CYCLIN-B INTERACTION WITH MICROTUBULE-ASSOCIATED PROTEIN-4 (MAP4) TARGETS P34(CDC2) KINASE TO MICROTUBULES AND IS A POTENTIAL REGULATOR OF M-PHASE MICROTUBULE DYNAMICS

We previously demonstrated (Ookata et al., 1992, 1993) that the p34(cd c2)/cyclin B complex associates with microtubules in the mitotic spind le and premeiotic aster in starfish oocytes, and that microtubule-asso ciated proteins (MAPs) might be responsible for this interaction. In t his study, we have investigated the mechanism by which p34(cdc2) kinas e associates with the microtubule cytoskeleton in primate tissue cultu re cells whose major MAP is known to be MAP4. Double staining of prima te cells with anti-cyclin B and anti-MAP4 antibodies demonstrated thes e two antigens were colocalized on microtubules and copartitioned foll owing two treatments that altered MAP4 distribution. Detergent extract ion before fixation removed cyclin B as well as MAP4 from the microtub ules. Depolymerization of some of the cellular microtubules with nocod azole preferentially retained the microtubule localization of both cyc lin B and MAP4. The association of p34(cdc2)/cyclin B kinase with micr otubules was also shown biochemically to be mediated by MAP4. Cosedime ntation of purified p34(cdc2)/cyclin B with purified microtubule prote ins containing MAP4, but not with MAP-free microtubules, as well as bi nding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interac tion between cyclin B and MAP4. Using recombinant MAP4 fragments, we d emonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34(cdc2)/cyclin B phy sically associated with MAP4, we examined the ability of the kinase co mplex to phosphorylate MAP4. Incubation of a ternary complex of p34(cd c2), cyclin B, and the COOH-terminal domain of MAP4, PA(4), with ATP r esulted in intracomplex phosphorylation of PA(4). Finally, we tested t he effects of MAP4 phosphorylation on microtubule dynamics. Phosphoryl ation of MAP4 by p34(cdc2) kinase did not prevent its binding to micro tubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targe ting the mitotic kinase to appropriate cytoskeletal substrates, for th e regulation of spindle assembly and dynamics.