Mitotic phosphorylation of the dynein light intermediate chain is mediatedby cdc2 kinase

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membr ane organelIes, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be contr olled, at least in part, by the phosphorylation of its light intermediate c hains (DLIC), since a 10-fold increase in light intermediate chain phosphor ylation correlates with a decrease in dynein-based membrane transport of si milar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will u ndergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wi ld-type DLIC is stoichiometrically phosphorylated at this site when incubat ed with metaphase but not interphase extracts. We also show that purified c dc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylate s dynein and thus may be an important regulator of dynein activity in the c ell cycle.