Dimerization Of CPAP Orchestrates Centrosome Cohesion Plasticity

Mammalian centrosomes,the major microtubule-organizing centers(MTOCs), consist of a pair of centrioles normally localized at the periphery of the nucleus and surrounded by a number of proteins collectively referred to as pericentriolar material (PCM).The coordination of the centrosome cycle and cell cycle ensures an accurate distribution of genetic material.Daughter cells resulting from cell division possess one centrosome that includes two tightly associated centrioles.During late mitosis or early G1 phase,the close link between the two centrioles is lost and a loose link between the proximal ends of the two centrioles develops.The centrosome cohesion is a prerequisite of centriole duplication and keeps the distance between the two centrioles less than 2μm.In G2/M phase,centrosome cohesion dissipates and centrosome splitting happened and two centrosomes separate into two functional units to form the bipolar spindle. Perturbation of the spatiotemporal dynamics of centrosome splitting can lead to genomic instability during cell division.Malfunctions in centrosome cohesion and separation,in fact,have been seen in many solid tumors.How the cohesion and separation of the centrosome are regulated throughout the cell cycle is not yet well understood.However,several proteins such as C-Nap1,Rootletin,RanBP1,Dynamin2, Cep68 and Cep215 have been reported to be involved with centrosome cohesion.CPAP(centrosomal P4.1 associated protein),also named CENP-J,is a centrosome protein which contains five short coiled-coil segments and a glycine repeated segment termed the G-BOX.Residues 311-422 AA of CPAP(including coiled-coil 2) is a MY-destabilizing domain,and residues 423-607 AA of CPAP (including coiled-coil 3) is a MT-binding domain.Mutations that disrupt theα-helical structure or the charge property significantly affect the MT-destabilizing ability of CPAP.Mutation of CPAP induces a neuro-developmental disorder named MCPH (autosomal recessive primary microcephaly) that causes a great reduction in brain growth in human.In this study,we found that both partial depletion of CPAP by CPAP siRNA and overexpression of the C-terminal of CPAP induced an increase in centrosome splitting, indicating that CPAP functions on centrosome cohesion.Moreover,the CC5 domain of CPAP was identified as being essential for the formation of polymers between CPAP by means of co-immunoprecipitation and pull-down experiments.Here we reported that CPAP forms a homodimer during interphase and the fifth coiled-coil domain of CPAP is required for its dimerization.Moreover,this self-interaction is required for maintaining centrosome cohesion and preventing the centrosome from splitting before G2/M phase. Our biochemical studies show that CPAP forms homo-dimers in vivo.In addition,both monomeric and dimeric CPAP are required for accurate cell division,suggesting that the temporal dynamics of CPAP homodimerization is tightly regulated during the cell cycle.Significantly,our results provide evidence that CPAP is phosphorylated during mitosis and this phosphorylation releases its inter-molecular interaction.Taken together, these results suggest that cell cycle regulated phosphorylation orchestrates the dynamics of CPAP molecular interaction and centrosome splitting to ensure genomic stability in cell division.Collectively,these results demonstrate that negative regulation of CPAP self-interaction by mitotic phosphorylation is required for accurate centrosome cohesion and splitting during the cell cycle.