Microtubule Association of the Neuronal p35 Activator of Cdk5

Abstract

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...

Suppression of Mutant Huntingtin Aggregate Formation by Cdk5/p35 through the Effect on Microtubule Stability

“…MTs in COS-7 cells disassembled and tubulin staining became diffuse in the cytoplasm when Cdk5/p35 was coexpressed with tNhtt-poly(Q)-EGFP, as occurred with nocodazole treatment. Although it was recently reported that p35 bound and stabilized MTs in vitro (Hou et al, 2007), MT stabilization was not observed in our cultured cells. Our results are consistent with previous reports demonstrating the requirement of MTs in cytoplasmic aggregate formation (Johnston et al, 1998;Iwata et al, 2005).…”

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

“…The p10 interaction in vivo with other cellular proteins, such as tubulin (microtubules) and calmodulin (31,74), may protect the activity of the Cdk5-p35 complex (and not the Cdk5-p25 complex) from p5 inhibition. In our experiments (Fig.…”

“…Effect of Tubulin Polymerization on Cdk5 Activity-Tubulin polymerization was performed as detailed in a previous report (31). Briefly, active kinase Cdk5-p35 (2 ng/l) or active Cdk5-p25 (1 ng/l) was incubated with ␤-tubulin (enzyme/tubulin at 1:100) in PEM buffer (80 mM PIPES, 2 mM MgCl 2 , 1 mM EGTA, pH 7.0) supplemented with 1 mM GTP at 35°C for 45 min.…”

“…A fundamental structural difference between the two activators offers a possible explanation; p25, a truncated version of p35, lacks the myristoylated p10 N-terminal domain of the intact p35. The p10 region is responsible for interaction of p35 with several cellular proteins, such as importins, Munc18, calmodulin, microtubules, and protein kinase CK2 (31,(53)(54)(55). Microtubules, for example, bind p35 due to its p10 domain, whereas they do not interact with the p25 due to the loss of the p10 region of the p35 truncated fragment (31).…”

Caspase-mediated cleavage of C53/LZAP protein causes abnormal microtubule bundling and rupture of the nuclear envelope

“…In vitro MT assembly assay clearly demonstrated that C53/LZAP was associated with the assembled MT but did not bind MT directly (Figure 6 proteins. Such mediators may include p35 [26] and other potential C53/LZAP-interacting protein such as Disrupted-in-Schizophrenia 1 (DISC1) protein, which is also known to bind the microtubule [27,28]. Meanwhile, a relatively large fraction of cytoplasmic C53/LZAP is associated with light membranes, such as the ER (Figure 6B and [12]), probably through its interaction with the ER protein RCAD (also known as Ufl1, NLBP and MAXER) [12,[15][16][17].…”