 (Cdk5) is one of the kinases that have been linked to neurodegeneration. Cdk5 along with its principal activator p35 is involved in multiple cellular functions ranging from neuronal differentiation and migration to synaptic transmission. However, during neurotoxic stress, intracellular rise in Ca²⁺ activates calpain, which cleaves p35 to generate p25. The long half-life of Cdk5/p25 results in a hyperactive, aberrant Cdk5 that hyperphosphorylates Tau, neurofilament and other cytoskeletal proteins. These hyperphosphorylated cytoskeletal proteins set the groundwork to forming neurofibrillary tangles and aggregates of phosphorylated proteins, hallmarks of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease and Amyotropic Lateral Sclerosis. Attempts to selectively target Cdk5/p25 activity without affecting Cdk5/p35 have been largely unsuccessful. A polypeptide inhibitor, CIP (Cdk5 inhibitory peptide), developed in our laboratory, successfully inhibits Cdk5/p25 activity in vitro, in cultured primary neurons, and is currently undergoing validation tests in mouse models of neurodegeneration. Here, we discuss the therapeutic potential of CIP in regenerating neurons that are exposed to neurodegenerative stimuli.     

Cdk5: a multifaceted kinase in neurodegenerative diseases

Since the identification of cyclin-dependent kinase-5 (Cdk5) as a tau kinase and member of the Cdk family almost 20 years ago, deregulation of Cdk5 activity has been linked to an array of neurodegenerative diseases. As knowledge on the etiopathological mechanisms of these diseases evolved through the years, Cdk5 has also been implicated in additional cellular events that are affected under these pathological conditions. From the role of Cdk5 in the regulation of synaptic functions to its involvement in autophagy deregulation, significant insights have been obtained regarding the role of Cdk5 as a key regulator of neurodegeneration. Here, we summarize recent findings on the involvement of Cdk5 in the pathophysiological mechanisms underlying various neurodegenerative diseases.