Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes.
Movement of various cargoes toward microtubule minus ends is driven by the microtubule motor cytoplasmic dynein (CD). Many cargoes are motile only during certain cell cycle phases, suggesting that CD function may be under cell cycle control. Phosphorylation of the CD light intermediate chain (DLIC) has been suggested to play a crucial role in modulating CD function during the Xenopus embryonic cell cycle, where CD-driven organelle movement is active in interphase but greatly reduced in metaphase. This down-regulation correlates with hyperphosphorylation of DLIC and release of CD from the membrane. Here we investigate the role of the key mitotic kinase, cdc2-cyclinB1, in this process. We show that DLIC within the native Xenopus CD complex is an excellent substrate for purified Xenopus cdc2-glutathione S-transferase (GST) cyclinB1 (cdc2-GSTcyclinB1) kinase. Mass spectrometry of native DLIC revealed that a conserved cdc2 site (Ser-197) previously implicated in the metaphase modulation of CD remains phosphorylated in interphase and so is unlikely to be the key regulatory site. We also demonstrate that incubating interphase membranes with cdc2-GSTcyclinB1 kinase results in substantial release of CD from the membrane. These data suggest that phosphorylation of DLIC by cdc2 kinase leads directly to the loss of membrane-associated CD and an inhibition of organelle movement.
University of Manchester, School of Biological Sciences, Manchester, M13 9PT, United Kingdom.
S G
SG
Addinall
P S
PS
Mayr
S
S
Doyle
J K
JK
Sheehan
P G
PG
Woodman
V J
VJ
Allan
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D056744:Cyclin B1; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D007399:Interphase; D008677:Metaphase; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000201:enzymology; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014982:Xenopus laevis
D056744:Cyclin B1; D011994:Recombinant Proteins; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D004398:Dyneins