Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation.

Helen J Whalley,Zoi Diamantopoulou,Gavin R M White,Eduardo Castañeda-Saucedo,Andrew P Porter,Angeliki Malliri

doi:10.1038/ncomms8437

Helen J Whalley, Zoi Diamantopoulou + Show 4 more

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https://doi.org/10.1038/ncomms8437

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Abstract

Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the development of Eg5 inhibitors as cancer therapeutics.

Highlights

Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis
Many mechanisms appear to contribute to centrosome separation after NEBD3, but most notable is the plus-end-directed kinesin Eg5, whose microtubule (MT)-sliding activity is essential for centrosome separation in prometaphase across many species[4] and which functions in the less-understood prophase pathway in mammalian cells[5,6,7]
We found that Cdk[1], cyclin A and cyclin B1 all co-precipitated with Tiam1-GFP from asynchronous and mitotically arrested HEK293T cells (Supplementary Fig. 1b,c), supporting our hypothesis that Tiam[1] interacts with active Cdk[1] complexes

Summary

Introduction

Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam[1] and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. The initiation of mitosis and regulation of centrosome separation is controlled by the mitotic kinases cyclin-dependant kinase 1 (Cdk1), polo-like kinase 1 (Plk1) and Aurora A15. Active Cdk[1] primes certain substrates (with the sequence S-S/T-P) for phosphorylation by Plk[1] by creating a docking site for its polo box domain (PBD)[23] One such protein is Nek[9], whose sequential phosphorylation by Cdk[1] and Plk[1] is required for it to activate Nek6/7, which promote Eg5 centrosomal localization by phosphorylating the kinesin on S1033 Aurora A is required for centrosome separation, probably through its pivotal roles in regulating centrosome maturation and MT organization[25]

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