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

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

The protein SET binds the neuronal Cdk5 activator p35nck5a and modulates Cdk5/p35nck5a activity.

The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35(nck5a) or its isoform, p39(nck5ai). To identify novel p35(nck5a)- and p39(nck5ai)-binding proteins, fragments of p35(nck5a) and p39(nck5ai) were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35(nck5a) and p39(nck5ai). Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35(nck5a) through its binding to p35(nck5a). The p35(nck5a)-interacting region was mapped to a predicted alpha-helix in SET. When cotransfected into COS-7 cells, SET and p35(nck5a) displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35(nck5a) from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35(nck5a), but not that of Cdk5/p25(nck5a), was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35(nck5a).     

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

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

The regulation of cyclin-dependent kinase 5 activity through the metabolism of p35 or p39 Cdk5 activator

Cyclin-dependent kinase 5 (Cdk5) displays kinase activity predominantly in post-mitotic neurons and its physiological roles are unrelated to cell cycle progression. Cdk5 is activated by its binding to a neuron-specific activator, p35 or p39. The protein amount of p35 or p39 is a primary determinant of the Cdk5 activity in neurons, with the amount of p35 or p39 being determined by its synthesis and degradation. The expression of p35 is induced in differentiated neurons and is enhanced by extracellular stimuli such as neurotrophic factors or extracellular matrix molecules, specifically those acting on the ERK/Erg pathway. p35 is a short-lived protein and its degradation determines the life span. Degradation is mediated by the ubiquitin/proteasome system, similar to that for cyclins in proliferating cells. Autophosphorylation of p35 by Cdk5 is a signal for ubiquitination/degradation, and the degradation of p35 is triggered by glutamate treatment in cultured neurons. p35 is cleaved to p25 by calpain at the time of neuronal cell death, and this limited cleavage is suggested to be the cause of neurodegenerative diseases such as Alzheimer's disease. Active Cdk5 changes the cellular localization by cleavage of p35 to p25; p35/Cdk5 is associated with membrane or cytoskeletons, but p25/Cdk5 is a soluble protein. Cleavage also increases the life span of p25 and changes the activity or substrate specificity of Cdk5. p25/Cdk5 shows higher phosphorylating activity to tau than p35/Cdk5 in a phosphorylation site-specific manner. Phosphorylation of p35 suppresses cleavage by calpain. Thus, phosphorylation of p35 modulates its proteolytic pattern, stimulates proteasomal degradation and suppresses calpain cleavage. Phosphorylation is age dependent, as p35 is phosphorylated in foetal brains, but unphosphorylated in adult brains. Therefore, foetal phosphorylated p35 is turned over rapidly, whereas adult unphosphorylated p35 has a long life and is easily cleaved to p25 when calpain is activated. p39 is also a short-lived protein and cleaved to the N-terminal truncation form of p29 by calpain. How the metabolism of p39 is regulated, however, is a future problem to be investigated.     

Microtubule association of the neuronal p35 activator of Cdk5

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.     

Resveratrol inhibits Cdk5 activity through regulation of p35 expression

Background

We have previously reported that cyclin-dependent kinase 5 (Cdk5) participates in the regulation of nociceptive signaling. Through activation of the ERK1/2 pathway, Tumor Necrosis Factor-α (TNF-α) induces expression of Egr-1. This results in the sustained and robust expression of p35, a coactivator of Cdk5, in PC12 cells, thereby increasing Cdk5 kinase activity. The aim of our present study was to test whether resveratrol, a polyphenolic compound with known analgesic activity, can regulate Cdk5/p35 activity.

Results

Here we used a cell-based assay in which a p35 promoter-luciferase construct was stably transfected in PC12 cells. Our studies demonstrate that resveratrol inhibits p35 promoter activity and also blocks the TNF-α mediated increase in Cdk5 activity in PC12 cells. Resveratrol also inhibits p35 expression and blocks the TNF-α mediated increase in Cdk5 activity in DRG neurons. In the presence of resveratrol, the MEK inhibitor decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK and NF-κB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The TNF-α-mediated increase in Egr-1 expression was decreased by resveratrol treatment with a concomitant reduction in p35 expression and protein levels, resulting in reduced Cdk5 kinase activity.

Conclusions

We demonstrate here that resveratrol regulates p35 promoter activity in PC12 cells and DRG neurons. Most importantly, resveratrol blocks the TNF-α-mediated increase in p35 promoter activity, thereby reducing p35 expression and subsequent Cdk5 kinase activity. This new molecular mechanism adds to the known analgesic effects of resveratrol and confirms the need for identifying new analgesics based on their ability to inhibit Cdk5 activity for effective treatment of pain.

     

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

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

Pctaire1 interacts with p35 and is a novel substrate for Cdk5/p35

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that plays important roles during central nervous system development. Cdk5 kinase activity depends on its regulatory partners, p35 or p39, which are prominently expressed in the central nervous system. We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse. Here we report the identification of Pctaire1, a member of the Cdk-related kinase family, as a p35-interacting protein in muscle. Binding of Pctaire1 to p35 can be demonstrated by in vitro binding assay and co-immunoprecipitation experiments. Pctaire1 is associated with p35 in cultured myotubes and skeletal muscle, and is concentrated at the neuromuscular junction. Furthermore, Pctaire1 can be phosphorylated by the Cdk5/p25 complex, and serine 95 is the major phosphorylation site. In brain and muscle of Cdk5 null mice, Pctaire1 activity is significantly reduced. Moreover, Pctaire1 activity is increased following preincubation with brain extracts and phosphorylation by the Cdk5/p25 complex. Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitro and in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity.     

Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that is activated by binding to its regulatory subunit, p35. The calpain-mediated cleavage of p35 to p25 and the resulting aberrant activity and neurotoxicity of Cdk5 have been implicated in neurological disorders, such as Alzheimer's disease. To gain further insight into the molecular mechanisms underlying the pathological function of Cdk5, we investigated the role of the calpain inhibitor protein calpastatin (CAST), in controlling the aberrant production of p25. For this purpose, brain tissue from wild-type, CAST-over-expressing (transgenic), and CAST knockout mice were analyzed. Cleavage of p35 to p25 was increased in extracts from CAST knockout mice, compared with wild-type. Conversely, generation of p25 was not detected in brain lysates from CAST-over-expressing mice. CAST expression was 5-fold higher in mouse cerebellum than cerebral cortex. Accordingly, p25 production was lower in the cerebellum than the cerebral cortex. Furthermore, the Ca(2+) -dependent degradation of p35 by proteasome was evident when calpain was inhibited. Taken together, these results suggest that CAST is a crucial regulator of calpain activity, the production of p25, and, hence, the deregulation of Cdk5. Therefore, impairment of CAST expression and its associated mechanisms may contribute to the pathogenesis of neurodegenerative disorders.     

Fas engagement induces neurite growth through ERK activation and p35 upregulation

Fas (also known as CD95), a member of the tumour-necrosis receptor factor family of 'death receptors', can induce apoptosis or, conversely, can deliver growth stimulatory signals. Here we report that crosslinking Fas on primary sensory neurons induces neurite growth through sustained activation of the extracellular-signal regulated kinase (ERK) pathway and the consequent upregulation of p35, a mediator of neurite outgrowth. In addition, functional recovery after sciatic nerve injury is delayed in Fas-deficient lpr mice and accelerated by local administration of antibodies against Fas, which indicates that Fas engagement may contribute to nerve regeneration in vivo. Our findings define a role for Fas as an inducer of both neurite growth in vitro and accelerated recovery after nerve injury in vivo.     

Drosophila lacking the Cdk5 activator, p35, display defective axon guidance, age-dependent behavioral deficits and reduced lifespan

The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. We show here that Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. We further show that, while viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan.     

The Expression of Cdk5, p35, p39, and Cdk5 Kinase Activity in Developing, Adult, and Aged Rat Brains

The expression of cyclin-dependent kinase 5 (Cdk5) and its regulatory subunits, p35 and p39, was investigated in rat brain from embryonic day 12 (E12) to postnatal 18 months (18M). The Cdk5 protein levels increased from E12 to postnatal day 7 (P7) and remained at this level until 18M. The Cdk5 kinase activity and the levels of both p35 mRNA and protein were low at E12, became prominent at E18-P14 but then decreased in the adult and aged rat brains of 3M to 18M. In comparison, the expression pattern of p39 appeared to have an inverse relationship to that of Cdk5 and p35. In regional distribution studies, p35 protein levels and Cdk5 kinase activity were significantly higher in the cerebral cortex and hippocampus, but lower in the cerebellum and striatum. These results suggested that Cdk5, p35 and p39 might have region-specific and developmental stage-specific functions in rat brain.     

ODF1 phosphorylation by Cdk5/p35 enhances ODF1-OIP1 interaction.

Cdk5 and p35 are integral components of the sperm tail outer dense fibers (ODFs), which contribute to the distinct morphology and function of the sperm tail. In this study, we sought to characterize and investigate the significance of Cdk5/p35 association with ODFs. We show that ODF2 interacts with Cdk5 and p35 but not with the Cdk5/p35 heterodimer. By using deletion mutants, the ODF2 binding region in p35 was mapped to residues 122 to 198. This overlaps the Cdk5 binding region in p35, explaining the inability of ODF2 to bind to the Cdk5/p35 complex. In vitro phosphorylation assay showed that although Cdk5/p35 does not phosphorylate ODF2, it phosphorylates ODF1. Mass spectrometry revealed that Cdk5/p35 specifically phosphorylates Ser193 in the ODF1 C-terminal region containing the Cys-X-Pro motif, the interaction site for the novel RING finger protein, ODF1 interacting protein (OIP1), a candidate E3 ubiquitin ligase, that also localizes in the sperm tail. Cdk5 phosphorylation of ODF1 Ser193 results in enhanced ODF1-OIP1 interaction. These findings suggest that Cdk5 may be important in promoting ODF1 degradation, and potentially, the detachment and fragmentation of the sperm tail following fertilization.     

Cdk5/p35 expression in the mouse ovary

Cyclin-dependent kinase 5 (Cdk5) is primarily associated with brain development but it is also implicated in lens and muscle differentiation. We found that Cdk5 is also expressed in mouse ovary, and explored the possibility that it plays a role in that tissue. We show by Western blotting and immunohistochemistry that the known Cdk5 activator, p35, is also present in the mouse ovary. Cdk5 and p35 were detected in oocytes at all stages of the follicle. While Cdk5 was present in the cytoplasm and nucleus of the oocyte, p35 was observed only in the cytoplasm. Both proteins were detected in the cytoplasm of luteinized cells in the corpus luteum. Immunoprecipitation and histone H1 kinase assays revealed that they form an ovarian complex with considerable kinase activity. Phosphorylation assays showed that several ovarian proteins are substrates for Cdk5/p35 in vitro. Together our findings suggest that p35-associated Cdk5 activity plays an important role in the ovary, where it may regulate cell differentiation and apoptosis as it does in the brain.     

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).     

Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology

The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.