Hence, ULK3 can interact with a specific subset of ESCRT-III proteins in cells
Hence, ULK3 can interact with a specific subset of ESCRT-III proteins in cells. Open in a separate window Figure 1. ULK3 binds ESCRT-III via tandem MIT domains.(A) Lysates from 293T cells overexpressing Myc-endosomal sorting complexes required for transport (ESCRT)-III proteins were mixed with lysates from cells non-transfected (?) or overexpressing One-strep-flag (OSF)-Unc-51-like kinase 3 (ULK3) (+). 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension causes at the midbody. Our structural and biochemical studies reveal an unusually tight conversation between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. DOI: http://dx.doi.org/10.7554/eLife.06547.001 (Webster et al., 2014). Tension causes applied by dividing cells around the midbody also regulate cytokinesis, with high-tension delaying abscission, and tension release triggering ESCRT-III assembly and membrane scission (Lafaurie-Janvore et al., 2013). How these different physiological inputs converge to influence abscission timing is not understood. Here, we investigate the function of Unc-51-like kinase 3 (ULK3), a poorly characterized member of the ULK family of serine/threonine kinases that is predicted to contain tandem MIT domains (Row et al., 2007). Live-cell imaging analysis revealed that ULK3 regulates abscission timing in response to lagging chromosomes, defects in nuclear pore complex assembly, and tension forces at the midbody. Furthermore, our biochemical Fluocinonide(Vanos) and structural studies show that this ULK3 MIT domains bind tightly to IST1, an ESCRT-III subunit required for cytokinesis (Agromayor et al., 2009; Bajorek et al., 2009a). Finally, we show that ULK3 phosphorylates IST1 and other ESCRT-III proteins and that IST1 phosphorylation provides an essential inhibitory transmission in the abscission checkpoint, thereby ensuring proper coordination of the final events in cell division. Results ULK3 binds to ESCRT-III via tandem MIT domains The predicted MIT domains in ULK3 suggested a novel mechanism of ESCRT regulation, Fluocinonide(Vanos) and we, therefore, surveyed potential ULK3CESCRT interactions using yeast two-hybrid (Y2H) experiments. ULK3 COL4A1 binding was observed for three ESCRT-III subunits: CHMP1A, CHMP1B, and CHMP2A, but not for other ESCRT complexes (Physique 1figure product 1A). These interactions were confirmed by co-immunoprecipitation of Myc-tagged ESCRT-III proteins from mixed 293T cell lysates that contained One-strep-flag (OSF)-tagged ULK3 (Physique 1A, note interactions in lanes 2, 4, 6, and 14). This approach revealed that ULK3 also bound the ESCRT-III subunit IST1 (lane 26), an conversation not tested by Y2H because IST1 fusion constructs activated transcription nonspecifically. Endogenous ULK3 also co-precipitated with overexpressed HA-tagged CHMP1A, CHMP1B, CHMP2A, or IST1, but not with CHMP2B (Physique 1B). Finally, endogenous IST1 was efficiently biotinylated in cells that expressed a biotin ligase BirA-ULK3 fusion protein, which promiscuously biotinylates proximal proteins (Physique 1C, lane 4, bottom panel) (Roux et al., 2012). Hence, ULK3 can interact with a specific subset of ESCRT-III proteins in cells. Open in a separate window Physique 1. ULK3 binds ESCRT-III via tandem MIT domains.(A) Lysates from 293T cells overexpressing Myc-endosomal sorting complexes required for transport (ESCRT)-III proteins were mixed with lysates from cells non-transfected (?) or overexpressing One-strep-flag (OSF)-Unc-51-like kinase 3 (ULK3) (+). OSF-ULK3 proteins were bound to streptactin resin and bound ESCRT-III Fluocinonide(Vanos) proteins were detected with -Myc antibody (top). (B) Lysates from 293T cells expressing HA-ESCRT-III were immunoprecipitated with -HA antibody and co-precipitated endogenous ULK3 protein was detected by Western blot with -ULK3 antibody. (C) HeLa cells expressing ULK3 fused to the biotin protein ligase BirA-113G or unfused BirA were treated overnight with biotin. Vicinal biotinylated proteins were isolated with streptavidin-coated beads, and endogenous IST1 was found to be biotinylated, implying that it was in close proximity with ULK3. Asterisks denote isolated BirA-Empty and BirA-ULK3, respectively, on -Avidin blot (lanes 3 and 4). Images shown for both lysate and pull-down samples were cropped from your same blot in all cases. (D) Co-immunoprecipitation of Myc-IST1 with different ULK3 constructs. Asterisks denote phosphorylated IST1 species. (E) Fluocinonide(Vanos) Top: overlaid 15N-HSQC NMR spectra of 15N,13C-IST1 (residues 303C366) alone (black) or with 2 equivalents of ULK3(MIT)2 (residues 277C449).