Cyclin-dependent kinase 5
Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, a dominant negative form, Crp-D144N, was overexpressed under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. These observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development (Sharma, 2002b).
Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon was identified. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors (Ou, 2010).
Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. A novel Cdc2-like kinase has been purified from bovine brain; it is capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25. When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function (Lew, 1994).
Cyclin-dependent kinase 5 (Cdk5) was originally isolated through its structural homology to human Cdc2, a key regulator of cell-cycle progression. In tissue samples from adult mice, Cdk5 protein is found at the highest level in brain, at an intermediate level in testis, and at low or undetectable levels in all other tissues, but brain is the only tissue that shows Cdk5 histone H1 kinase activity. No equivalent kinase activity has been found in tissue culture cell lines despite high levels of Cdk5. This raised the possibility that a Cdk5 regulatory subunit was responsible for the activation of Cdk5 in brain. The cloning and characterization of a regulatory subunit for Cdk5, known as p35, is described. p35 displays a neuronal cell-specific pattern of expression; it associates physically with Cdk5 in vivo and activates the Cdk5 kinase. p35 differs from the mammalian cyclins and thus represents a new type of regulatory subunit for cyclin-dependent kinase activity (Tsai, 1994).
Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit that is derived from a 35-kDa brain- and neuron-specific protein called the neuronal Cdk5 activator (p35/p25nck5a). Upon screening of a human hippocampus library with a bovine Nck5a cDNA, a distinct clone encoding a 39-kDa isoform of Nck5a was uncovered. The isoform, designated the neuronal Cdk5 activator isoform (p39nck5ai), shows a high degree of sequence similarity to p35nck5a, with 57% amino acid identity. Northern blot analysis detected its mRNA transcript in bovine and rat cerebrum and cerebellum, but not in any other rat tissues examined. In situ hybridization has shown that Nck5ai is enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of hippocampal formation. Among seven cell lines in proliferating cultures, only PC12 and N2A, two cell lines capable of differentiating into neuron-like cells, were found to contain Nck5ai mRNA. A 30-kDa truncated form of Nck5ai (expressed as a glutathione S-transferase fusion protein in Escherichia coli) was found to associate with Cdk5 to form an active Cdk5 kinase. Thus, the isoform shares many common characteristics with p35nck5a, including Ckd5 activating activity and brain- and neuron-specific expression. Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins (Tang, 1995).
Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. The proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109-291, is sufficient to bind and activate Cdk5. Distinct regions in p35 required for binding to Cdk5 or activation of Cdk5 have been identified. Residues approximately 150-200 of p35 are sufficient for binding to Cdk5, but residues approximately 279-291 are needed in addition for activation of Cdk5 in vitro (Poon, 1997).
Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2 gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association with and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. The regulation of the p35/Cdk5 kinase has been investigated. p35, the activator of Cdk5, is a short-lived protein with a half-life (t1/2) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2- to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2- to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays an autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis (Patrick, 1998).
The ubiquitously expressed cyclin-dependent kinase 5 (cdk5) is essential for brain development. Bioactivation of cdk5 in the brain requires the presence of one of two related regulatory subunits, p35 and p39. Since either protein alone can activate cdk5, the significance of their coexistence as cdk5 kinase activators is unclear. To determine whether the two activators are expressed in different cells throughout the nervous system and during development, the tissue distributions of cdk5, p35, and p39 mRNAs in the rat were compared using in situ hybridization. In the adult rat, expression levels of p35 mRNA are generally higher in the brain than in the spinal cord, while the converse is observed for p39 mRNA. During neurogenesis, both p35 and p39 transcripts can be detected as early as embryonic day 12 (E12) in the marginal zone, but are absent from the ventricular zone, which may restrict cdk5 activation to the postmitotic neural cells in the developing brain. The expression levels of p35 and p39 mRNAs in the marginal zone increase by E15 and E17, paralleling the neurogenetic timetable. One exception is in the rostral forebrain, where p35 mRNA expression levels are high, suggesting that p35 may be the major activator for cdk5 during telencephalic morphogenesis. A significant level of p35 mRNA is present in the myotome at E12 and p35 expression persists in the premuscle mass and mature musculature at later stages, suggesting that p35 may also activate cdk5 during myogenesis (Zheng, 1998).
Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, it is reported that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 is cleaved to p25 by incubation with Ca(2+). This cleavage is inhibited by a calpain inhibitor peptide derived from calpastatin and is ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation can then be cleaved to p25 by purified calpain. Cleavage of p35 is also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changes the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but does not affect the histone H1 kinase activity. Increased cleavage is detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death (Kusakawa, 2000).
CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. The crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator, is reported in this study. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Evidence is provided that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity (Tarricone, 2001).
