Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain

Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35-/- mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35-/-Cdk5 +/- mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35-/- mice compared with Cdk5-/- mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35-/- causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.     

Expression of the neuronal cyclin-dependent kinase 5 activator p35Nck5a in human monocytic cells is associated with differentiation

Although cyclin-dependent kinase 5 (Cdk5) is widely expressed in human tissues, its activator p35Nck5a is generally considered to be neuron specific. In addition to neuronal cells, active Cdk5 complexes have been reported in developing tissues, such as the embryonic muscle and ocular lens, and in human leukemia HL60 cells induced to differentiate by an exposure to 1,25-dihydroxyvitamin D(3); however, its activator in these cells has not been demonstrated. The results of this study indicate that p35Nck5a is associated with Cdk5 in monocytic differentiation of hematopoietic cells. Specifically, p35Nck5a is expressed in normal human monocytes and in leukemic cells induced to differentiate toward the monocytic lineage, but not in lymphocytes or cells induced to granulocytic differentiation by retinoic acid. It is present in a complex with Cdk5 that has protein kinase activity, and when ectopically expressed together with Cdk5 in undifferentiated HL60 cells, it induces the expression of CD14 and "nonspecific" esterase, markers of monocytic phenotype. These observations not only indicate a functional relationship between Cdk5 and p35Nck5a, but also support a role for this complex in monocytic differentiation. (Blood. 2001;97:3763-3767)     

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in 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. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. 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.     

Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo

The mechanisms involving neuronal death after ischemic/hypoxic insult are complex, involving both rapid (excitotoxic) and delayed (apoptotic-like) processes. Recent evidence suggests that cell cycle regulators such as cyclin-dependent kinases are abnormally activated in neuropathological conditions, including stroke. However, the function of this activation is unclear. Here, we provide evidence that inhibition of the cell cycle regulator, Cdk4, and its activator, cyclinD1, plays critical roles in the delayed death component of ischemic/hypoxic stress by regulating the tumor suppressor retinoblastoma protein. In contrast, the excitotoxic component of ischemia/hypoxia is predominately regulated by Cdk5 and its activator p35, components of a cyclin-dependent kinase complex associated with neuronal development. Hence, our data both characterize the functional significance of the cell cycle Cdk4 and neuronal Cdk5 signals as well as define the pathways and circumstances by which they act to control ischemic/hypoxic damage.     

The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons.

Cell cycle withdrawal in postmitotic cells involves cyclin-dependent kinase (Cdk) inhibitors that repress cell cycle Cdk activity. During mouse neurogenesis, cortical postmitotic neurons are shown here to accumulate high levels of the p27 Cdk inhibitor compared with their progenitor neuroblasts. Elevated p27 levels in staged embryo brain extracts correlate with p27 binding to Cdk2, and Cdk inactivation. Yet, Cdk5, which is associated with the noncyclin activator p35 in neurons, remains active in the presence of high p27 levels. Both in vitro and in vivo, p27 and related inhibitors can recognize a cyclin D-Cdk5 complex but not a p35-Cdk5 complex. The results indicate that the choice of activator determines the susceptibility of Cdk5 to p27 and related Cdk inhibitors, and thus its ability to act in postmitotic cells.     

Cyclin-dependent kinase 5 regulates steroidogenic acute regulatory protein and androgen production in mouse Leydig cells

The roles of cyclin-dependent kinase 5 (Cdk5) in central nervous system and neurodegenerative diseases have been intensely investigated in recent decades. Because protein expressions of Cdk5 and its regulator, p35, have been identified in Leydig cells, it is informative to further explore the novel function of Cdk5/p35 in male reproduction. Here we show that Cdk5/p35 protein expression and kinase activity in mouse Leydig cells are regulated by human chorionic gonadotrophin (hCG) in both dose- and time-dependent manners. Blocking of Cdk5 by molecular inhibitors or small interfering RNA resulted in reduction of testosterone production by Leydig cells. cAMP, a second messenger in LH signaling, was identified as a factor in hCG-dependent regulation of Cdk5/p35. Importantly, Cdk5 protein and kinase activity could support accumulation of steroidogenic acute regulatory (StAR) protein, a crucial component of steroidogenesis. We additionally addressed the protein interaction between Cdk5/p35 and StAR. The Cdk5-dependent serine phosphorylation of StAR indicated a possible mechanism by which Cdk5 induced accumulation of StAR protein. In conclusion, Cdk5 modulates hCG-induced androgen production in mouse Leydig cells, possibly through regulation of StAR protein levels. These results indicate that Cdk5 may play an important role in male reproductive endocrinology and is a potential therapeutic target in androgen-related diseases.     

Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.     

Cyclin-dependent kinase 5 activity regulates pain signaling

Several molecules and cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase implicated in the development and disease of the mammalian nervous system. The precise role of this kinase in sensory pathways has not been well characterized. Here we report a molecular role for Cdk5 in nociception. We identified the expression of Cdk5 and its activator p35 in nociceptive neurons, which is modulated during a peripheral inflammatory response. Increased calpain activity in sensory neurons after inflammation resulted in the cleavage of p35 to p25, which forms a more stable complex with Cdk5 and, consequently, leads to elevation of Cdk5 activity. p35 knockout mice (p35(-/-)), which exhibit significantly decreased Cdk5 activity, showed delayed responses to painful thermal stimulation compared with WT controls. In contrast, mice overexpressing p35, which exhibit elevated levels of Cdk5 activity, were more sensitive to painful thermal stimuli than were controls. In conclusion, our data demonstrate a role for Cdk5/p35 activity in primary afferent nociceptive signaling, suggesting that Cdk5/p35 may be a target for the development of analgesic drugs.     

