Centralspindlin assembly and 2 phosphorylations on MgcRacGAP by Polo-like kinase 1 initiate Ect2 binding in early cytokinesis

Abstract

Keywords: Cyk4, Ect2, MKLP1, Plk1, RACGAP1Abbreviations: BRCT, BRCA1 C-terminal; Ect2, Epithelial cell transforming sequence 2; MgcRacGAP, Male germ cell RacGAP; MKLP1, Mitotic kinesin-like protein 1; Plk1, Polo-like kinase 1.Cytokinesis is the final step of cell division which partitions genetic and cytosolic content into daughter cells. Failed cytokinesis causes polyploidy, genetic instability, and cancer. Kinases use phosphorylation to regulate the timing and location of the cytokinetic furrow. Polo-like kinase 1 (Plk1) is an essential mitotic kinase that triggers cytokinesis by phosphorylating MgcRacGAP to create a docking site for Ect2 at the central spindle. Ect2 binds to MgcRacGAP via its N-terminal BRCT domain (BRCA1 C-terminal), which docks at specific phosphorylated residues. Here we investigate the minimal Plk1-dependent phosphorylation sites required for cytokinesis onset. We demonstrate that phosphorylation of the major MgcRacGAP site, S157, is necessary but not sufficient to bind the Ect2 BRCT domain. Phosphorylation of an additional residue on MgcRacGAP at S164 is also required to elicit efficient binding. Surprisingly, BRCT binding additionally requires MKLP1 and its cognate interacting N-terminal domain of MgcRacGAP. Our findings indicate that central spindle assembly and 2 Plk1-dependent phosphorylations are required to establish efficient binding of the Ect2 BRCT in early cytokinesis. We propose that these requirements establish a high threshold to restrain premature or ectopic cytokinesis.

PP2A-B′ holoenzyme substrate recognition, regulation and role in cytokinesis

“…Cyk4 contains a coiled-coil domain near the N terminus that is required for oligomerization and mitotic kinesin-like protein 1 binding, a long disordered region following coiled-coil that harbors a high density of phosphorylation sites, and a RhoGAP domain near the C terminus reversibly controls RhoA activation ( Figure 4a ). We previously showed that Plk1 catalyzes phosphorylation at multiple sites of Cyk4; two of the phosphorylation sites are crucial for recruitment of Ect2, a guanine nucleotide exchange factor required for activation of RhoA, cleavage furrow formation and ingression [ 38 ] ( Figure 4a ). The phosphorylation-responsive B′-interaction motif of Cyk4 (143-LSTIDESGS-151) predicted by bioinformatic search ( Table 2 ) is located upstream of the Ect2-binding site, which harbors several potential Plk1 phosphorylation sites ( Figure 4a ).…”

“…Expression and purification of full-length PP2A Aα, Cα, B′γ1 and PR70 (B″ family), and assembly of the PP2A core enzyme (Aα–Cα heterodimer) and PP2A-B′ holoenzymes followed procedures described previously [ 11 , 51 ]. Expression and purification of GST-Cyk4 (1–177), Ect2 (1–321) and Plk1-phosphorylated Cyk4 (GST-pCyk4 or pCyk4), and the assembly of pCyk4–Ect2 complex were performed as previously described [ 38 ].…”

RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer

“…The cytokinesis regulators ECT2 and RACGAP1 (ref. 33 ) were top hits for both cell lines for multinucleate cell percentage Z-scores. The high number of multinucleate cells for these siRNAs was also observed by looking at the images ( Fig.…”

Progressive loss of RacGAP1/ ogre activity has sequential effects on cytokinesis and zebrafish development

“…Second, RacGAP1 provides specific binding sites for a number of key effectors of cytokinesis and abscission. Notably, ECT2 (Burkard et al, 2009;Kim et al, 2014;Somers and Saint, 2003;Wolfe et al, 2009;Zavortink et al, 2005), the guanine nucleotide exchange factor (GEF) needed to activate the small GTPase RhoA (Prokopenko et al, 1999;Su et al, 2011;Yüce et al, 2005), which controls formation and contraction of the actomyosin ring (Matsumura, 2005;Otomo et al, 2005;Piekny and Glotzer, 2008). Finally, RacGAP1 possesses an evolutionary conserved atypical C1 domain promoting protein-lipid interactions (Colon-Gonzalez and Kazanietz, 2006), a domain essential for linking the central spindle to the plasma membrane and successful abscission (Lekomtsev et al, 2012).…”