The interaction of p25 with CDK5 stabilizes an active conformation of the T loop, which is indistinguishable from those observed in phosphorylated CDK2 and ERK2. Evidence that Ile153 and Ser159 in the T loop of CDK5 are critical for p25 and p35 recognition and might contribute to the selectivity of the CDK5-p35 interaction. Retention of serine in all CDK5 homologs from yeast to human suggests that the presence of a phosphate acceptor at position 159 might be relevant for CDK5 regulation. It is predicted that phosphorylation of Ser159CDK5, if occurring, would negatively regulate kinase activity. Phosphorylation of Tyr15 on CDK5 by Abl is stimulatory, while phosphorylation of Tyr15 and Thr14 by Wee1 family kinases is inhibitory for CDK1 and 2. When taken together, these observations indicate that phosphorylation and dephosphorylation events similar to those impinging on mitotic CDKs regulate CDK5 in a completely distinct fashion. Structural and biochemical evidence is provided that the CDK5-p25 complex has devised a novel and distinct mechanism for substrate recognition and specificity entailing the participation of the activator subunit. An important difference between the activation mechanism of CDK5 and that of other proline-directed kinases, such as ERK2 and CDK2, is that the active T loop conformation of CDK5 is not stabilized by phosphorylation but by extensive interactions with the regulatory moiety (Tarricone, 2001).
Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity of Cdk5-deficient mice and p35-deficient mice. Cdk5-null mutants show a more severe disruption of lamination in the cerebral cortex, hippocampus, and cerebellum. In addition, Cdk5-null mice display perinatal lethality, whereas p35-null mice are viable. These discrepancies have been attributed to the function of other Cdk5 activators, such as p39. To understand the roles of p39 and p35, p39-null mice and p35/p39 compound-mutant mice were created. Interestingly, p39-null mice show no obvious detectable abnormalities, whereas p35-/-p39-/- double-null mutants are perinatal lethal. The p35-/-p39-/- mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow for the determination of the distinct and redundant functions between p35 and p39. The data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5 (Ko, 2001).
Cyclin-dependent kinase 5 (Cdk5) null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering attributable to abnormal neuronal migration, lack of cerebellar foliation, and chromatolytic changes of neurons in the brainstem and the spinal cord. Because Cdk5 is expressed in both neurons and astrocytes, it has been unclear whether this phenotype is primarily attributable to defects in neurons or in astrocytes. Cdk5 expression has been reconstructed in neurons in Cdk5 null mice, and its effect on the null phenotype was examined. Unlike the Cdk5 null mice, the reconstituted Cdk5 null mice that express the Cdk5 transgene under the p35 promoter (TgKO mice) are viable and fertile. Because Cdk5 expression is mainly limited to neurons in these mice and rescues the defects in the nervous system of the Cdk5 null phenotype, it clearly demonstrates that Cdk5 activity is necessary for normal development and survival of p35-expressing neurons (Tanaka, 2001).
Cultures of cerebellar macroneurons were used to study the expression, activity, subcellular localization, and function of cdk5 during neuronal morphogenesis. The results obtained indicate that in non-polarized neurons, cdk5 is restricted to the cell body but as soon as polarity is established it becomes highly concentrated at the distal tip of growing axons where it associates with microtubules and the subcortical cytoskeleton. In addition, laminin, an extracellular matrix molecule capable of stimulating axonal extension and promoting MAP1b phosphorylation, accelerates the redistribution of cdk5 to the axonal tip and dramatically increases its activity. Finally, these results indicate that cdk5 suppression by antisense oligonucleotide treatment selectively reduces axonal elongation and decreases the phosphorylation status of MAP1b, as well as its binding to microtubules. Taken collectively, these observations suggest that cdk5 may serve as an important regulatory linker between environmental signals (e.g. laminin) and constituents of the intracellular machinery (e.g. MAP1b) involved in axonal formation (Pigiono, 1997).
Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced axonal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association with the subcortical cytoskeleton, and accelerates its redistribution to the axonal growth cones. In addition, suppression of p35, (but not of a highly related isoform designated as p39), by antisense oligonucleotide treatment selectively reduces cdk5 activity, laminin-enhanced axonal elongation, and MAP1b phosphorylation. Taken collectively, the present results suggest that cdk5/p35 may serve as an important regulatory linking agent between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation (Paglini, 1998).
Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increases Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, is blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 are blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, is abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhances voltage-dependent Ca2+ currents. This enhancement is eliminated by either butyrolactone or CK1-7 and is absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca2+ channels (Liu, 2001).
Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. This study shows that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development (Mokalled, 2010).
Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. The p35/Cdk5 kinase is shown to co-localize with Rac (see Drosophila Rac1) in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth (Nikolic, 1998).
The p35-Cdk5 kinase has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex. In order to identify molecules important for p35-Cdk5-dependent function in the cortex, a screen was undertaken for p35-interacting proteins using the two-hybrid system. In this study, the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin is reported. The p35 and beta-catenin proteins interact in vitro and colocalize in transfected COS cells. In addition, the p35-Cdk5 kinase is associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine leads to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35(-/-) cortical neurons, which aggregate to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells leads to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion. The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35-Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N-cadherin-mediated adhesion in cortical neurons (Kwon, 2000).
