Cyclin-dependent kinase 5 (Cdk5) is required for proper development of the mammalian central nervous system. To be activated, Cdk5 has to associate with its regulatory subunit, p35. We have found that p25, a truncated form of p35, accumulates in neurons in the brains of patients with Alzheimer's disease. This accumulation correlates with an increase in Cdk5 kinase activity. Unlike p35, p25 is not readily degraded, and binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location and alters its substrate specificity. In vivo the p25/Cdk5 complex hyperphosphorylates tau, which reduces tau's ability to associate with microtubules. Moreover, expression of the p25/Cdk5 complex in cultured primary neurons induces cytoskeletal disruption, morphological degeneration and apoptosis. These findings indicate that cleavage of p35, followed by accumulation of p25, may be involved in the pathogenesis of cytoskeletal abnormalities and neuronal death in neurodegenerative diseases.

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.

Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2 gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. We have investigated the regulation of the p35/Cdk5 kinase. Here we show that p35, the activator of Cdk5, is a short-lived protein with a half-life (t 1/2 ) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2-to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2-to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays a autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis.

The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.