Cdk5 is essential for adult hippocampal neurogenesis

The molecular factors regulating adult neurogenesis must be understood to harness the therapeutic potential of neuronal stem cells. Although cyclin-dependent kinase 5 (Cdk5) plays a critical role in embryonic corticogenesis, its function in adult neurogenesis is unknown. Here, we assessed the role of Cdk5 in the generation of dentate gyrus (DG) granule cell neurons in adult mice. Cre recombinase-mediated conditional knockout (KO) of Cdk5 from stem cells and their progeny in the DG subgranular zone (SGZ) prevented maturation of new neurons. In addition, selective KO of Cdk5 from mature neurons throughout the hippocampus reduced the number of immature neurons. Furthermore, Cdk5 gene deletion specifically from DG granule neurons via viral-mediated gene transfer also resulted in fewer immature neurons. In each case, the total number of proliferating cells was unaffected, indicating that Cdk5 is necessary for progression of adult-generated neurons to maturity. This role for Cdk5 in neurogenesis was activating-cofactor specific, as p35 KO but not p39 KO mice also had fewer immature neurons. Thus, Cdk5 has an essential role in the survival, but not proliferation, of adult-generated hippocampal neurons through both cell-intrinsic and cell-extrinsic mechanisms.     

Combining chemical genetics and proteomics to identify protein kinase substrates

Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.     

Movement of yeast cortical actin cytoskeleton visualized in vivo.

Fusion proteins between the green fluorescent protein (GFP) and the cytoskeleton proteins Act1p (actin), Sac6p (yeast fimbrin homolog), and Abp1p in budding yeast (Saccharomyces cerevisiae) localize to the cortical actin patches. The actin fusions could not function as the sole actin source in yeast, but fusions between the actin-binding proteins Abp1p and Sac6p complement fully the phenotypes associated with their gene deletions. Direct observation in vivo reveals that the actin cortical patches move. Movement of actin patches is constrained to the asymmetric distribution of the patches in growing cells, and this movement is greatly reduced when metabolic inhibitors such as sodium azide are added. Fusion protein-labeled patches are normally distributed during the yeast cell cycle and during mating. In vivo observation made possible the visualization of actin patches during sporulation as well.     

Striatal dysregulation of Cdk5 alters locomotor responses to cocaine, motor learning, and dendritic morphology

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.     

Early activation of the multicomponent signaling complex associated with focal adhesion kinase induced by pressure overload in the rat heart

Mechanical overload elicits functional and structural adaptive mechanisms in cardiac muscle. Signaling pathways linked to integrin/cytoskeleton complexes may have a function in mediation of the effects of mechanical stimulus in myocardial cells. We investigated the tyrosine phosphorylation and the assembly of the multicomponent signaling complex associated with focal adhesion kinase (Fak) and the actin cytoskeleton in the overloaded myocardium of rats. Pressure overload induced a 3-fold increase in Fak tyrosine phosphorylation within 3 minutes after a 60-mm Hg rise in aortic pressure. A pressure stimulus that lasted for 60 minutes was accompanied by a 5-fold increase in the amount of tyrosine-phosphorylated Fak, and a stimulus as low as 10 mm Hg doubled the amount of tyrosine-phosphorylated Fak in the myocardium within 10 minutes. Pressure overload also induced a time-dependent association of actin with Fak and an increase in the amount of Fak detected in the cytoskeletal fraction of the myocardium. These events were paralleled by c-Src activation and binding to Fak and by an association of Grb2 and p85 subunit of phosphatidylinositol 3-kinase with Fak. Erk1/2 and Akt, two possible downstream effectors of Fak via Grb2 and phosphatidylinositol 3-kinase, were also shown to be activated in parallel with Fak. These findings show that pressure overload induced a rapid activation of the Fak multiple signaling complex in the myocardium of rats, which suggests that this mechanism may have a role in mechanotransduction in the myocardium.     

PKCδ regulates cortical radial migration by stabilizing the Cdk5 activator p35

Cyclin-dependent kinase 5 (Cdk5) and its activator p35 are critical for radial migration and lamination of cortical neurons. However, how this kinase is regulated by extracellular and intracellular signals during cortical morphogenesis remains unclear. Here, we show that PKCδ, a member of novel PKC expressing in cortical neurons, could stabilize p35 by direct phosphorylation. PKCδ attenuated the degradation of p35 but not its mutant derivative, which could not be phosphorylated by PKCδ. Down-regulation of PKCδ by in utero electroporation of specific small interference RNA (siRNA) severely impaired the radial migration of cortical neurons. This migration defect was similar to that caused by down-regulation of p35 and could be prevented by cotransfection with the wild-type but not the mutant p35. Furthermore, PKCδ could be activated by the promigratory factor brain-derived neurotrophic factor (BDNF) and was required for the activation of Cdk5 by BDNF. Both PKCδ and p35 were required for the promigratory effect of BDNF on cultured newborn neurons. Thus, PKCδ may promote cortical radial migration through maintaining the proper level of p35 in newborn neurons.     

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr(774) in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Czeta, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.