Several models can be proposed to account for the regulation of cadherin-mediated adhesion by the p35-Cdk5 kinase. As p35-Cdk5 is a protein serine/threonine kinase, it may phosphorylate one or more components of the cadherin-adhesion complex, ultimately leading to decreased cell adhesion. Indeed, beta-catenin contains three minimal consensus sites for phosphorylation by Cdks. However, there is little evidence to support a role for serine/threonine phosphorylation as a modulator of cadherin-mediated adhesion in cortical neurons. On the other hand, evidence of regulation of tyrosine phosphorylation of beta-catenin and the cadherins by the EGF receptor, the kinase Src, and the phosphatases PTP1B and LAR suggests that tyrosine phosphorylation may serve as an important mechanism to regulate cadherin-mediated adhesion. The role of tyrosine phosphorylation in cadherin-mediated adhesion is interesting in light of the recent identification of a Cdk5-interacting protein, Cables, which bridges Cdk5 and the non-receptor tyrosine kinase Abl (L. Zukerberg, G. Patrick, M. Nicolic, S. Humbert, L. Lanier, F. Gertler, et al., unpublished observations cited in Kwon, 2000). Additionally, the p35-Cdk5 kinase may function as a scaffold to assemble molecules that act to destabilize N-cadherin-mediated adhesion. For instance, p35-Cdk5 interacts with the active form of the small GTPase Rac, and modulates Pak1 kinase activity. As Rac activity is necessary for cadherin-mediated adhesion, it is possible that the p35-Cdk5 kinase may regulate cadherin-mediated adhesion by regulating a Rac-dependent signaling pathway (Kwon, 2000).
Only about 1% of endogenous beta-catenin binds to p35, whereas more than 10% of total p35 binds to beta-catenin in the embryonic brain lysates. This observation suggests that whereas the beta-catenin-N-cadherin complex may be one of the major targets for the p35-Cdk5 kinase, only a small fraction of the beta-catenin-N-cadherin complex in the developing cortex is actually regulated by p35-Cdk5. Indeed, no difference was detected in the overall levels and association of beta-catenin and N-cadherin in membrane extracts derived from the embryonic and adult cortices of p35-/- mice. Thus, it is possible that p35-Cdk5 regulation of N-cadherin mediated adhesion is only crucial in a very specific population of migrating cortical neurons. In fact, it may be that p35-Cdk5 regulation of N-cadherin-mediated adhesion is relevant to neurons only when they traverse the intermediate zone of the developing cortex (Kwon, 2000).
Members of the N-methyl-D-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs (Li, 2001).
The Munc-18/syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologs in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18, when it has been phosphorylated by Cdk5, has a significantly reduced affinity for syntaxin 1A. Cdk5 can also bind to syntaxin 1A and a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18/syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism (Shuang, 1998).
Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulates Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbes Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrates that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicates that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons shows that p67 is highly enriched in axons. It is proposed that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components (Shetty, 1999).
Disruption of one allele of the LIS1 gene (see Drosophila Lissencephaly-1)causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, it has been demonstrated that mammalian LIS1 regulates dynein functions. NUDEL is a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. These findings point to cross-talk between two prominent pathways that regulate neuronal migration (Niethammer, 2000).
Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables (Cdk5 and Abl enzyme substrate), a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Cables displays little sequence homology to other known proteins in the databases. It does, however, show weak homology to cyclin A and weaker homology to cyclin C over an ~200 amino acid stretch in the C-terminal third of the protein, which may be the Cdk-interacting region. Cables also contains six PXXP motifs, defined as the minimal consensus for SH3 domain binding, and two tyrosine-based sorting motifs (YXXLE), which have been implicated in axonal growth cone sorting. It contains three serine proline/threonine proline minimal Cdk phosphorylation sites and at least one potential c-Abl phosphorylation site (YXXP). Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibits neurite outgrowth. Furthermore, expression of active Abl results in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system (Zukerberg, 2000).
These data suggest that Cables serves as an adaptor molecule, facilitating Cdk5 tyrosine phosphorylation and regulation by c-Abl. Phosphorylation of key substrates involved in actin and microtubule dynamics by active Cdk5 is likely to contribute to its role in neuronal migration and neurite outgrowth. Furthermore, Cdk5 has been shown to downregulate N-cadherin-mediated cell adhesion. Data presented in this communication suggest that Cables mediates an interaction between c-Abl and Cdk5, and may positively affect brain development and neurite outgrowth by enhancing Cdk5 tyrosine phosphorylation and upregulation of kinase activity. Cables may also mediate an interaction between Cdk5 and mDab1 by binding to both Cdk5 and c-Abl (Zukerberg, 2000).
