Characterization ofp35, a neuronal activator of Cdk5, as a novel microtubule-associated protein.

摘要

Microtubules, a major type of the cytoskeleton, play an important role in a wide variety of cellular functions, such as cell mobility, morphogenesis, and intracellular transport. Microtubule cytoskeleton is dynamic in cells. A number of factors regulate microtubule dynamic instability through a sophisticated mechanism. One major regulator is the microtubule associated proteins (MAPs), which bind to microtubule polymers and promote microtubule polymerization. p35, the primary activator of Cdk5, plays an important role in a variety of activities in the central nervous system, such as neuronal migration, and neurite outgrowth. Suppression of p35 expression by RNAi reduces lamnin-enhanced axonal elongation and p35-/- mice display a defect neocortex through the brain, indicating the critical role of p35 during nervous system development. The role of Cdk5/p35 kinase complex in the central nervous system is due to its various substrates with diverse functions, ranging from neuronal migration to synaptic plasticity and neuronal differentiation. For examples, Tau, a major MAP, regulates microtubule dynamics in neurons; synapsin 1 regulates synaptic transmission and pRb is an important factor in neuronal differentiation and apoptosis.; In this study, we have demonstrated that p35 is a MAP that can directly bind to alpha/beta tubulin and microtubules. The tubulin- and microtubule-binding domains are located at the amino-terminus of p35, a region rich in positive charge. We further demonstrated the activity of p35 to promote microtubule assembly by microtubule polymerization assay. The morphology of microtubules assembled by p35 or its fragments was examined by fluorescence or electron microscopy. Interestingly, the microtubule polymerization assay shows that the charged mutation from Lys to Ala still has the activity to polymerize microtubules, even thought the mutation significantly reduced tubulin-binding activity compared to the wild type, suggesting that the tubulin-binding activity is not required for microtubule assembly or the weak tubulin-binding affinity is enough to polymerize microtubules. This finding will shed new light on the mechanism of microtubule assembly. Moreover, p35 full-length protein has a strong activity to bundle microtubules and to stabilize microtubules against cold-induced depolymerization. The microtubule-stabilizing activity of p35 is thought to be partly dependent on the bundling ability. We also demonstrated that p35 is intermolecular self-association in vitro, which may facilitate microtubule nucleation through its homodimerization/oligomerization, and subsequently, stabilize the microtubules in the form of bundles. We also demonstrated that microtubules disrupt p35 association with Cdk5, indicating that microtubules negatively regulate Cdk5 kinase activity via binding to p35. As p25 lacks the microtubule-binding activity, the Cdk5/p25 complex avoids the regulation by microtubules.; Like other MAPs, phosphorylation of p35 affects its association with and assembly of microtubules. We demonstrated that phosphorylation of p35 by Cdk5 disassociates it from microtubules. Phosphomimetic mutation of Thr138 abolishes the microtubule-assembly ability of p35 as well as impairs neurite outgrowth promoted by p35, whereas phosphomimetic mutation of Ser8 has little change on microtubule assembly and neurite outgrowth compared to the wild type, indicating that the residue Thr138 is a critical phosphorylation site in the regulation of p35-promoted microtubule assembly and neurite outgrowth. In addition, calmodulin binds to a p35 region overlapping with microtubules and blocks p35 association with microtubules.; In conclusion, p35 is a MAP that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation. Two modes of the microtubule associated p35 functions are demonstrated. CaM-binding and Cdk5 catalyzed phosphorylation. Also p35 can promote NGF-induced neurite outgrowth i