EMBO/soda-nel huggingface dataset:
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 2

Figure 2. Plx1 bound Apc1 promotes APC/C-Cdc20 complex formation.

A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant WT or Apc-loop500 mutant APC/Cs with mutations (1-T532A, 1-T539A, 1-T532A/T539A (1-2A) and 1-S558A) was incubated with APC/C depleted (ΔAPC) interphase extract (Inter) or ΔAPC anaphase extracts supplemented with non-degradable cyclin B (Ana) at 23˚C for 1 h. 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.

B. Quantification of (A). The bar graph is quantification of bound Cdc20. The intensities of WT control in anaphase were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

C. Plx1-binding deficient APC/Cs are less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or mutant APC/C (1- 2A or 1-S558A) was incubated with ΔAPC anaphase extract and the recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay (Fig S3). Ubiquitylation was quantified and the activity of WT at 45 min was arbitrarily set to 1.0. Error bars, SEM from three independent experiments. A representative result is shown in Fig S3.

D. In vitro kinase assay of Apc3-loop using purified Plx1. The purified Apc3-loop (182-451 in Xenopus) fragment was incubated with Plx1 in the presence of ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by Coomassie brilliant blue (CBB).

E. In vitro phosphorylation by Plx1. Apc3-loop fragment, α-Casein, N-terminus of Cdc20 (N159) or MBP protein was incubated with purified Plx1 in the presence of [γ32P]-ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by autoradiography and Coomassie brilliant blue (CBB) staining.

F. Phosphorylation of Apc3-loop by Plx1 in the presence of [γ32P]-ATP. WT Apc3-loop fragment or its CDK-sites mutant (9A) was incubated with purified Plx1 in the presence of [γ32P]-ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by autoradiography.
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 3

Figure 3. Interplay between Plx1 and PP2A-B56 on Apc1-loop500.

A. Apc1-loop500 is the hub for both Plx1 and PP2A-B56 phosphatase. CDK sites (T or S) and B56-binding site (green) on Apc1-loop500 (yellow bar) are shown. Bindings of Plx1 and B56 to Apc1-loop500 depend on phosphorylation of CDK sites. The PP2A-B56 associated on Apc1-loop500 may dephosphorylate CDK sites.

B. Phosphorylation status of T532 and T539 in Apc1-loop500 varies depending on mutations in the B56-binding site. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A and E562A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with phospho-specific (pAb) or MBP antibody.

C. Plx1-binding assay using MBP-fused Apc1-loop500 fragments with mutations in B56-binding site. The Plx1-binding to Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was analysed as described in Fig 1B.

D. Quantification of (C). The bar graph is quantification of bound Plx1. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

E. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, T532A/T539A (2A) and S558A) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The recovery of WT (x1/2 and x1) was run as a standard.

F. Quantification of (E). The bar graph is quantification of bound B56γ. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

G. Summary of Plx1 and B56 binding to Apc1-loop500 derivatives. The abilities of Apc1-loop500 fragments carrying the indicated mutations are shown. The B56-bindings to ∆11 (deletion of 11 residues including B56 binding site) and 3A (T532A/T539A/S558A) were tested previously (Fujimitsu & Yamano, 2020).
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 4

Figure 4. Mutual regulation of Plx1 and PP2A-B56 on Apc1-loop500.

A. Plx1 and PP2A-B56 competes for Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining.

B. Dephosphorylation of T532, T539 and S558 on Apc1 by PP2A-B56. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A and E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, isolated by amylose beads and further incubated in the presence (78 nM) or absence of purified PP2A-B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with phospho-specific or MBP antibodies. The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was very faint in L557A/V560A mutant probably because these two residue are close to S558, interfering with pS558 antibody recognition.

C. Dephosphorylation of Apc1-loop500 by PP2A-B56 in the presence of Plx1-PBD. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, and analysed as described in (B).
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 5

Figure 5. Phosphorylation and function of Apc1-loop500 in Xenopus egg extracts.

A. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 in anaphase extracts. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with Xenopus egg extracts in the presence of non-degradable cyclin B at 23˚C. The samples taken as indicated were analysed by SDS-PAGE and immunoblotting using phospho-specific antibodies (pT532, pT539 and pS558) or MBP antibody. To monitor CDK activity, Apc3 antibody was used (bottom). The signals of a phospho-specific antibody and MBP antibody on the same membrane were detected in the 800- and 700-nm channels, respectively. The two channels were pseudo-coloured (green; pT532, pT539 and pS558, red; MBP) and overlaid in the bottom panel (Overlay). The signal of pS558 was not detected in ∆11 mutant because ∆11 mutation includes S558.

B. Quantification of (A). The intensities of WT at 50 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

C. Phosphorylation kinetics of T532, T539 and S558 on Apc1-loop500 of the endogenous APC/C in Xenopus egg extract. Endogenous APC/C was immunoprecipitated with Apc3 MAb (AF3.1) beads at the indicated time points after mitotic induction by adding non-degradable cyclin B, analysed by SDS-PAGE and immunoblotting with phospho-specific antibodies, pT532, pT539 and pS558. Quantification of Fig EV5A. The intensities at 45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

D. Dissociation of Plx1-PBD from Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 30 or 60 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie brilliant blue (CBB) staining. The intensities at 0 min were arbitrarily set to 1.0.
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 6

Figure 6. Increased Plx1 binding to Apc1-loop500 induces premature APC/C activation

and delays dephosphorylation of APC/C during mitotic exit.

A. Cdc20 binding assay in Xenopus egg extracts. The Cdc20-binding activities of WT APC/C and Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) were analysed as described in Fig 2A.

B. Quantification of (A). The bar graph is quantification of bound Cdc20.The intensities of WT control at Ana-45 min were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.

C. Premature activation of a mutant APC/C (1-E562A) in Xenopus egg extracts. The purified recombinant WT APC/C or Apc-loop500-mutant APC/C (1-E562A) was incubated with its substrates (35S-labelled cyclin B and a version of cyclin B lacking the N-terminal 67 residues, ∆67) in APC/C-depleted (ΔAPC) interphase extracts supplemented with non-degradable cyclin B at 23˚C. Samples taken at indicated time points after addition of substrates were analysed by SDS-PAGE and autoradiography (Fig S7A). The relative cyclin levels are shown, normalized with reference to the intensities found at time 0 for each time point. Error bars, SEM from three independent experiments. A representative result is shown in Fig S7A.

D. APC/C dephosphorylation after inactivation of CDK. The purified recombinant WT or Apc-loop500-mutant APC/Cs with mutations (1-L557A/V560A or 1-E562A) was incubated with APC/C depleted (ΔAPC) in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1h (Ana). After addition of p27 (0.3 µM), the samples taken at time points shown were analysed by SDS-PAGE and immunoblotting with indicated antibodies. 'Inter' denotes interphase.
Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop (2021)
Figure 7

Figure 7. A model: Plx1 and PP2A-B56 creates feedback-loop.

A. The mutations in CDK sites on Apc3-loop reduces phosphorylation of T532 on Apc1. The purified recombinant WT or a mutant APC/C with mutations at nine CDK sites in Apc3-loop (3-9A) was incubated with APC/C-depleted (ΔAPC) anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 60 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the phosphorylation status of T532 in Apc1 was analysed by SDS-PAGE and immunoblotting with phospho-specific (pT532) or Apc1 (pan-Apc1) antibodies.

B. Similar to (A), but the experiments were performed at three time points, 45 min, 60 min or 75 min in anaphase extracts. Quantification of phosphorylation levels of T532 are shown. The intensities of pT532 signals in WT at each time point (45 min, 60 min or 75 min) were arbitrarily set to 1.0. The level of T532-phosphorylation was normalised to pan-Apc1. Error bars, SEM from three independent experiments.

