Abstract
During interphase, centrosomes are held together by a proteinaceous linker that connects the proximal ends of the mother and daughter centriole. This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation. The NIMA (never in mitosis A)-related kinase Nek2A is implicated in disconnecting the centrosomes through disjoining the linker proteins C-Nap1 and rootletin. However, the mechanisms controlling centrosome disjunction remain poorly understood. Here, we report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1), directly interact with Nek2A and regulate its ability to localize to centrosomes, and phosphorylate C-Nap1 and rootletin. Furthermore, we demonstrate that the hSav1-Mst2-Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation on partial inhibition of the kinesin-5 Eg5. We propose that hSav1-Mst2-Nek2A and Eg5 have distinct, but complementary functions, in centrosome disjunction.
Highlights
This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation
We report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador, directly interact with Nek2A regulating its ability to localize to centrosomes and phosphorylate C-Nap[1] and rootletin
We show that the hSav1-Mst2Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation upon partial inhibition of the kinesin-5 Eg5
Summary
Mammalian expression vectors for Nek2A and C-Nap1-CTD were as described 3, 6, 7, 37. Nek2A mutants were generated by site directed mutagenesis using pRcCMV-myc-Nek2A as template 7. CDNAs of hSav[1] and Mst[2] and their corresponding mutants were subcloned into the mammalian expression vector pCMV-HA (Clontech). For yeast two-hybrid analysis, cDNAs were cloned into pGADT7 and pGBKT7 DNA (Clontech). For bacterial expression cDNA of hSav[1] was subcloned into a derivative of pQE vector (Qiagen) carrying a NusA tag. GST-tagged Nek2A-ΔN was cloned into pGEX-5X-1 vector (GE Healthcare). CDNAs of wild-type and kinase dead versions of Mst[2] were cloned into pFastBac-HT-A vector (Invitrogen) for expression in insect cells. Sequences and details of the plasmids are available upon request
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