MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK

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Abstract

Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.

Section snippets

Experimental procedures

Plasmids. Plasmids were prepared using Endo Free plasmid purification kits (Qiagen). Oligonucleotides containing multicloning sites were inserted into pcCAG [9] to generate pCAG-MCS2. MAP1B [8], Cdk5 [14], p35 or p25 [15] cDNA was inserted into the pCAG-MCS2 vector. JNK expression vector was a generous gift from Dr. S. Tamura [16].

Antibodies and chemical regents. Primary antibodies used in this study were SMI31 (Sternberger Monoclonals), anti-MAP1B (Santa Cruz N-19), anti-phospho-Ser732 FAK

Results and discussion

E15 cerebral cortices were dissociated and after 1 day in culture, inhibitors or solvent was added for another 24 h. Cells were harvested and subjected to immunoblot analysis with SMI31 antibody which recognizes mode I phosphorylated MAP1B [19]. As previously reported [9], [20], treatment with a JNK inhibitor, SP600125, resulted in decreased mode I phosphorylation of MAP1B (Fig. 1A, SP25 and SP50). In contrast, treatment with a Cdk5 inhibitor, roscovitine, had no effect (Fig. 1A, R100), although

Acknowledgments

We thank A. Takashima, S. Tamura, L.H. Tsai, and Y. Uchida for providing plasmids. We also thank R. Yu and M. Sone for helpful comments and technical help. This work was supported by the center of excellence (COE) grant and Grant-in-Aid for Scientific Research on Priority Areas, “Elucidation of glia–neuron network mediated information processing systems (#16047220),” and “Membrane Traffic (#16044225)” from the Ministry of Education, Culture, Sports and Science and Technology, Japan, and by

References (29)

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