The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. A single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase (Bibb, 1999).
The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Overexpression of Xenopus Pak5 enhances microtubule stabilization, and Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin. A specific phosphorylation site has been identified on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase. Pak1 phosphorylated on T212, Pak1T212(PO4), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected. Cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO4) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. It is proposed that the region surrounding phosphorylated T212 contains a protein binding site, since the phosphorylated peptide is enriched in spindles and MTOCs and competes with endogenous Pak1 for this location.Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division (Banerjee, 2002).
Neurotoxic insults deregulate Cdk5 activity, which leads to neuronal apoptosis and may contribute to neurodegeneration. The biological activity of Cdk5 has been ascribed to its phosphorylation of cytoplasmic substrates. However, its roles in the nucleus remain unknown. The mechanism by which Cdk5 promotes neuronal apoptosis has been investigated. The prosurvival transcription factor MEF2 has been identified as a direct nuclear target of Cdk5. Cdk5 phosphorylates MEF2 at a distinct serine in its transactivation domain to inhibit MEF2 activity. Neurotoxicity enhances nuclear Cdk5 activity, leading to Cdk5-dependent phosphorylation and inhibition of MEF2 function in neurons. MEF2 mutants resistant to Cdk5 phosphorylation restore MEF2 activity and protect primary neurons from Cdk5 and neurotoxin-induced apoptosis. These studies reveal a nuclear pathway by which neurotoxin/Cdk5 induces neuronal apoptosis through inhibiting prosurvival nuclear machinery (Gong, 2003).
Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules (Tanaka, 2004).
The relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway has been studied. cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 results in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) -/- mice, which lack appreciable cdk5 activity, an increase is observed in the phosphorylation of NF-M subunit of neurofilament proteins that correlate with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) is not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data have prompted the proposal of a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1 (Sharma, 2002a).
Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. This study shows that Cdk5 phosphorylation of p27(Kip1) at Thr187 is crucial to neural differentiation because (A) neurogenesis is specifically suppressed by transfection of p27(Kip1) siRNA into Cdk5(+/+) NSCs; (B) reduced neuronal differentiation in Cdk5(-/-) compared with Cdk5(+/+) NSCs; (C) Cdk5(+/+) NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; (D) transfection of mutant p27(Kip1) (p27/187A) into Cdk5(+/+) NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27(Kip1) at the Thr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27(Kip1), a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth and migration (Zheng, 2010).
Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases, including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control mice in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases (Ahlijanian, 2000).
Phosphorylation of tau (a heat-stable neuron-specific microtubule-associated protein) by cdk5 is stimulated in the presence of microtubules (MTs). This stimulation is due to an increased phosphorylation rate but there is no increase in the total amount of phosphorylation. Two-dimensional phosphopeptide map analysis shows that MTs stimulate phosphorylation of a specific peptide. Using Western blotting with antibodies that recognize phosphorylation-dependent epitopes within tau, the phosphorylation sites stimulated by the presence of MTs were found to be Ser202 and Thr205 (numbered according to the human tau isoform containing 441 residues). MT-dependent phosphorylation at Thr205 is observed in situ in rat cerebrum primary cultured neurons. Stimulated phosphorylation at Ser202 and Thr205 decreases the MT-nucleation activity of tau, which is in contrast to MT-independent phosphorylation at Ser235 and Ser404 (Wada, 1998).
Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, namely, Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. The structural basis for this type of substrate selectivity was probed by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats are phosphorylated on serine with a recombinant CDK-5/p25 complex, whereas those with KSPXXX repeats are unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O reveal a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicates the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide, which includes two such KSPXK repeats, suggests a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yield an unusually stable, folded structure with a double S-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments (Sharma, 1998).
Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35nck5a and other protein components. The yeast two-hybrid system was employed to identify p35nck5a-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment reveals significant homology between the p35nck5a-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35nck5a and the Cdk5/p35nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35nck5a (Qi, 1998).
Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, it has been demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 are associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, a P13 complex was obtained from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, are consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibit co-elution of phosphorylated NF-H, tubulin, tau and cdk5. It is suggested that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon (Veeranna, 1999).
During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. Signal transduction pathways were investigated in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) show that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 reveal that growth cone collapse by direct activation of G(o/i) proteins with mastoparan does not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induces growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevents mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, it is concluded that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins (Nakayama, 1999).
