Phosphorylated Astrin regulated kinetochore function mediated by TRAIP in mitosis

Abstract

Protein-protein interaction post-translational modification and protein degradation are important for the mitotic progression such as chromosome segregation to opposite spindle poles and the stable attachment of spindle microtubules (MTs) to kinetochores (KTs). Error-free chromosome segregation required precisely timed search and capture of chromosomes by spindles in early mitosis. However, the molecular mechanism underlying conversions from labile to stable attachment are poorly understood. Here, I show that the sequential action of the kinase Cdk1 (Cyclin-Dependent Kinase 1) or Plk1 (Polo-Like Kinase 1), TRAIP (TRAF-interacting protein), and the phosphatase CDC14A (Cell Division Cycle 14A) controls the spindle attachment of Astrin to KTs.

In previously study, the mitotic spindle protein SPAG5/Astrin phosphorylated during mitosis. However, unfortunately there's no evidence which site of Astrin phosphorylated. This study contains that Astrin is transported into centrosomes by Kinastrin and I identified phosphorylation sites of Astrin at S135 and S249 using PhosphoSite program (http://www.phosphosite.org), and it's phosphorylated by Cdk1 or Plk1. Somehow, what protein loaded Astrin onto the spindle and targeted to KTs is unknown. I found TRAIP as a new Astrin interacting protein by using yeast two hybrid screening and confirm that TRAIP binds and co-localizes with Astrin during early mitotic progression by immunoprecipitation and immunofluorescence assay. Depletion of TRAIP resulted in hyper-stabilized and multipolar spindles, accompanied by aberrant chromosomal alignment measured by phosphor-Histone H3 (p-Histone H3) staining for Fluorescence-Activated Cell Sorting (FACS), Live cell imaging observation for time-lapse microscopy and Fluorescence Loss on Photobleaching (FLIP) experiment. Mechanistically, TRAIP regulates Astrin phosphorylation and targets phospho-Astrin to KTs, leading to the attachment of spindles to KTs. I also demonstrate that CDC14A dephosphorylates Astrin and therefore, overexpression of CDC14A sequestrates Astrin in the centrosome and results in aberrant chromosome alignment. These comprehensive findings uncover a regulatory circuit for protein targeting to KTs that controls the change of KT composition for stable spindle attachment and chromosome integrity.

In summary, this study reveals a new agent involved in search and capture, the signal messenger phospho-Astrin, which is generated by Cdk1 and targeted to KTs by TRAIP through spindle MTs, and is involved in stable KT attachment to spindle. It elucidates a key mechanism in mitotic progression. This study provides additional information about both the mechanism underlying bi-oriented spindle formation and chromosome instability in cancer cells.