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Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre-synaptic terminals triggered by Ca(2+) influx into the pre-synaptic cytoplasm through voltage-dependent Ca(2+) channels (VDCCs). It is reported that Cdk5 regulates L-, P/Q-, or N-type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca(2+) -channel property of VDCCs, using PC12 cells expressing endogenous, functional L-, P/Q-, and N-type VDCCs. The Ca(2+) influx, induced by membrane depolarization with high K(+) , was monitored with a fluorescent Ca(2+) indicator protein in both undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells. Overall, Ca(2+) influx was increased by expression of Cdk5-p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5-p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5-p35 regulates L-, P/Q-, or N-type VDCCs in a cellular context-dependent manner. Calcium (Ca(2+) ) influx through voltage-dependent Ca(2+) channels (VDCCs) triggers neurotransmitter release from pre-synaptic terminal of neurons. The channel activity of VDCCs is regulated by Cdk5-p35, a neuronal Ser/Thr kinase. However, there have been debates about the regulation of VDCCs by Cdk5. Using PC12 cells, we show that Cdk5-p35 regulates VDCCs in a type (L, P/Q, and N) and differentiation-dependent manner. NGF = nerve growth factor.

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

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca(2+)/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

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.

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

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

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase of increasingly recognized importance in a large number of fields, ranging from neuronal migration to synaptic plasticity and neurodegeneration. However, little is known about its mechanism of activation beyond its requirement for binding to p35 or p39. We have examined membrane interactions as one method of regulating the Cdk5-p35 complex. The kinase activity of Cdk5-p35 is low when it is bound to membranes. The Cdk5-p35 found in rat brain extract associates with membranes in two ways. Approximately 75% of complexes associate with membranes via ionic interactions only, and the remaining 25% associate with membranes via ionic interactions together with lipidic interactions. Solubilization with detergent or high-salt solution activates Cdk5-p35 several fold, and this activation is reversible. Therefore, membrane interactions represent a novel mechanism for the regulation of Cdk5-p35 kinase activity.

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca(2+)/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.