Phosphorylation of the neurofilament-H subunit (NF-H) was investigated in rat embryonic brain neurons in culture. A portion of the NF-H is phosphorylated in vivo by embryonic day 17 when brain neurons were prepared. When the neurons were isolated and cultured, the NF proteins disappear and then reappear over the next several days in the following order: (1) NF-L/NF-M; (2) dephosphorylated NF-H and (3) phosphorylated NF-H. Phosphorylation of NF-H began around 4 days after cell plating, at about the time of synapse formation. Treatments that appear to modulate the timing of synapse formation also affect the timing of NF-H phosphorylation: (1) earlier phosphorylation is observed at higher neuronal cell density; (2) earlier phosphorylation is observed in neurons cultured on a coating substrate that promotes rapid neurite extension, and (3) phosphorylation is suppressed when neurite extension is inhibited by brefeldin A. Three possible synapse formation-induced events, excitation, cell-cell contact through adhesion proteins and elevated concentrations of neurotrophic factors, were examined for their possible involvement in generating the signal for NF-H phosphorylation. Neither excitation nor cell contact enhances NF-H phosphorylation. Neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) stimulate phosphorylation of NF-H. The BDNF-stimulated phosphorylation is inhibited by an anti-BDNF antibody and K252a, an inhibitor of BDNF receptor TrkB tyrosine kinase. Among known NF-H kinases of cyclin-dependent kinase 5 (CDK5), external signal-regulated protein kinase (ERK) and stress-activated protein kinase (SAPK), CDK5 and SAPK show an increase in kinase activity or an active form with a time course similar to NF-H phosphorylation in control culture. BDNF stimulates the kinase activity of CDK5 and induces appearance of an active form of ERK transiently. These results suggest a possibility that synapse formation induces NF-H phosphorylation, at least in part, through activation of CDK5 by BDNF (Tokuoka, 2000).
Cyclin-dependent kinase 5(cdk5) is highly homologous to other members of the cdk family that are known to function in proliferating cells. Despite the structural similarity, cdk5-associated histone H1 kinase activity is only detectable in postmitotic neurons of the central nervous system (CNS). p35 is a neuronal-specific cdk5 regulator that activates cdk5 kinase activity upon association. The cdk5/p35 kinase activity increases during the progression of CNS neurogenesis, suggesting a function of cdk5 in neuronal differentiation. Both cdk5 and p35 proteins are present in the growth cones of developing neurons. The staining pattern of cdk5 in the growth cones is similar to that of actin filaments but not microtubules. To address the functional significance of the cdk5/p35 kinase in neurogenesis, wild-type or mutant kinases were ectopically expressed in cortical cultures. Expression of dominant-negative mutants of cdk5 (cdk5N144 and cdk5T33) inhibits neurite outgrowth, which is rescued by coexpression of the wild-type proteins. A similar extent of neurite outgrowth inhibition is obtained by transfection of an antisense p35 construct, which in turn is only rescued by p35 but not cdk5 coexpression. In contrast, longer neurites were elaborated in neurons that coexpressed exogenous cdk5 and p35. These observations suggest that the cdk5/p35 kinase plays a critical role in neurite outgrowth during neuronal differentiation (Nikolic, 1996).
The expression, activity and localization of cyclin dependent kinase 5 (cdk5) during myogenesis was examined. Cdk5 protein is expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 shows a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level is specific for differentiation and not simply related to cell cycle arrest since it is not observed in fibroblasts grown for 48 hours in low serum medium. Anti-cdk5 antibodies showe a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhances differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibits the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis (Lazaro, 1997).
The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an 'inside-out' manner to produce six cortical laminae. p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase (Chae, 1997).
Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in the developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. Cdk5(-/-) mice were generated through gene targeting. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization (Ohshima, 1996).
The cerebral cortex of mice with a targeted disruption in the gene for cyclin-dependent kinase 5 (cdk5) is abnormal in its structure. Bromodeoxyuridine labeling reveals that the normal inside-out neurogenic gradient is inverted in the mutants; earlier born neurons are most often found superficial to those born later. Despite this, the early preplate layer separates correctly and neurons with a normal, pyramidal morphology can be found between true marginal zone and subplate. Consistent with their identity as layer VI corticothalamic neurons, they can be labeled by DiI injections into thalamus. The DiI injections also reveal that the trajectories of the cdk5(-/-) thalamocortical axons are oblique and cut across the entire cortical plate, instead of being oriented tangentially in the subcortical white matter. A model is proposed in which the cdk5(-/-) defect blocks cortical development at a heretofore undescribed intermediate stage, after the splitting of the preplate, but before the migration of the full complement of cortical neurons (Gilmore, 1998).
The p35/cdk5 neuronal-specific kinase complex has been shown to play an important role in the laminar configuration of cortical neurons. Mice lacking either p35 or cdk5 exhibit a disrupted cortical lamination pattern. It has previously been shown that nstead of the normal 'inside-out' layering pattern of cortical neurons, cortical neurons are layered from 'outside-in' in p35 mutant mice. To gain insight into the mechanisms that underlie these defects, the organization of landmark structures formed during cortical development and the migratory behavior of p35(-/-) cortical neurons were examined by using bromodeoxyuridine labeling. Reelin localization in the marginal zone is normal in p35 mutant mice. Furthermore, the preplate properly splits into the marginal zone and subplate, a developmental event that fails to occur in reeler mice. Finally, the migration of the earliest born cortical plate neurons is normal in p35 mutant mice; cortical neurons subsequently generated remain underneath these neurons. These data suggest that the p35/cdk5 kinase is required for cortical plate neurons to migrate past preexisting neurons and take up superficial positions to constitute the inside-outside layering order of cortical lamination (Kwon, 1998).
