Multisite phosphorylation of C-Nap1 releases it from Cep135 to trigger centrosome disjunction.
During mitotic entry, centrosomes separate to establish the bipolar spindle. Delays in centrosome separation can perturb chromosome segregation and promote genetic instability. However, interphase centrosomes are physically tethered by a proteinaceous linker composed of C-Nap1 (also known as CEP250) and the filamentous protein rootletin. Linker disassembly occurs at the onset of mitosis in a process known as centrosome disjunction and is triggered by the Nek2-dependent phosphorylation of C-Nap1. However, the mechanistic consequences of C-Nap1 phosphorylation are unknown. Here, we demonstrate that Nek2 phosphorylates multiple residues within the C-terminal domain of C-Nap1 and, collectively, these phosphorylation events lead to loss of oligomerization and centrosome association. Mutations in non-phosphorylatable residues that make the domain more acidic are sufficient to release C-Nap1 from the centrosome, suggesting that it is an increase in overall negative charge that is required for this process. Importantly, phosphorylation of C-Nap1 also perturbs interaction with the core centriolar protein, Cep135, and interaction of endogenous C-Nap1 and Cep135 proteins is specifically lost in mitosis. We therefore propose that multisite phosphorylation of C-Nap1 by Nek2 perturbs both oligomerization and Cep135 interaction, and this precipitates centrosome disjunction at the onset of mitosis.
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Tara
T
Hardy
Department of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea.
Miseon
M
Lee
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Rebecca S
RS
Hames
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Suzanna L
SL
Prosser
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Donna-Marie
DM
Cheary
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Mugdha D
MD
Samant
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Francisca
F
Schultz
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Joanne E
JE
Baxter
Department of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea.
Kunsoo
K
Rhee
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK amf5@le.ac.uk.
Andrew M
AM
Fry
D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002502:Centrioles / Q000378:metabolism*; D018385:Centrosome / Q000502:physiology*; D020090:Chromosome Segregation / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D042822:Genomic Instability; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D009154:Mutation / Q000235:genetics; D000072379:NIMA-Related Kinases; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D008941:Spindle Apparatus / Q000378:metabolism*
D001324:Autoantigens; C534330:CEP135 protein, human; C499237:CROCC protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C111329:CEP250 protein, human; D003598:Cytoskeletal Proteins; D034741:RNA, Small Interfering; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases
C-Nap1; Centriolar linker; Centrosome disjunction; Cep135; Nek2