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Microtubule Association of the Neuronal p35 Activator of Cdk5

2007; Elsevier BV; Volume: 282; Issue: 26 Linguagem: Inglês

10.1074/jbc.c700052200

1083-351X

Zhibo Hou, Qing Li, Lisheng He, Hui‐Ying Lim, Xinrong Fu, Nam Sang Cheung, Donna X. Qi, Robert Z. Qi,

Ubiquitin and proteasome pathways

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 α/β-tubulin and microtubules. Microtubule polymers but not the α/β-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 cold-induced 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. 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 α/β-tubulin and microtubules. Microtubule polymers but not the α/β-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 cold-induced 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 (1Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759Crossref PubMed Scopus (956) Google Scholar). Both Cdk5 and p35 are required for neurite outgrowth (2Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (536) Google Scholar). 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 (3Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 4Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (815) Google Scholar). 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 (1Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759Crossref PubMed Scopus (956) Google Scholar). In living cells, the dynamic properties of microtubules are modulated through a sophisticated mechanism involving microtubule-associated proteins (MAPs), 2The abbreviations used are: MAP, microtubule-associated protein; GST, glutathione S-transferase; DTT, dithiothreitol; K-PIPES, potassium 1,4-piperazinediethanesulfonic acid. which bind microtubule polymers and promote microtubule polymerization by stabilizing the polymer structure (5Mandelkow E. Mandelkow E.M. Curr. Opin. Cell Biol. 1995; 7: 72-81Crossref PubMed Scopus (384) Google Scholar). Cdk5 phosphorylates several MAPs including MAP1b, MAP2, tau, and doublecortin, mediating their association with microtubules and their microtubule-stabilizing functions (1Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759Crossref PubMed Scopus (956) Google Scholar, 6Lim A.C. Qu D. Qi R.Z. Neurosignals. 2003; 12: 230-238Crossref PubMed Scopus (29) Google Scholar, 7Tanaka T. Serneo F.F. Tseng H.C. Kulkarni A.B. Tsai L.H. Gleeson J.G. Neuron. 2004; 41: 215-227Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). 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 (8Tang D. Chun A.C. Zhang M. Wang J.H. J. Biol. Chem. 1997; 272: 12318-12327Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Tarricone C. Dhavan R. Peng J. Areces L.B. Tsai L.H. Musacchio A. Mol. Cell. 2001; 8: 657-669Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The binding of p35 highly stimulates Cdk5 activity (10Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Several proteins, including C42, protein kinase CK2, and three importin family members (importin-β, importin-5, and importin-7), show inhibitory effects toward Cdk5 activation via binding to p35 (11Lim A.C. Hou Z. Goh C.P. Qi R.Z. J. Biol. Chem. 2004; 279: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 12Ching Y.P. Pang A.S. Lam W.H. Qi R.Z. Wang J.H. J. Biol. Chem. 2002; 277: 15237-15240Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Fu X. Choi Y.K. Qu D. Yu Y. Cheung N.S. Qi R.Z. J. Biol. Chem. 2006; 281: 39014-39021Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Under neurotoxic conditions, p35 is transformed into the N-terminally truncated p25 protein, which causes sustained activation and mislocalization of Cdk5 (14Kusakawa G. Saito T. Onuki R. Ishiguro K. Kishimoto T. Hisanaga S. J. Biol. Chem. 2000; 275: 17166-17172Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 15Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (922) Google Scholar, 16Patrick G.N. Zukerberg L. Nikolic M. de la M.S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1338) Google Scholar). Moreover, p25 deregulation of Cdk5 has been linked to neuronal cell death and pathogenesis of neurodegenerative diseases such as Alzheimer disease (1Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759Crossref PubMed Scopus (956) Google Scholar). In this report, we have identified direct association of p35 with tubulin and microtubules and have shown the function of p35 as a MAP as well as the regulation of Cdk5 activation by microtubules. Antibodies—The following antibodies were purchased: anti-α-tubulin from Abcam; anti-β-tubulin (TUB2.1) from Sigma; anti-tau (H-150), anti-p35 (C-19), and anti-Cdk5 (C-8 and J-3) from Santa Cruz Biotechnology; and anti-GST from GE Healthcare. Recombinant Protein Production—Recombinant proteins of Cdk5, p35, and p35 fragments were expressed in Escherichia coli BL21(DE3) and were purified (11Lim A.C. Hou Z. Goh C.P. Qi R.Z. J. Biol. Chem. 2004; 279: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The expression and purification of the largest human tau isoform hT40 was performed as reported (18Qi Z. Zhu X. Goedert M. Fujita D.J. Wang J.H. FEBS Lett. 1998; 423: 227-230Crossref PubMed Scopus (15) Google Scholar). Recombinant proteins used in microtubule sedimentation and polymerization assays were dialyzed in PEM buffer (80 mm K-PIPES, pH 6.9, 1 mm MgCl2, and 1 mm EGTA) supplemented with 1 mm EDTA, 1 mm DTT, and 50 mm NaCl. Microtubule Isolation from Rat Brain—Brain microtubules were isolated by temperature-dependent assembly/disassembly experiments (19Vallee R.B. Methods Enzymol. 1986; 134: 89-104Crossref PubMed Scopus (146) Google Scholar). Rat brain was homogenized on ice in 2-folds (v/w) of homogenization buffer (PEM buffer plus 100 mm NaCl, 0.1% Triton X-100, 1 mm DTT, 10 mm NaF, 0.1 mm Na3VO4, 10 mm β-glycerophosphate, and the protease inhibitor mixture (Roche Applied Science)). The homogenate was cleared by centrifugation at 4 °C first at 20,000 × g for 45 min and then at 100,000 × g for 45 min. Microtubule assembly was initiated in the extract by the addition of 30% (v/v) glycerol and 1 mm GTP and was conducted at 35 °C for 45 min. After microtubules were spun down at 35 °C (100,000 × g; 45 min), the supernatant was removed. The pelleted microtubules were resuspended by homogenization in the ice-cold homogenization buffer and were allowed to disassemble on ice for 45 min. The suspension was then centrifuged at 4 °C (100,000 × g;45 min) to pellet undisrupted microtubules, which were designated as cold-stable microtubules. The resulting supernatant, which was derived from cold-labile microtubules, was used for the following cycles of microtubule assembly/disassembly. Isolation of MAP-free Tubulin—Tubulin was purified from porcine brain by two cycles of microtubule assembly/disassembly followed by phosphocellulose chromatography (19Vallee R.B. Methods Enzymol. 1986; 134: 89-104Crossref PubMed Scopus (146) Google Scholar). The temperature-dependent assembly and disassembly were performed as described above except that PEM buffer was used instead of the homogenization buffer, and 1 mm each of ATP and GTP was applied for microtubule polymerization in the first cycle. After two assembly/disassembly cycles, the pre-cleared tubulin sample was applied at less than 3 mg protein/ml resin to a phosphocellulose (Whatman) column, which was pre-equilibrated with the buffer of 50 mm K-PIPES, pH 6.9, 1 mm EGTA, and 0.2 mm MgCl2. After column washing by the buffer, peak tubulin fractions were pooled, aliquoted into a small volume, and quickly frozen by liquid nitrogen for storage at –80 °C. Coomassie Blue staining of the purified tubulin proteins separated by SDS-PAGE did not detect any contaminating protein, and anti-tau Western blotting did not detect tau in the samples. Microtubule Sedimentation—Microtubule cosedimentation was performed as described previously (20Lim A.C. Tiu S.Y. Li Q. Qi R.Z. J. Biol. Chem. 2004; 279: 4433-4439Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Proteins were centrifuged at 4 °C (100,000 × g; 30 min) to remove aggregates before experiments. Immunofluorescence Microscopy—Five-day cultures of mouse cortical neurons were immunostained as described previously (13Fu X. Choi Y.K. Qu D. Yu Y. Cheung N.S. Qi R.Z. J. Biol. Chem. 2006; 281: 39014-39021Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and were analyzed on a Nikon microscope (Eclipse TE2000). Extraction of Microtubule Proteins from Cell Cultures—Microtubule proteins and the remaining cytoplasm were differentially extracted from 5-day cultures of cortical neurons as described previously (17Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Both extracted fractions were clarified by centrifugation before subjected to Western blotting. Cdk5 Binding Assay and Immunoprecipitation—GST-Cdk5 (2 μg), prereconstituted with p35-His6 or p25-His6 (2 μg), was incubated with α/β-tubulin or taxol-stabilized microtubules at 25°C for 1 h in 800 μl of PEM buffer supplemented with 100 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.1% Nonidet P-40, and protease inhibitors. GST-Cdk5 was retrieved using GSH beads to detect bound p35/p25 by Western blotting. To perform Cdk5 immunoprecipitation, rat brain extract prepared in the homogenization buffer was added with 2 mm GTP and 10 μm taxol or nocodazole and was then incubated at 35 °C for 1 h. Following the incubation, anti-Cdk5 (J-3) immunoprecipitation was carried out to analyze co-precipitation of p35. Cdk5 Kinase Assay—Cdk5 kinase activity was determined by phosphorylation of a histone H1 peptide PKTPKKAKKL as detailed in a previous report (10Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Microtubule Assembly—Microtubule assembly was examined by the following methods. The light scattering assay was performed with 2 mg/ml α/β-tubulin at 35 °C in PEM buffer supplemented with 1 mm GTP and 10 mm MgCl2 (20Lim A.C. Tiu S.Y. Li Q. Qi R.Z. J. Biol. Chem. 2004; 279: 4433-4439Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 21Gaskin F. Methods Enzymol. 