Mitotic phosphorylation of PRC1 at Thr470 is required for PRC1 oligomerization and proper central spindle organization.
During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with the centromere. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, the chromosomal passenger protein complex, and microtubule bundling protein PRC1. PRC1 is phosphorylated by Cdc2 at Thr470 and Thr481 during mitosis. However, the functional relevance of PRC1 phosphorylation at Thr470 has remained elusive. Here we show that expression of the non-phosphorylatable mutant PRC1(T470A) but not the phospho-mimicking mutant PRC1(T470E) causes aberrant organization of the central spindle. Immunoprecipitation experiment indicates that both PRC1(T470A) and PRC1(T470E) mutant proteins associate with wild-type PRC1, suggesting that phosphorylation of Thr470 does not alter PRC1 self-association. In addition, in vitro co-sedimentation experiment showed that PRC1 binds to microtubule independent of the phosphorylation state of Thr470. Gel-filtration experiment suggested that phosphorylation of Thr470 promotes oligomerization of PRC1. Given the fact that prevention of the Thr470 phosphorylation inhibits PRC1 oligomerization in vitro and causes an aberrant organization of central spindle in vivo, we propose that this phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriate time in the cell cycle.
Fu
Chuanhai
C
Laboratory of Cellular Dynamics, Hefei National Laboratory and the University of Science and Technology of China, Hefei 230027, China.
Yan
Feng
F
Wu
Fang
F
Wu
Quan
Q
Whittaker
Joseph
J
Hu
Haiying
H
Hu
Renming
R
Yao
Xuebiao
X
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D008941:Spindle Apparatus / Q000187:drug effects / Q000502:physiology*; D013912:Threonine / Q000378:metabolism*
D018797:Cell Cycle Proteins; C116662:PRC1 protein, human; D013912:Threonine