Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase.
Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar 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 undergo 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 wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 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 phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.
Dell
K R
KR
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.
Turck
C W
CW
Vale
R D
RD
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001692:Biological Transport; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000378:metabolism*; D010766:Phosphorylation; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D020409:Molecular Motor Proteins; D016203:CDC2 Protein Kinase; D004398:Dyneins