1982; 85: 433-439Crossref PubMed Scopus (26) Google Scholar). To examine polymerized microtubules by electron microscopy, samples were deposited onto electron microscopic grids (carbon-coated copper grids) and were negatively stained in 2% uranyl acetate (21Gaskin F. Methods Enzymol. 1982; 85: 433-439Crossref PubMed Scopus (26) Google Scholar). The specimens were examined on a Philipps CM-20 electron microscope. Quantitative Western Blotting—Images of anti-Cdk5 (C-8) and anti-p35/p25 (C-19) Western blots were acquired on the ChemiDoc XRS system (Bio-Rad) and were analyzed using the Quantity One software (Bio-Rad). Known amounts of recombinant Cdk5 and p25 proteins were used as standards on all blots. The quantities of Cdk5 and p35/p25 in the samples were calculated from within the linear range of standard curves from each Western blot done in triplicate. p35 Binds to Tubulin and Microtubules—In brain, Cdk5 and p35 appear in various molecular complexes (1Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759Crossref PubMed Scopus (956) Google Scholar, 6Lim A.C. Qu D. Qi R.Z. Neurosignals. 2003; 12: 230-238Crossref PubMed Scopus (29) Google Scholar). We sought for p35-interacting proteins by biochemical affinity isolation from rat brain using immobilized p35 fragments (11Lim A.C. Hou Z. Goh C.P. Qi R.Z. J. Biol. Chem. 2004; 279: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). After separation of the isolated proteins by electrophoresis, the most prominent band that appeared specifically in the pull-down of p10, but not in that of p25, was revealed by mass spectrometry to be α- and β-tubulins. We then examined pull-downs from brain extract by Western blotting. Both p10 and p35 were bound to α- and β-tubulins in the extract, whereas p25 did not exhibit any detectable tubulin-binding activity (Fig. 1A). The identical results were obtained when the binding was tested using purified MAP-free α/β-tubulin (Fig. 1B). Thus, the N-terminal region of p35 interacts directly with the α/β-tubulin heterodimer. To examine microtubule association, p35 and its fragments were incubated with microtubules polymerized from MAP-free tubulin and stabilized with taxol. After centrifugation to sediment microtubules, almost all p35 and p10 were found in the microtubule fraction, whereas p25 and GST were exclusively detected in the supernatant (Fig. 1C). In the control assays in which tubulin was omitted, all of the recombinant proteins were not pelleted by the centrifugation (Fig. 1C). We conclude that the N-terminal region of p35 can bind directly to microtubules. p35 Localizes to Microtubules in Brain—When p35 was examined in cultured cortical neurons, immunofluorescence revealed the co-localization of p35 and the microtubule structure in neurites including the growth cones (Fig. 1D). These data were in agreement with the observations in a previous report (2Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (536) Google Scholar). To assess p35 distribution, cultured neurons were fractionated to sequentially extract cytoplasmic and microtubule proteins. Approximately one-third of p35 associated with microtubule polymers (Fig. 1E). Under the extraction conditions, p25 was undetectable in the extracts (data not shown). Cdk5 was only detected in the cytoplasmic fraction (Fig. 1E), implying that microtubule-associated p35 is free of Cdk5 binding. To further analyze the microtubule association, we isolated microtubules from adult rat brain through three cycles of temperature-induced assembly and disassembly. When microtubules were assembled in the brain extract during the first cycle, the majority of p35 associated with the polymers, including the cold-stable polymers (Fig. 1F). Most of Cdk5 remained in the supernatant, where Cdk5 was in abundance when compared with p35 and p25 (Fig. 1, F and G). p25 was detected in the cold-labile but not cold-stable fraction of microtubules (Fig. 1F). In the following cycles, p35 continued to assemble with both cold-labile and cold-stable microtubules; Cdk5 and p25 existed with cold-labile microtubules (Fig. 1F). When probed as a control, tau was found to be present almost exclusively with cold-labile microtubules of each cycle (Fig. 1F). After three cycles of assembly/disassembly, microtubules were essentially free of contaminants. In the isolated cold-labile microtubules, the amount of p35 and p25 combined was more than Cdk5 (Fig. 1G). In the cold-stable microtubules, p25 was undetectable, and p35 was greatly in excess of Cdk5 (p35:Cdk5 was 6:1; Fig. 1, F and G). Thus, p35 associated with the microtubules was free of Cdk5 binding. Taken together, the microtubule co-assembly demonstrates prominent association of p35 with microtubules including cold-stable microtubules. In a previous report, the Cdk5-p25 kinase is shown to associate indirectly with microtubules via binding to tau (22Sobue K. garwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We reason that the sedimentation of Cdk5 and p25 with cold-labile microtubules was attributed to their association with tau and possibly some other MAPs in the brain extract. Microtubules Disrupt the Association of p35 with Cdk5—We investigated how Cdk5, p35, and microtubules interact with one another. Cdk5 does not bind directly to microtubules (22Sobue K. garwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In a binding assay, the preformed complex of Cdk5 and p35 was incubated with taxol-stabilized microtubules or with the α/β-tubulin heterodimer. Interestingly, the microtubules disrupted the interaction between p35 and Cdk5 in a dose-dependent manner, whereas the tubulin dimer did not exhibit any effect (Fig. 2A). In addition, the microtubules did not affect p25 binding to Cdk5 (Fig. 2A). In accordance with these findings, the microtubule polymers but not the tubulin dimer inhibited the activity of Cdk5-p35, and the polymers did not inhibit the activity of Cdk5-p25 (Fig. 2B). Next, we conducted co-immunoprecipitation of Cdk5 and p35 from a rat brain extract that was pretreated with taxol to induce microtubule assembly or with nocodazole to inhibit the assembly. In the presence of taxol, p35 failed to co-precipitate with Cdk5 (Fig. 2C). In contrast, the co-immunoprecipitation was readily detected under the inhibitory condition of microtubule assembly (Fig. 2C). Therefore, microtubules sequester p35 but not p25 from Cdk5 and thus inhibit p35 activation of Cdk5. p35 Induces Microtubule Assembly and Bundling—The microtubule and tubulin association prompted us to investigate whether p35 alters microtubule assembly characteristics. In the absence of p35, there was a minimal polymerization of tubulin even after a prolonged incubation because tubulin was below the concentration required for spontaneous polymerization (Fig. 3A). The addition of 3 μg/ml p35 (p35:tubulin at ∼1:400) resulted in significant polymerization of tubulin (Fig. 3A). Both the rate and the extent of polymerization were significantly enhanced in a manner dependent on the input of p35. With 12 μg/ml p35 (p35:tubulin at 1:100), most of the tubulin was polymerized into microtubule polymers. Thus, p35 exhibited a strong activity in inducing microtubule assembly. We also tested the p35 fragments p10 and p25 in the assay. In contrast to p35, neither p10 nor p25 induced microtubule assembly when applied at the same amount as p35 or even at much higher concentrations (data not shown). Given that p10 retains the microtubule and tubulin binding activities (Fig. 1), the microtubule-polymerizing function either involves additional domains from the p25 region or requires it for protein folding. Conceivably, the neurotoxin-induced cleavage of p35 abrogates its microtubule-polymerizing property. To observe polymerized microtubules, samples from the assembly assay were negatively stained for electron microscopy. In agreement with the turbidimetric assay results, almost no microtubules were found in the assay sample without p35 (Fig. 3B, panel a). Microtubules were readily seen in those polymerized by using taxol or p35 (Fig. 3B, panels b and c). Interestingly, most of the p35-assembled microtubules existed in the form of bundles (Fig. 3B, panel c). As seen in the micrograph, several microtubules were closely attached to each other to form a bundle (Fig. 3B, panel d). In the control, taxol-polymerized microtubules did not form bundles (Fig. 3B, panel b), consistent with the observation in a previous report (23Turner P.F. Margolis R.L. J. Cell Biol. 1984; 99: 940-946Crossref PubMed Scopus (55) Google Scholar). It appeared that p35 cross-bridges microtubules in addition to the promotion of microtubule assembly. Given the co-isolation of p35 and cold-stable microtubules from rat brain, we tested whether p35 can stabilize microtubules