Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

Mammalian Cdk5 is a member of the cyclin-dependent kinase family that is activated by a neuron-specific regulator, p35, to regulate neuronal migration and neurite outgrowth. p35/Cdk5 kinase colocalizes with and regulates the activity of the Pak1 kinase in neuronal growth cones and likely impacts on actin cytoskeletal dynamics through Pak1. Here, we describe a functional homologue of Cdk5 in budding yeast, Pho85. Like Cdk5, Pho85 has been implicated in actin cytoskeleton regulation through phosphorylation of an actin-regulatory protein. Overexpression of CDK5 in yeast cells complemented most phenotypes associated with pho85Δ, including defects in the repression of acid phosphatase expression, sensitivity to salt, and a G1 progression defect. Consistent with the functional complementation, Cdk5 associated with and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast cells. In a reciprocal series of experiments, we found that Pho85 associated with the Cdk5 activators p35 and p25 to form an active kinase complex in mammalian and insect cells, supporting our hypothesis that Pho85 and Cdk5 are functionally related. Our results suggest the existence of a functionally conserved pathway involving Cdks and actin-regulatory proteins that promotes reorganization of the actin cytoskeleton in response to regulatory signals.

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.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

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.

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.