Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration

Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. We found that beta-amyloid (Abeta42) initiated Abl kinase activity and that blockade of Abl kinase rescued both Drosophila and mammalian neuronal cells from cell death. We also found activated Abl kinase to be necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 was not observed in Abeta42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. Our genetic studies also showed that abl mutations repressed Abeta42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Abeta42 induced conversion of p35 to p25 in mammalian cells, it did not sufficiently induce Cdk5 activation when c-Abl kinase activity was suppressed. Therefore, we propose that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Abeta42-triggered neurodegeneration.     

Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling

The molecular mechanism behind what causes an infection of Enterovirus 71 (EV71) in young children to result in severe neurological diseases is unclear. Herein, we show that Cdk5, a critical signalling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71-induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71-induced Cdk5 activation was modulated by c-Abl. The suppression of c-Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non-neuronal cells, it did not affect Abl and Cdk5 activities in several non-neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggest a serotype- and cell type-specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5-signalling for neuronal apoptosis.     

Cell-autonomous role of Presenilin in age-dependent survival of cortical interneurons

Studies link c-Abl activation with the accumulation of pathogenic α-synuclein (αS) and neurodegeneration in Parkinson’s disease (PD). Currently, c-Abl, a tyrosine kinase activated by cellular stress, is thought to promote αS pathology by either directly phosphorylating αS or by causing autophagy deficits. αS overexpressing transgenic (Tg) mice were used in this study. A53T Tg mice that express high levels of human mutant A53TαS under the control of prion protein promoter. Two different approaches were used in this study. Natural aging and seeding model of synucleinopathy. In seeding model, intracortical/intrastriatal (IC/IS) stereotaxic injection of toxic lysates was done using tissue lysates from end-stage symptomatic mice. In this study, nilotinib and pifithrin-α was used as a c-Abl and p53 inhibitor, respectively. Both Tg and non-transgenic (nTg) mice from each group were subjected to nilotinib (10 mg/kg) or vehicle (DMSO) treatment. Frozen brain tissues from PD and control human cases were analyzed. In vitro cells study was implied for c-Abl/p53 genetic manipulation to uncover signal transduction. Herein, we show that the pathologic effects of c-Abl in PD also involve activation of p53, as c-Abl activation in a transgenic mouse model of α-synucleinopathy (TgA53T) and human PD cases are associated with the increased p53 activation. Significantly, active p53 in TgA53T neurons accumulates in the cytosol, which may lead to inhibition of autophagy. Thus, we hypothesized that c-Abl-dependent p53 activation contributes to autophagy impairment in α-synucleinopathy. In support of the hypothesis, we show that c-Abl activation is sufficient to inhibit autophagy in p53-dependent manner. Moreover, inhibition of either c-Abl, using nilotinib, or p53, using pifithrin-α, was sufficient to increase autophagic flux in neuronal cells by inducing phosphorylation of AMP-activated kinase (AMPK), ULK1 activation, and down-regulation of mTORC1 signaling. Finally, we show that pharmacological attenuation of c-Abl activity by nilotinib treatment in the TgA53T mouse model reduces activation of p53, stimulates autophagy, decreases accumulation αS pathology, and delays disease onset. Collectively, our data show that c-Abl activation by α-synucleinopathy causes p53 dependent autophagy deficits and both c-Abl and p53 represent therapeutic target for PD.     

Stimulation of p53 DNA binding by c-Abl requires the p53 C terminus and tetramerization

The carboxyl terminus of p53 is a target of a variety of signals for regulation of p53 DNA binding. Growth suppressor c-Abl interacts with p53 in response to DNA damage and overexpression of c-Abl leads to G(1) growth arrest in a p53-dependent manner. Here, we show that c-Abl binds directly to the carboxyl-terminal regulatory domain of p53 and that this interaction requires tetramerization of p53. Importantly, we demonstrate that c-Abl stimulates the DNA-binding activity of wild-type p53 but not of a carboxyl-terminally truncated p53 (p53Delta363C). A deletion mutant of c-Abl that does not bind to p53 is also incapable of activating p53 DNA binding. These data suggest that the binding to the p53 carboxyl terminus is necessary for c-Abl stimulation. To investigate the mechanism for this activation, we have also shown that c-Abl stabilizes the p53-DNA complex. These results led us to hypothesize that the interaction of c-Abl with the C terminus of p53 may stabilize the p53 tetrameric conformation, resulting in a more stable p53-DNA complex. Interestingly, the stimulation of p53 DNA-binding by c-Abl does not require its tyrosine kinase activity, indicating a kinase-independent function for c-Abl. Together, these results suggest a detailed mechanism by which c-Abl activates p53 DNA-binding via the carboxyl-terminal regulatory domain and tetramerization.     

Activation of latent cyclin-dependent kinase 5 (Cdk5)-p35 complexes by membrane dissociation

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.     

Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism.

Cyclin-dependent kinase 5 (Cdk5), a complex of Cdk5 and its activator p35 (Cdk5/p35), phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. Cdk5, acting together with other kinases, phosphorylates numerous KSPXK consensus motifs in diverse cytoskeletal protein target molecules, including neurofilaments, and microtubule associated proteins, tau and MAPs. Phosphorylation regulates the dynamic interactions of cytoskeletal proteins with one another during all aspects of neurogenesis and axon radial growth. In this review we shall focus on Cdk5 and its regulation as it modulates neurofilament metabolism in axon outgrowth, cytoskeletal stabilization and radial growth. We suggest that Cdk5/p35 forms compartmentalized macromolecular complexes of cytoskeletal substrates, other neuronal kinases, phosphatases and activators ('phosphorylation machines') which facilitate the dynamic molecular interactions that underlie these processes.     

p53, Apaf-1, caspase-3, and -9 are dispensable for Cdk5 activation during cell death

Cyclin-dependent kinase 5 (Cdk5) is a member of the cyclin-dependent kinase family that is mostly seen in neurons, does not vary with cell cycle, and is activated in many neurodegenerative disorders and other non-neuronal pathologies, but its relationship to non-neuronal apoptosis is not understood, nor is the control of the activation of Cdk5 by its activators. The most widely studied activator of Cdk5, p35, is cleaved to p25 by calpain, an event that has been linked with activation of Cdk5 and neuronal death. Here we report that calpain-mediated Cdk5/p25 activation accompanies non-neuronal as well as neuronal cell death, suggesting that the p35/calpain/p25/Cdk5 activation sequence is a general feature of cell death. We further demonstrate that Cdk5 can be activated in the absence of p53, Apaf-1, caspase-9, and -3 during cell death, indicating that its activation relates more to cell death than to a specific pathway of apoptosis.     

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.     

Interplay between the p53 tumor suppressor protein family and Cdk5: novel therapeutic approaches for the treatment of neurodegenerative diseases using selective Cdk inhibitors

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Interplay Between the p53 Tumor Suppressor Protein Family and Cdk5: Novel Therapeutic Approaches for the Treatment of Neurodegenerative Diseases Using Selective Cdk Inhibitors.

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Interplay between the p53 tumor suppressor protein family and Cdk5

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper, control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as τ protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability

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.     

Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. 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 inhibited neurite outgrowth. Furthermore, expression of active Abl resulted 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.