The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

Abstract

AbstractGlobal regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function. The mass spectrometry data are available from ProteomeXchange, identifier <ID to be provided when available, PXD000xxx>.Author SummaryAccurate segregation of chromosomes during cell division is fundamental for genome stability. Chromosome segregation is mediated by the spindle, which is made of dynamic microtubules and associated proteins that regulate microtubule behaviour. How these microtubule-associated proteins are regulated is not well understood. Furthermore, as oocytes have an exceptionally large volume of cytoplasm and lack centrosomes, regulation of microtubule-associated proteins is especially crucial for organisation and function of the meiotic spindle. In this study, we showed that 14-3-3, a protein that binds to phosphorylated proteins, plays an important role to regulate multiple microtubule-associated proteins in fly oocytes. The regulated proteins include subunits of the conserved kinase complex called the chromosomal passenger complex. We further found that interaction of one of the subunits with 14-3-3 is regulated by two phosphorylations, and that these phosphorylations are important for localisation and function of this subunit. As these proteins are widely conserved including humans, this study may provide an insight into chromosome mis-segregation in human oocytes, which is very frequent and a major cause of human infertility, miscarriages and congenital birth conditions.