The complex of Cdk5 and its neuronal activator p35 is a proline-directed Ser/Thr kinase that plays an important role in various neuronal functions. Deregulation of the Cdk5 enzymatic activity was found to associate with a number of neurodegenerative diseases. To search for regulatory factors of Cdk5-p35 in the brain, we developed biochemical affinity isolation using a recombinant protein comprising the N-terminal 149 amino acids of p35. The catalytic ␣-subunit of protein kinase CK2 (formerly known as casein kinase 2) was identified by mass spectrometry from the isolation. The association of CK2 with p35 and Cdk5 was demonstrated, and the CK2-binding sites were delineated in p35. Furthermore, CK2 displayed strong inhibition toward the Cdk5 activation by p35. The Cdk5 inhibition is dissociated from the kinase function of CK2 because the kinase-dead mutant of CK2 displayed the similar Cdk5 inhibitory activity as the wild-type enzyme. Further characterization showed that CK2 blocks the complex formation of Cdk5 and p35. Together, these findings suggest that CK2 acts as an inhibitor of Cdk5 in the brain.Cdk5 was identified independently on the basis of its sequence similarity to the family of Cdks, its interaction with cyclin D, and its protein kinase activity toward a prolinedirected Ser/Thr sequence (1-3). Despite having 60% sequence identity with Cdk1 and Cdk2, a role for Cdk5 in cell cycle regulation has yet to be identified. Nevertheless, Cdk5 has been implicated in the regulation of neuronal differentiation, degeneration, and cytoskeletal dynamics (4). Monomeric Cdk5 shows no enzymatic activity because its activation is dependent on its association with the regulatory subunit p35 or p39, neither of which is a cyclin protein (5-7). Mammalian p35 and p39 are primarily expressed in post-mitotic neurons, thereby restricting Cdk5 activity predominantly to the nervous system (8). Cdk5 and p35 are essential for proper brain development. Mice deficient of Cdk5 or p35 display cell-positioning defects in the cerebral cortex (9, 10). In the brain, Cdk5-p35 phosphorylates a number of cytoskeletal proteins that are thought to play important roles in the reassembly of cytoskeletal elements, thereby mediating neurite outgrowth and neuronal migration during the development (11-16). In addition, there are several lines of evidence implicating aberrant regulation of Cdk5 in neurodegeneration and cell death (17). Indeed, p25, a truncated C-terminal fragment of p35, was found to accumulate in Alzheimer's disease brains; its associated-Cdk5 kinase activity was shown to lead to cytoskeletal disruption, morphological degeneration, and apoptosis (18 -21).Cdk5 is involved in the regulation of many vital functions in the brain and, hence, its activity needs to be tightly controlled in the cells. Upon association with its activator p35 or p39, Cdk5 is regulated through various means, and many of its regulatory properties are distinct from those of the authentic Cdk-cyclin. First, p35 is an unstable protein with a half-life of 20 -30 min ...