Tumor and Stem Cell Biology BRCA2 Phosphorylated by PLK1 Moves to the Midbody to Regulate Cytokinesis Mediated by Nonmuscle Myosin IIC

Cytokinesis is the critical final step in cell division. BRCA2 disruption during cytokinesis is associated with chromosome instability, but mechanistic information is lacking that could be used to prevent cancer cell division. In this study, we report that BRCA2 phosphorylation by the mitotic polo-like kinase (PLK1) governs the localization of BRCA2 to the Flemming body at the central midbody, permitting an interaction with nonmuscle myosin IIC (NM-IIC). Formation of an NM-IIC ring-like structure at the Flemming body shows that the IIC-ring relies on its ATPase activity stimulated by interaction with BRCA2 and associated proteins. Notably, inhibiting this binding inactivated the ATPase activity, causing disassembly of the IIC-ring, defective formation of the midbody, and interruption of cytokinesis. An analysis of cancer-associated mutations in BRCA2 at the PLK1-binding site suggests that they may contribute to cytokinetic defects by altering BRCA2 localization. Our findings suggest that BRCA2-dependent IIC-ring formation is a critical step in proper formation of the midbody, offering an explanation for how chromosome instability may arise in breast cancer. Cancer Res; 74(5); 1518–28. 2014 AACR. Introduction Germline mutations in the BRCA2 gene have been reported to increase the risk of developing breast and ovarian cancer. The BRCA2 protein has multiple functions, including DNA double-strand break repair (1–3), and the regulation of centrosome amplification and localization (4, 5). BRCA2 also contributes to the regulation of cytokinesis (6, 7). During anaphase, constriction of the actomyosin ring leads to formation of a cleavage furrow (8–10). Continued furrowing results in the formation of a narrow intercellular bridge, which contains the midbody, consisting of a bundled microtubule and a ringlike structure called the Flemming body within its central portion (11). Several studies have implicated various protein factors in the modulation of cytokinesis by BRCA2 (12–14). Disruption of BRCA2 during cytokinesis leads to disorganization of myosin-II at the cleavage furrow and the intercellular bridge (6). Mitotic polo-like kinase 1 (Plk1) is a key regulator of mitosis from mitotic initiation to cytokinesis. Plk1 contains a serine/ threonine kinase domain followed by the carboxy-terminal polo-box domain (PBD), which binds to phosphopeptides within a consensus motif of S-[pS/pT]-[P/X] (15, 16). The PBD regulates cellular function, the interactionwith substrates, and the subcellular localization of Plk1 (17). Plk1 is also required for appropriate localization of substrates (18). Previous studies revealed that Plk1 binds to the N-terminal region of BRCA2 and phosphorylates Ser193, and that this phosphorylation is enhanced as mitosis progresses (19, 20). However, the specific Plk1-binding site within this region of BRCA2 has not been identified. The role of BRCA2 phosphorylation in the maintenance of genome stability also remains unclear. Nonmuscle myosin-II (NM-II) proteins in humans are hexamers, consisting of a pair of heavy chains and two pairs of light chains that hydrolyze MgATP. They are members of a family of actin-binding motor proteins that play essential roles in cellular processes such as cell division and embryonic development. The NM-II family comprises three isoforms: NM-IIA, NM-IIB, and NM-IIC. These contain different nonmuscle myosin heavy chains (NMHC-IIA, NMHC-IIB, and NMHC-IIC) that are encoded by MYH (myosin heavy chain) 9, MYH10, and MYH14, respectively (21, 22). The N-terminal region of the NMHC-II protein consists of a globular head containing the actin-binding region, an ATPase domain, and a Src homology 3 (SH3)-like domain (myosin head; refs. 23, 24). NM-IIC is alternatively spliced both in loop-1 and loop-2. Isoform NMIIC0 contains no inserts in either of the loops. An 8–amino acid extension in the loop-1 region is present in isoforms NM-IIC1 and NM-IIC1C2. Isoform NM-IIC1C2 displays a 33–amino acid extension in the loop-2 region. The presence of 8 amino acid insert in NM-IIC increases the actin-activated ATPase activity (25). The C-terminal deletion isoform of NM-IIC is already present in the Ensembl database (http://www.ensembl.org/ index.html; MYH14-007, Protein ID: ENSP00000469573). Jana Authors' Affiliations: Department of Molecular Genetics, Medical Research Institute, Tokyo Medical & Dental University, Bunkyo-Ku; and Department of Genetic Diagnosis, the Cancer Institute of JFCR, Koto-Ku, Tokyo, Japan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Yoshio Miki, Department of Molecular Genetics, Medical Research Institute, TokyoMedical & Dental University, Yushima 15-45, Bunkyo-Ku, Tokyo 113-8510, Japan. Phone: 81-3-5803-5825; Fax: 81-3-5803-0242; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-0504 2014 American Association for Cancer Research. Cancer Research Cancer Res; 74(5) March 1, 2014 1518 on April 14, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 21, 2014; DOI: 10.1158/0008-5472.CAN-13-0504