Journal: EMBO Reports

Article Title: PP2A‐B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis

doi: 10.15252/embr.201948503

Figure Lengend Snippet: (left panel) Cdk‐dependent in vitro kinase assay of Apc1‐loop 300 . 3xFlag‐tagged WT or 7T Apc1‐loop 300 fragment was incubated with Cdk2‐cyclin A in the presence of [γ‐ 32 P]‐ATP at 30°C for 30 min, and separated by SDS–PAGE and detected by autoradiography. (right panel) 32 P‐phosphorylated WT or 7T Apc1‐loop 300 fragment was incubated in anaphase extract, and removal of radioactivity was analysed by SDS–PAGE followed by autoradiography. Quantification of removal of radioactivity in (A). Error bars, SEM from three independent experiments. Cdc20‐5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1‐loop 500 mutant APC/C (1‐L557A/V560A) was incubated with WT Cdc20 or non‐phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23°C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS–PAGE and immunoblotting with indicated antibodies. Quantification of (C). The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A‐B56γ at 23°C. Samples were taken at the indicated time points and analysed by SDS–PAGE and immunoblotting with antibodies, including phospho‐site‐specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318). A model for Apc1‐loop 500 ‐mediated Cdc20‐APC/C complex formation in mitosis. Apc1‐loop 500 is phosphorylated by Cdk1 and binds to PP2A‐B56 in anaphase. PP2A‐B56 dephosphorylates inhibitory phosphorylation sites in N‐Cdc20 and promotes the formation of active APC/C‐Cdc20 complex. Apc1‐loop 500 may control phosphorylation of other APC/C subunits and possibly interacting proteins although the mechanisms involved remain elusive. The indicated numbers on the schematic view of the APC/C (the back view is rotated by 180° around the vertical axis from the front view) represent APC/C subunits.

Article Snippet: The cleared lysate was incubated with GSH beads (GE Healthcare) at 4°C for 1 h. The beads were washed twice with PP2A wash buffer 1 (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM DTT, 0.1% Tween‐20, 1 mM EDTA and 1 mM EGTA) and once with PP2A wash buffer 2 (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM DTT, 1 mM EDTA and 5% glycerol) and suspended into PP2A wash buffer 2 containing PreScission protease and incubated at 4°C for 3.5 h with gently mixing.

Techniques: In Vitro, Kinase Assay, Incubation, SDS Page, Autoradiography, Radioactivity, Binding Assay, Mutagenesis, Purification, Recombinant, Western Blot, Immunoprecipitation