Mice lacking p35, an activator of cdk5 in the central nervous system (CNS), exhibit defects in a variety of CNS structures, most prominently characterized by a disruption in the laminar structure of the neocortex. In addition, alterations of certain axonal fiber tracts are found in the cortex of p35 mutant mice. Notably, the corpus callosum appears bundled at the midline, but dispersed lateral to the midline. Tracer injection experiments in adult p35 mutant mice reveal that projecting cortical axons fail to assimilate into the corpus callosum, and take oblique paths to the midline. After crossing the midline, cortical axons defasciculate prematurely from the corpus callosum and take similarly oblique paths through the cortex. This callosal phenotype is not detected in reeler mice, which also exhibit defects in cortical lamination, suggesting that the lack of fasciculation of callosal axons is not an inherent manifestation of a disruption of cortical lamination. The embryonic callosal axon tract is defasciculated before crossing the midline, suggesting that axon guidance may be affected during embryonic development of the corpus callosum. In addition, embryonic thalamocortical afferents also exhibit a defasciculated phenotype. These results suggest that defective axonal fasciculation and guidance may be primary responses to the loss of p35 in the cortex. Furthermore, this study postulates a role for the p35/cdk5 kinase in molecular signaling pathways necessary for proper guidance of selective axons during embryonic development (Kwon, 1999).
In spite of the clarification in the temporal and spatial expression pattern of cyclin-dependent kinase 5 (Cdk5) and its neuron-specific activator, p35, in the CNS, these expression patterns remain to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, Cdk5 and p35 expression and activity in the PNS were examined by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicates the expression of Cdk5 and p35 proteins in both the dorsal root ganglion (DRG) and sciatic nerve in the CNS. By immunohistochemistry, both proteins are present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicates the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity is found only in DRG, and 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 (Terada, 1998).
Cyclin-dependent kinase-5 (cdk-5) is a serine/threonine kinase that displays neuron-specific activity. Experimental manipulation of cdk-5 expression in neurons has shown that cdk-5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk-5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk-5, the yeast two-hybrid system was used to search for proteins that interact with cdk-5. In two independent yeast transformation events, cyclin D2 interacts with cdk-5. Immunoprecipitation experiments confirm that cyclin D2 and cdk-5 interact in mammalian cells. Cyclin D2 did not activate cdk-5 as assayed using three different substrates; this is in contrast to a known cdk-5 activator, p35. However, cyclin D2 expression leads to a decrease in cdk-5/p35 activity in transfected cells. Since cyclin D2 and cdk-5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk-5 activity in postmitotic developing neurons (Guidato, 1998).
The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When GFP was introduced into migrating neurons at E14.5 by in utero electroporation, migrating neurons were observed in wild-type but not in Cdk5-/- embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons (Ohshima, 2007).
The role of cyclin-dependent kinases in cell death has been investigated and the expression of cyclin-dependent kinase 5 (Cdk5) has been found to be associated with apoptotic cell death in both adult and embryonic tissues. By double labeling immunohistochemistry and confocal microscopy, the expression of Cdk5 was specifically associated with dying cells. The association of Cdks with cell death is unique to Cdk5, since this association is not found with the other Cdks (Cdk 1-8) and cell death. The differential increase in Cdk5 expression is at the level of protein only, and no differences can be detected at the level of mRNA. Using the limbs of mutant mice detective in the pattern of interdigital cell death and limbs with increased interdigital cell death as a result of retinoic acid treatment, the specificity of Cdk5 protein expression in dying cells has been confirmed. To investigate the regulation of Cdk5 during cell death, the expression of a regulatory protein of Cdk5, p35, was examined. p35 was found to be expressed in the dying cells as well. Similar to Cdk5, there is also no specific differential expression of the p35 mRNA in dying cells. These results suggest a role for Cdk5 and p35 proteins in cell death. This protein complex may function in the rearrangement of the cytoskeleton during apoptosis (Ahuja, 1998).
Cyclin-dependent kinase 5 (Cdk5) is required for proper development of the mammalian central nervous system. To be activated, Cdk5 has to associate with its regulatory subunit, p35. p25, a truncated form of p35, accumulates in neurons in the brains of patients with Alzheimer's disease. This accumulation correlates with an increase in Cdk5 kinase activity. Unlike p35, p25 is not readily degraded, and binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location and alters its substrate specificity. In vivo the p25/Cdk5 complex hyperphosphorylates tau, which reduces tau's ability to associate with microtubules. Moreover, expression of the p25/Cdk5 complex in cultured primary neurons induces cytoskeletal disruption, morphological degeneration and apoptosis. These findings indicate that cleavage of p35, followed by accumulation of p25, may be involved in the pathogenesis of cytoskeletal abnormalities and neuronal death in neurodegenerative diseases (Patrick, 1999).
Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5-/- and p35-/- mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival have been unknown. This study shows that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis (Li, 2002).
Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. In this study, inducible transgenic mouse lines were generated overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directs Cdk5 to pathological substrates. These animals exhibit neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau is hyperphosphorylated at many epitopes, aggregated tau accumulates, and neurofibrillary pathology develops progressively in these animals. These cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology (Cruz, 2003).
Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins was investigated in a mammalian system. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. It was found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-α. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis (Samuels, 2007).
Homologs of liprin-α proteins are essential for presynaptic terminal formation in C. elegans and Drosophila . Mutations in C. elegans syd-2 result in a diffuse localization of several presynaptic proteins and abnormally sized active zones, and loss- and gain-of-function experiments demonstrate that presynaptic organization is dependent on syd-2. Likewise, Dliprin-α is required for normal synaptic morphology including the size and shape of the presynaptic active zone in Drosophila . Cdk5-dependent phosphorylation of CASK occurs in both the CaMK and L27 domains, and only mutation of both sites yields a localization phenotype. Since liprin-α proteins require the presence of both domains to interact with CASK, the phosphorylation sites are in a prime spot to mediate the interaction. According to the model described in this study, liprin-α is required for initial CASK localization to presynaptic terminals. Since, liprin-α binds directly to the kinesin motor KIF1A and in Drosophila liprin-α mutant axons there is decreased anterograde processivity resulting in reduced levels of presynaptic markers at terminals, it is feasible that liprin-α acts as a cargo receptor that delivers CASK, as well as other components, to and within the developing synapse. Cdk5-dependent phosphorylation could then act to coordinate distinct pools of CASK that are bound to liprin-α or are bound to other components of the presynaptic machinery. Importantly, it is not believed that Cdk5 loss of function generally affects liprin-α-mediated transport since synaptophysin, a marker of synaptic vesicles, is still properly localized within synaptosomes. In this model, there would be advantages of having locally enhanced Cdk5 activity within the presynaptic terminal relative to some other cellular compartments. Supporting this idea, phospho-CASK is particularly enriched at synaptic membranes, and Cdk5 has been shown to phosphorylate and regulate several proteins, including Munc-18, Dynamin-1, Amphiphysin-1, and Synaptojanin-1, that function to control multiple rounds of the synaptic vesicle cycle. Synapsin-1 is also a Cdk5 substrate. With regard to the role of liprin-α, it will ultimately be essential to assay synapse formation and CASK localization in mammalian liprin-α loss-of-function models (Samuels, 2007).
Learning is accompanied by modulation of postsynaptic signal transduction pathways in neurons. Although the neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) has been implicated in cognitive disorders, its role in learning has been obscured by the perinatal lethality of constitutive knockout mice. Conditional knockout of Cdk5 in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice is attributed to reduced NR2B degradation, which causes elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. Cdk5 facilitates the degradation of NR2B by directly interacting with both it and its protease, calpain. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and learning (Hawasli, 2007).
Homeostatic plasticity keeps neuronal spiking output within an optimal range in the face of chronically altered levels of network activity. Little is known about the underlying molecular mechanisms, particularly in response to elevated activity. In hippocampal neurons experiencing heightened activity, the activity-inducible protein kinase Polo-like kinase 2 (Plk2, also known as SNK) is required for synaptic scaling-a principal mechanism underlying homeostatic plasticity. Synaptic scaling also requires CDK5, which acts as a 'priming' kinase for the phospho-dependent binding of Plk2 to its substrate SPAR, a postsynaptic RapGAP and scaffolding molecule that is degraded following phosphorylation by Plk2. RNAi knockdown of SPAR weakens synapses, and overexpression of a SPAR mutant resistant to Plk2-dependent degradation prevents synaptic scaling. Thus, priming phosphorylation of the Plk2 binding site in SPAR by CDK5, followed by Plk2 recruitment and SPAR phosphorylation-degradation, constitutes a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity (Seeburg, 2008).
Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an antioncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes, including cell cycle exit and shape changes. Senescence is accompanied by increased expression and altered localization of 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 (Yang, 2003).
Normal human somatic cells do not divide indefinitely, but rather, have a limited capacity to replicate in culture. The finite replicative lifespan of cells leads to an arrest of cell division by a process termed senescence, clearly distinct from differentiation, in which cells remain metabolically active indefinitely. The irreversible arrest of cell division that accompanies cellular senescence may be tumor suppressive, and escape of cells from senescence accompanies immortalization and oncogenesis. Indeed, a premature senescence is observed following oncogene introduction into primary human cells, and this antiproliferative response must be overcome if cells are to become transformed. Further, senescence may play a significant role in response to cancer therapy (Yang, 2003 and references therein).
