PPP1R2, a Protein Phosphatase‐1 Regulatory Subunit, Balances Aurora A and PP1 Activities to Maintain Centrosome Number

Abstract

Centrosomes are the primary microtubule organizing centers in the cell and nucleate spindle microtubules at mitosis. A balance of kinase and phosphatase activity controls centrosome number and mitotic spindle function. PPP1R2 (R2) is a negative regulator of protein phosphatase 1 (PP1) and an activator of Aurora A Kinase (AURKA). Both PP1 and AURKA play critical roles in centrosome regulation, however R2’s role at the centrosome has not been examined. Given that R2 interacts with PP1 and AURKA, critical regulators of the centrosome, we hypothesized that disruption of R2 function would impact centrosome behavior and mitosis. We overexpressed R2 in cultured cells and then measured centrosome number and nuclear size. R2 overexpression caused an increase in centrosome number compared to controls. R2 cooverexpression with either AURKA or PP1 returned centrosome number to normal levels. Since inhibition of PP1 by R2 is dependent on phosphorylation of R2, we tested the role of R2 phosphorylation on centrosome number. We transfected cells with R2 phosphorylation site mutants and measured centrosome number as well as nuclear size. Phosphomimetic R2 increased the percentage of cells with supernumerary centrosomes while cells transfected with phospho‐null R2 exhibited normal centrosome number. Overexpression of R2 and its phosphomimetic caused significantly larger nuclei than controls suggesting that R2 overexpression may interfere with cytokinesis. We also examined the localization of these proteins in dividing cells by indirect immunofluorescence and discovered that R2, its phosphorylated isoform pR2, AURKA, and PP1 were all localized to the midbody. Together these findings suggest that R2, AURKA and PP1 regulate cytokinesis. To test the role of specific protein domains of R2 in recruitment of PP1 to the midbody, we overexpressed R2 deletion mutants lacking either the N or C terminus of R2 and measured PP1 localization during cytokinesis. Overexpression of R2 truncation mutants resulted in a dramatic reduction of PP1 at the midbody supporting an important role for R2 in directing PP1 to the midbody during cytokinesis. We conclude that R2 balances opposing activities of AURKA and PP1 to regulate centrosome number through control of cytokinesis.