SUMO targets the APC/C in a phosphorylation-dependent manner to regulate transition from metaphase to anaphase
Signal transduction by Small Ubiquitin-like Modifier (SUMO) regulates a myriad of nuclear processes. Here, we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery resulted in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identified the APC/C complex, a master regulator of metaphase to anaphase transition, as a SUMO target. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation increased APC/C ubiquitylation activity towards a subset of its targets, including the newly identified target KIF18B. Intriguingly, SUMOylation of APC/C was regulated by casein kinase driven phosphorylation of serines at positions 777 and 779. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal a triad of PTMs, cooperating to drive mitosis.
Sample Processing Protocol
Purification of the APC/C complex exhibiting GFP-tagged APC4 One gram of HeLa cells expressing GFP-APC WT and GFP-APC4 K772,798R mutant was lysed in a similar manner as described for the endogenous complex. Lysates were incubated with 20 µl GFP-trap (Chromotek) and incubated for 1 hour at 4°C. Beads were washed four times in 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% glycerol and proteins were eluted in 2x NuPAGE LDS sample buffer (Thermo Fisher Scientific). Purification of endogenous APC/C complex CDC27 antibody (65 µg) was coupled to 50 µl Protein A beads as described above. One gram of HeLa cells was resuspended in 1 ml of lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.05% Tween 20, 5% glycerol) and lysed using a dounce homogenizer. After centrifuging for 30 min at 20000 x g and 4°C, the supernatant was incubated with CDC27 antibody coupled Protein A beads for 1 h at 4°C. Beads were then washed four times with 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% glycerol. The APC/C complex was eluted in elution buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20, 2.5% glycerol, 1 mg/ml CDC27 peptide). Co-immunoprecipitation of binding partners of recombinant APC/C Recombinant APC/C WT and APC/C mutant complex was SUMOylated in vitro by adding 50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP, 3.5 U/ml Creatine kinase, 10 mM Creatine phosphate, 0.6 U/ml inorganic pyrophosphate, protease inhibitor cocktail (Roche), 6.4 µg SAE1/2, 40 µg UBC9 and 40 µg SUMO-3 to 10 µg samples of APC/C in a volume of 180 µl and incubating for 3 hours at 4°C. The SUMOylated recombinant complex was purified by adding Strep-tactin beads (GE Healthcare) and incubating for 2 hours at 4°C in 50 mM Tris pH 7.5 and 150 mM NaCl. Five 15-cm plates of HeLa cells per sample were lysed in 50 mM Tris pH 7.5, 150 mM NaCl and 20 mM NEM (Sigma), sonicated to reduce viscosity and centrifuged at 20000 x g for 30 min at 4°C. The Strep-tactin beads were washed three times in lysis buffer to eliminate the SUMO machinery and incubated with the supernatant of the HeLa lysates for two hours at 4°C. Afterwards the beads were washed three times with 50 mM Tris pH 7.5 and 150 mM NaCl and three times with 50 mM ammonium bicarbonate. The bound proteins were incubated with 2 µg of trypsin on the beads overnight at 37°C. The samples were then passed through a pre‐washed 0.45 μm filter (Millipore) and acidified with 2% Trifluoroacetic acid (Sigma). For subsequent mass spectrometric analysis the peptides were desalted by C-18 stage tips as described by Hendriks et al. 2014.
Data Processing Protocol
Desalted peptide samples were measured by nanoscale LC ‐ MS / MS on an Orbitrap Q ‐ Exactive (Thermo) and raw data analysis was performed using MaxQuant Software and Perseus Software version 1. 5. 4. 1.
Sabine Cuijpers, Leiden University Medical Center
Alfred C.O. Vertegaal, Department of Molecular Cell Biology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands ( lab head )
Eifler K, Cuijpers SAG, Willemstein E, Raaijmakers JA, El Atmioui D, Ovaa H, Medema RH, Vertegaal ACO. SUMO targets the APC/C to regulate transition from metaphase to anaphase. Nat Commun. 2018 9(1):1119 PubMed: 29549242