Despite this potentially critical role for senescence in tumor formation, knowledge of the biochemical pathways responsible for the acquisition of cellular senescence is rudimentary. The retinoblastoma tumor suppressor protein, pRb, plays a fundamental role in cellular senescence, consistent with a critical role for pRb in the cellular machinery that controls passage from G1 into S phase of the cell cycle. Senescent cells accumulate active pRb, fail to inactivate pRb upon mitogenic stimulation, and consequently cannot enter S phase. Indeed, reintroduction of pRb into Rb-/- tumor cell lines induces senescence, even in cells that do not contain wild-type p53. Similarly, overexpression of p16INK4a can induce senescence in pRb-positive tumor cells. Loss of p16INK4a or pRb function appears to be required for immortalization of at least some human cell types, apparently as an obligate step in preventing senescence (Yang, 2003 and references therein).
RB-transfected SAOS-2 osteosarcoma cells serve as a model system of senescence. Reintroduction of pRb into SAOS-2 cells results in an immediate G1 arrest and subsequent expression of characteristic markers of senescence. The first indication of pRb-induced senescence to be recognized in this system was 'flat cell' formation, typified by an increased cell area and a flattened appearance. This phenotype appears identical to that observed during classical senescence, where a morphological alteration from spindle shape to an enlarged, flattened, and irregular shape is taken as an indicator of the senescent state (Yang, 2003 and references therein).
Despite the universality of morphological changes observed in a wide variety of senescent cells, little is known about the induction of this phenotype nor about its potential contribution to the establishment or maintenance of the irreversible growth arrest that accompanies senescence. Nevertheless, considerable work has clearly indicated a significant role for cell shape in cellular proliferation, largely as a consequence of communication between the cytoskeleton and its associated proteins. One such set of cytoskeletal-associated proteins that has recently emerged as important in proliferation control is the ezrin-radixin-moesin (ERM) family of cytoskeleton-membrane crosslinking proteins, including the related protein NF-2/merlin, an established tumor suppressor. These proteins play a role in the formation of microvilli, cell-cell junctions, and membrane ruffles, and also regulate substrate adhesion and motility. It has most recently become clear that the ERM proteins regulate and respond to proliferative signals, both in a positive and negative manner (Yang, 2003 and references therein).
ERM proteins possess two conserved domains that have been termed N- and C- ERM association domains, or ERMADs. The NH2-terminal domain associates with several transmembrane adhesion molecules, whereas the COOH-terminal domain contains an F-actin binding site. These binding sites are masked in cytoplasmic, inactive ERMs due to an intramolecular N/C-ERMAD interaction. Regulation of ERMs is thought to occur through conformational changes consequent to posttranslational modifications that inhibit association of the N-ERMAD with the C-ERMAD. This scheme has been supported by solution of the crystal structure of the relevant domains of moesin. This work reveals a globular conformation for the N-ERMAD domain and an extended conformation for the C-ERMAD, which mutually mask binding sites for other cellular proteins (Yang, 2003 and references therein).
Phosphorylation has been proposed to regulate ERM activation, since phosphorylation of ERM proteins correlates with their cytoskeletal association. Several observations have suggested that phosphorylation of serine/threonine residues is important for the activity of ERM proteins. Phosphorylation of T567 in ezrin has been found to be critical for conversion of ezrin to the active, open form competent for membrane localization and actin binding. Indeed, structural studies suggest that phosphorylation of T567 would sterically interfere with N-ERMAD/C-ERMAD interactions. Furthermore, induction of apoptosis induces a serine/threonine dephosphorylation of ezrin. This dephosphorylation is essential for the translocation of ezrin from the plasma membrane to the cytoplasm. Thus, regulation of ERM proteins through phosphorylation is likely critical to membrane-cytosekeleton signaling, and this in turn will have a pleiotropic impact on cell shape, motility, and proliferation (Yang, 2003 and references therein).
Ezrin regulation has been linked to pRb function in the senescent phenotype. Ezrin expression increases upon pRb-induced senescence, and more significantly, ezrin becomes membrane associated concomitant with acquisition of the senescent phenotype. This membrane association appears to be the consequence of direct phosphorylation of T235 of ezrin by CDK5, which is activated in response to pRb expression. Phosphorylation of T235 prevents the intermolecular N/C ERMAD association in a manner analogous to and cooperative with phosphorylation of T567, likely allowing ezrin to participate in cytoskeleton-related signaling events germane to senescence (Yang, 2003).
Calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. This study reports a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. A mechanistic model is proposed by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinson's disease patients when compared to control, suggesting the relevance of this pathway in the human condition (Qu, 2007).
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