Dynamic recruitment of Nek2 kinase to the centrosome involves microtubules, PCM-1, and localized proteasomal degradation.
Centrosomes undergo dramatic changes in composition and activity during cell cycle progression. Yet mechanisms involved in recruiting centrosomal proteins are poorly understood. Nek2 is a cell cycle-regulated protein kinase required for regulation of centrosome structure at the G2/M transition. Here, we have addressed the processes involved in trafficking of Nek2 to the centrosome of human adult cells. We find that Nek2 exists in small, highly dynamic cytoplasmic particles that move to and from the centrosome. Many of these particles align along microtubules and a motif was identified in the Nek2 C-terminal noncatalytic domain that allows both microtubule binding and centrosome localization. FRAP experiments reveal that 70% of centrosomal Nek2 is rapidly turned over (t(1/2) approximately 3 s). Microtubules facilitate Nek2 trafficking to the centrosome but only over long distances. Cytoplasmic Nek2 particles colocalize in part with PCM-1 containing centriolar satellites and depletion of PCM-1 interferes with centrosomal recruitment of Nek2 and its substrate C-Nap1. Finally, we show that proteasomal degradation is necessary to allow rapid recruitment of new Nek2 molecules to the centrosome. Together, these data highlight multiple processes involved in regulating the abundance of Nek2 kinase at the centrosome including microtubule binding, the centriolar satellite component PCM-1, and localized protein degradation.
Hames
Rebecca S
RS
Department of Biochemistry, University of Leicester, UK.
Crookes
Renarta E
RE
Straatman
Kees R
KR
Merdes
Andreas
A
Hayes
Michelle J
MJ
Faragher
Alison J
AJ
Fry
Andrew M
AM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D018385:Centrosome / Q000201:enzymology* / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D000072379:NIMA-Related Kinases; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment
D001324:Autoantigens; D018797:Cell Cycle Proteins; C086074:PCM1 protein, human; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex