Centrosome-targeting region of CG-NAP causes centrosome amplification by recruiting cyclin E-cdk2 complex.
Centrosome duplication occurs once per cell cycle and is thought to be triggered by cyclin E-cdk2. However, it is largely unknown how the duplication is regulated. Here, we found that the expression of the centrosome-targeting region of CG-NAP (centrosome and Golgi-localized PKN-associated protein), which we designate as CG-NAP/D, increased the number of centrosomes in Chinese hamster ovary (CHO)-K1 cells. The amplified centrosomes co-localized with centrosome markers gamma-tubulin, centrin-2 and kendrin as well as endogenous CG-NAP. When CG-NAP/D was dislocated from centrosomes by deleting the centrosome-targeting domain or by fusing with a membrane-targeting sequence, centrosome amplification was suppressed. CG-NAP/D interacted with exogenously expressed cyclin E, which co-localized at centrosomes. The immunoprecipitates of CG-NAP/D exhibited histone H1 kinase activity, suggesting the co-immunoprecipitation of active cyclin-cdk complexes. Furthermore, centrosome fractions prepared from cells expressing CG-NAP/D contained increased amount of cdk2 compared with those from control cells. Centrosome amplification by CG-NAP/D was suppressed by co-expression of a mutant cyclin E unable to interact with cdk2. These results suggest that CG-NAP/D causes centrosome amplification by anchoring excess amount of cyclin E-cdk2 to centrosomes and, possibly, CG-NAP participates in centrosome duplication by recruiting cyclin E-cdk2 to centrosomes in normal cell cycle.
Nishimura
Tamako
T
Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan.
Takahashi
Mikiko
M
Kim
Hon-Song
HS
Mukai
Hideyuki
H
Ono
Yoshitaka
Y
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism*; D006224:Cricetinae; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D054758:A Kinase Anchor Proteins; C119719:AKAP9 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002148:Calmodulin-Binding Proteins; D019927:Cyclin E; D003598:Cytoskeletal Proteins; D011993:Recombinant Fusion Proteins; C410004:kendrin; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2