Broken DNA bridges or intercellular canals are indicated by dotted arrows

Broken DNA bridges or intercellular canals are indicated by dotted arrows. of S51 to alanine reduces Src catalytic activity and impairs formation of actin patches, whereas expression of a phosphomimicking Src-S51D protein rescues actin patches and prevents chromatin breakage in Chk1-deficient cells. We propose that Chk1 phosphorylates Src-S51 to fully induce Src kinase activity and that phosphorylated Src promotes formation of actin patches and stabilizes chromatin bridges. These results identify proteins that regulate formation of actin patches in cytokinesis. Graphical Abstract Open in a separate window Introduction Chromatin bridges are strands of incompletely segregated chromatin that connect anaphase poles or daughter nuclei and have been linked to tumorigenesis (Hoffelder et al., 2004; Ganem and Pellman, 2012). In the presence of chromatin bridges, eukaryotic cells delay abscission, the final cut of the narrow cytoplasmic canal that connects the daughter cells, to prevent tetraploidization by regression of the Monomethyl auristatin F (MMAF) cleavage furrow or chromatin breakage (Steigemann et al., 2009; N?hse et al., 2017). In mammals, this abscission delay is called the abscission checkpoint and relies on the Aurora B protein kinase (Steigemann et al., 2009; N?hse et al., 2017). Activated Aurora B phosphorylates the endosomal sorting complex required for transport-III (ESCRT-III) subunit charged multivesicular body protein 4C (Chmp4c; Capalbo et al., 2012; Carlton et al., 2012; Petsalaki and Zachos, 2016). In turn, phosphorylated Chmp4c can cooperate with several proteins to inhibit the ATPase Vps4 at the midbody and prevent its activity on ESCRT-III filaments in order to inhibit abscission (Morita et al., 2007; Thoresen et al., 2014; Caballe et al., 2015). Furthermore, cells with chromatin bridges form and retain actin-rich structures called actin patches at the base of the chromatin bridge (Chen and Doxsey, 2009; Steigemann et al., 2009). It is suggested that actin patches stabilize the intercellular canal until the DNA bridge is resolved; however, how actin patches are formed has not been previously reported. Src is a nonreceptor tyrosine kinase that is involved in a diverse spectrum of biological activities including cell proliferation, adhesion, spreading, Rabbit polyclonal to CD14 and migration (Playford and Schaller, 2004). Src is located at the plasma membrane and is also found at late endosomes, the Golgi apparatus, and the nucleus (Takahashi et al., 2009). Src family kinases share a conserved domain structure consisting of an amino-terminal membrane-binding SH4 domain with a myristoylation sequence, followed by a Unique region that is divergent among family members (amino acids 20C85 of human Src), consecutive Src homology 3 (SH3) and SH2 domains, and a kinase domain that is followed by a short C-terminal tail (Maffei et al., 2015; Roskoski, 2015). The C-terminal tail contains an Monomethyl auristatin F (MMAF) autoinhibitory phosphorylation site (tyrosine 530 [Y530] in human Src), and phosphorylation at this site promotes Monomethyl auristatin F (MMAF) assembly of the SH2, SH3, and kinase domains into an autoinhibited closed conformation (Xu et al., 1997; Brbek et al., 2002). Displacement of the SH3- and SH2-mediated intramolecular interactions by Src binding to downstream substrates or higher-affinity ligands allows dephosphorylation of Src-Y530, followed by autophosphorylation of tyrosine 419 (Y419) inside the human Src catalytic loop, and leading to conversion of the enzyme into an active open form (Bernad et al., 2008; Roskoski, 2015). In addition, the Unique domain of Src contains phosphorylation residues that activate Src by promoting dephosphorylation of the autoinhibitory site (Shenoy et al., 1992; Stover et al., 1994) or regulate Src binding to lipids (Prez et al., 2013; Amata et al., 2014). Activating mutations in cellular Src or infection with the Src encoding Rous sarcoma virus can cause oncogenic transformation that is accompanied by dramatic changes in Monomethyl auristatin F (MMAF) the actin cytoskeleton (Frame, 2002). Src binds to FAK at focal adhesions and phosphorylates FAK at various residues including tyrosine 925 (Y925) to activate FAK or create binding sites for adaptor proteins (Brunton et al., 2005; Mitra et al., 2005). In turn, the FAKCSrc signaling complex promotes changes in actin cytoskeleton and regulates focal adhesion turnover (Goldberg et al., 2003; Brown et al., 2005; Mitra et al., 2005). Src phosphorylates cortactin to enhance actin nucleation and binds to formins to induce formation of stress fibers (Tominaga et al., 2000; Tehrani et al., 2007). In addition, Src signaling is involved in the completion of cytokinesis (Kasahara et al., 2007a; Kamranvar et al., 2016). Chk1 kinase was first identified to regulate the DNA damage response (Smith Monomethyl auristatin F (MMAF) et al., 2010); however, it is also required for proper mitotic cell division (Zachos et al., 2007; Peddibhotla et al., 2009). Chk1 phosphorylates the mitotic kinase Aurora B in prometaphase and metaphase to induce Aurora B catalytic activity and promote correction of misattached kinetochoreCmicrotubules (Petsalaki et al., 2011; Petsalaki and Zachos, 2013). Also, Chk1 is required for successful chromosome.