at low temperature. In the assay, microtubules polymerized in vitro were placed on ice, and solution turbidity was monitored. Most of tau-stabilized microtubules depolymerized within a few minutes (Fig. 3C) as tau does not confer the cold stability (20Lim A.C. Tiu S.Y. Li Q. Qi R.Z. J. Biol. Chem. 2004; 279: 4433-4439Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 24Baas P.W. Pienkowski T.P. Cimbalnik K.A. Toyama K. Bakalis S. Ahmad F.J. Kosik K.S. J. Cell Sci. 1994; 107: 135-143PubMed Google Scholar). The turbidity of the p35-polymerized sample barely changed even after 30 min of incubation (Fig. 3C). Thus, microtubules stabilized by p35 are resistant to cold-induced disassembly. This report describes for the first time the role of p35 as a MAP and the inactivation of Cdk5-p35 by microtubules. The p10 region of p35, which contains microtubule- and tubulin-binding domains, is rich in basic residues, reminiscent of the microtubule-binding sequences of conventional MAPs. However, scanning of the p10 sequence did not yield any recognizable microtubule-binding motif, implicating that p35 may contain novel microtubule- and tubulin-binding domains. The binding of p35 to microtubules does not confer Cdk5 attachment to microtubules. Instead, microtubules segregate p35 from Cdk5, acting as an inhibitor of Cdk5. Given that a significant proportion of p35 localizes to microtubules, the microtubule cytoskeleton may play an important role in the control of Cdk5 activity. Similar to microtubules, importin-β, importin-5, and importin-7 have been shown to block p35 association with Cdk5 via binding to the N-terminal region of p35 (13Fu X. Choi Y.K. Qu D. Yu Y. Cheung N.S. Qi R.Z. J. Biol. Chem. 2006; 281: 39014-39021Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Therefore, this p35 region confers Cdk5 inhibition via interaction with certain protein factors or subcellular structures, which sequester p35 from Cdk5. The truncation of p35 to p25, which loses the N-terminal domain, is a way to relieve the inhibition. Indeed, the production of p25 causes aberrant activation of Cdk5 (16Patrick G.N. Zukerberg L. Nikolic M. de la M.S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1338) Google Scholar). p35 has strong microtubule-polymerizing activity. The current model of microtubule polymerization involves the formation of microtubule nuclei (i.e. nucleation) from several tubulin dimers (25Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (2006) Google Scholar). Our preliminary results showed the intermolecular self-association of p35. 3L. He and R. Z. Qi, unpublished data. Therefore, p35 may facilitate microtubule nucleation through its homodimerization/oligomerization, and subsequently, stabilize the microtubules in the form of bundles. In addition, our results suggest that p35 may be one of the microtubule cold stabilizers and that the stabilization is at least partially due to the effect of bundling. In many cell types, there is a family of MAPs called stable tubule-only polypeptides, which render microtubules cold-stable (26Bosc C. Andrieux A. Job D. Biochemistry. 2003; 42: 12125-12132Crossref PubMed Scopus (68) Google Scholar). Although studies using transgenic mice have revealed a role of stable tubule-only polypeptides in synaptic plasticity, the precise function of microtubule cold stability is still unclear (27Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Faure S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (146) Google Scholar). The function of p35 as a MAP is highly relevant to its role in neuronal migration or morphogenesis. The localization of p35 to microtubules is readily found in axons and dendrites including the growth cone at the leading edge of an extending neurite. As the leading edge extends, microtubules undergo active polymerization to grow into the protrusion. Also, microtubules play a critical role in the movement of the nucleus into the leading process of migrating cells. The results presented here suggest that p35 directly participates in microtubule assembly and stabilization during these processes. It has been shown that the Cdk5-p35 kinase modulates the microtubule architecture through its action toward several molecular targets such as doublecortin and focal adhesion kinase (7Tanaka T. Serneo F.F. Tseng H.C. Kulkarni A.B. Tsai L.H. Gleeson J.G. Neuron. 2004; 41: 215-227Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 28Xie Z. Sanada K. Samuels B.A. Shih H. Tsai L.H. Cell. 2003; 114: 469-482Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Therefore, p35 may play a multifunctional role in the regulation of microtubule dynamics. We thank Dr. Jerry H. Wang for helpful discussions.