Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity of Cdk5-deficient mice and p35-deficient mice. Cdk5-null mutants show a more severe disruption of lamination in the cerebral cortex, hippocampus, and cerebellum. In addition, Cdk5-null mice display perinatal lethality, whereas p35-null mice are viable. These discrepancies have been attributed to the function of other Cdk5 activators, such as p39. To understand the roles of p39 and p35, we created p39-null mice and p35/p39 compound-mutant mice. Interestingly, p39-null mice show no obvious detectable abnormalities, whereas p35(-/-)p39(-/-) double-null mutants are perinatal lethal. We show here that the p35(-/-)p39(-/-) mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow us to determine the distinct and redundant functions between p35 and p39. Our data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5.

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), whose function remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly small autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.

Members of the N-methyl-D-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine͞threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDAevoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs. The N-methyl-D-aspartate (NMDA) class of glutamate receptors (NMDAR) are essential for learning, memory, and development in the central nervous system (1-5). NMDARs are multimeric complexes formed from both NMDA receptor subunit (NR1) and modulatory NR2 subunits (6-9). A single gene encodes the NR1 subunit. Eight possible alternative RNA-splice variants provide molecular diversity of NMDARs (10). NR2A-NR2D are encoded by four separate genes (11,12). NMDARs consist of NR1͞NR2 heteromeric complexes (13-15). The activation of NMDARs and the influx of Ca NMDA-channel activity is dynamically modulated in both intracellular and extracellular sites (3). Phosphorylation sites have been identified on the NR1, NR2A, and NR2B subunits, but not on the NR2C-NR2D subunits. Protein kinase C phosphorylates Ser-890 and Ser-896, and protein kinase A phosphorylates Ser-897 within the C1 exon of the NR1 subunit (17-19). Calcium͞calmodulin protein kinase II mediates phosphorylation of NR2B, but not NR2A (20). More recently, phosphorylation at tyrosine residues of NR2A and NR2B has been described as an important determinant for NMDAR functions (19)(20). In particular, Fyn, a member of the Src family of nonreceptor protein tyrosine kinases, was shown to phosphorylate the NR2A (7, 21-23). However, no information is available concerning which kinases phosphorylate NR2A at serine͞threonine sites. Cyclindependent kinase-5 (Cdk5) is a serine͞threonine kinase that is activated by neuron-specific p35 and p39 proteins (24-27). It exists as a large, multimeric complex associated with cytoskeletal proteins in the neurons. Cdk5 has been shown to phosphorylate a wide variety of proteins, all of which have serine͞threonine sites in (K͞RT͞SPXK)-type motifs (28,29). A number of synaptic proteins have been identified as Cdk5 substrates (30-32). Cdk5 and p35, predominantly expressed in postmitotic neurons, play essential roles in neuronal migration, neurite outgrowth, and laminar configuration of the cerebral cortex (25,27,33). Cdk5 in association with p25, a truncated form of p35, hyperphosphorylates the microtubule-associated protein tau. This hyperphosphorylation is thought to disrupt the neuronal cytoskeleton and ultimately contributes to neurodegeneration in Alzheimer's disease (34).Because of its virtue of phosphoryl...