PPP1R42 is a positive regulator of PP1 in centrosome duplication and separation and cilia dynamics

Abstract

The centrosome is a dynamic structure that undergoes structural and functional transitions, which are coordinated with the cell cycle. It functions as the microtubule organizing center (MTOC) in interphase, duplicates and separates in S-G ₂ forms the bipolar spindle poles in mitosis, and forms the basal body that assembles and sustains the primary cilium in G₀. Transitions of the functional state of the centrosome, here termed centrosome dynamics, must be carefully regulated. Errors in centrosome function result in mitotic defects promoting cancer as well as a class of disorders termed ciliopathies. Coordinated activity of phosphatases and kinases is required for proper control of centrosome function during the cell cycle. This study investigates a regulatory subunit of protein phosphatase 1 (PP1), phosphoprotein phosphatase regulatory subunit 42 (PPP1R42), and its role in control of centrosome dynamics by analyzing its regulatory effects on centrosome function and downstream targets of PP1, the centrosomal kinases never in mitosis gene-A related kinase 2 (Nek2) and Aurora A. In addition, this work investigates how these two kinases interact to control cilia assembly and disassembly. PPP1R42 is localized to the centrosome in human retinal pigmented epithelial (ARPE-19) cells and positively regulates PP1. Manipulation of cellular PPP1R42 protein levels causes changes in centrosome number, number of ciliated cells, cilia structure, and Nek2 kinase activity. Overexpression of Nek2 and Aurora A causes a decrease in cilia number. However, by using kinase dead versions of these kinases, we were able to determine that the effect of each individual kinase on cilia growth is dependent on whether cilia are assembling or disassembling. Nek2 and Aurora A negatively regulate one another during cilia assembly; however, both Nek2 and Aurora A are jointly required for cilia disassembly. Together, these data have uncovered a novel role for a positive PP1 regulator and provided evidence that PP1 negatively regulates Nek2 and Aurora A to inhibit centrosome duplication/separation and promote cilia assembly. Centrosomes are necessary for cell proliferation and function and undergo transitions linked with the cell cycle. The centrosome transitions from the MTOC to the basal body or mitotic spindle poles and must duplicate once per cell cycle. Misregulation of these events leads to cancers or ciliopathies. Centrosome amplification is a hallmark of many cancers and results in chromosome segregation errors and genomic instability. Defects in the structure and function of primary cilia are the root cause of a class of inherited disorders classified as ciliopathies. These diseases manifest in a variety of symptoms throughout the body, which reflects the ubiquitous presence of primary cilia and their crucial role in cell function and development. This work provides new information that will support improvements in the diagnosis and treatments of both cancers and ciliopathies.