Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to ␣/␤-tubulin and microtubules. Microtubule polymers but not the ␣/␤-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to coldinduced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.As a distinct member of the CDK family, Cdk5 is activated by a neuron-specific protein p35 or the p39 homologue of p35 in the central nervous system (1). Both Cdk5 and p35 are required for neurite outgrowth (2). Studies in animal models have revealed their crucial involvements in neuronal migration during nervous system development as mice deficient of Cdk5 or p35 display abnormal brain cortex (3, 4). To date, a wide range of evidence has been accumulated indicating that Cdk5-p35 is a multifunctional kinase that acts in the regulation of various neuronal activities, including organization of the microtubule cytoskeleton (1). In living cells, the dynamic properties of microtubules are modulated through a sophisticated mechanism involving microtubule-associated proteins (MAPs), 2 which bind microtubule polymers and promote microtubule polymerization by stabilizing the polymer structure (5). Cdk5 phosphorylates several MAPs including MAP1b, MAP2, tau, and doublecortin, mediating their association with microtubules and their microtubule-stabilizing functions (1, 6, 7).It is poorly understood how Cdk5 activity is regulated. Although p35 shows little apparent sequence homology to cyclins, it resembles the cyclin A structure with distinct features to bind specifically to Cdk5 (8,9). The binding of p35 highly stimulates Cdk5 activity (10). Several proteins, including C42, protein kinase CK2, and three importin family members (importin-␤, importin-5, and importin-7), show inhibitory effects toward Cdk5 activation via binding to p35 (11-13). Under neurotoxic conditions, p35 is transformed into the N-terminally truncated p25 protein, which causes sustained activation and mislocalization of Cdk5 (14 -16). Moreover, p25 deregulation of Cdk5 has been linked to neuronal cell death and pathogenesis of neurodegenerative diseases such as Alzheimer disease (1). In this report, we have identified direct association of p35 with tubulin and microtubules a...

In the nervous system, Cdk5 and its neuronal activator p35 are involved in the control of various activities, including neuronal differentiation and migration. Recently, we have reported that p35 is a microtubule-associated protein that regulates microtubule dynamics ( 2؉ -specific manner, suggesting that p35 may be involved in the Ca 2؉ /CaMmediated inhibition of microtubule assembly. Second, p35 phosphorylation by Cdk5 interferes with the microtubule-binding and polymerizing activities of p35. Using a mutational approach, we found that only phosphorylation at Thr-138, one of the two residues primarily phosphorylated in vivo, inhibits the polymerizing activity. In PC12 cells, expression of p35 promotes nerve growth factor-induced neurite outgrowth under a Cdk5 inhibitory condition. Such p35 activity is impaired by the phosphomimetic mutation of Thr-138. These data suggest that Thr-138 phosphorylation plays a critical role in the control of the p35 functions in microtubule assembly and neurite outgrowth.

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 ...