C. PP2A-B56 preferentially binds to T532-phoshorylated Apc1-loop500. Flag-tagged B56γ was incubated with the MBP-fused Apc1-loop500 phosphorylated by Cdk2/cyclin A. The bound proteins were recovered by anti-flag affinity M2 beads, separated by SDS-PAGE and detected by immunoblotting with phospho-specific (pT532), MBP or Flag antibody. The signals of a phospho-specific antibody (pT532) and MBP were pseudo-coloured (green; pT532, red; MBP) and overlaid (Overlay).

D. A feedback control of APC/C by Plx1 and PP2A-B56 on Apc1-loop500. CDK phosphorylates Apc1-loop500 at T539 and S558 and promotes Plx1-binding onto Apc1-loop500 via its PBD. Two PBD interactions, possibly dimerisation of Plx1, may stabilise the complex between Plx1 and Apc1-loop500. Plx1-binding blocks PP2A-B56 recruitment to Apc1-loop500 and stimulates Apc3-loop phosphorylation, which may induce Cdk1-CyclinB-p9/Cks2 recruitment and subsequent Apc1-loop300 phosphorylation. Cdk1 recruited onto phosphorylated Apc3-loop promotes phosphorylation of T532, a residue important for the recruitment of PP2A-B56 on Apc1-loop500. PP2A-B56 recruitment promotes dissociation of Plx1 by competitive binding and by dephosphorylation of T532 and T539, which diminishes Plx1-mediated phosphorylation of Apc3-loop. PP2A-B56 may quickly dissociate from Apc1-loop500 once T532 is dephosphorylated, but Apc1-loop500 is phosphorylated again by Cdk1 while Cdk1 is active. The dynamic phosphorylation-dephosphorylation cycle may continue until Cdk1 is inactivated, allowing swift response of the APC/C during the cell cycle.

Expanded View Figure legends
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 1

Figure 1. Identification of 3,4-Dimethoxychalcone (3,4-DC) as a potential caloric restriction mimetic (CRM). (A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h (compounds are listed in Appendix Tab. S1). And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame. Human osteosarcoma U2OS cells (B,F) or neuroglioma H4 cells (D,H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm. Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap, abbreviations are listed in Appendix Table S2 (G, I). (J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 2

Figure 2. 3,4-DC induced autophagic flux. (A-D) Hepatoma HepG2 cells were treated with the indicated concentrations of 3,4-DC for 8 h (A,B) or with 30 µM 3,4-DC for the indicated time points (C,D). Then, cells were processed to measure LC3 and p62 protein levels by SDS-PAGE and immunoblot (A, C). GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in (B, D). Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05). (E,F) HepG2 were treated with 30 µM 3,4-DC for 8 h in the presence or absence of chloroquine (CQ, 50µM) for 4 h. SDS-PAGE and immunoblot were performed as in (A). Band intensities of LC3-II and GAPDH were assessed and their ratio (LC3-II/GAPDH) was calculated in (F). Data are means ± SEM of at least three independent experiments (* = p < 0.05, ** = p < 0.01 versus untreated control; ##=p<0.01 versus CQ). (G,H) U2OS-GFP-LC3 cells were treated with 30 µM 3,4-DC, 10 µM rapamycin, or left untreated for 16 h in the presence or absence of CQ for 4 h. GFP-LC3 dots were measured. Data are means ± SD (*** = p < 0.001 versus untreated; ###=p<0.001 versus CQ). (I,J) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP-RFP proteins (GFP-RFP-LC3) were treated with the indicated raising doses of 3,4-DC or 50 µM chloroquine (CQ) for 16 h. After fixation, GFP-LC3 and RFP-LC3 dots were measured by automated image acquisition and analysis. GFP and RFP double positive LC3 dots indicating autophagosomes (GFP+), and GFP negative but RFP positive LC3 dots indicating autolysosomes (GFP-) were counted in (J). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in I. Scale bar equals 10 µm. Immunoblots in A,C and E were run together on the same gel, then blots were cut into horizontal stripes, and probed separately.
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 3

Figure 3. 3,4-DC induces autophagy in a transcription or translation dependent mechanism. (A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. (C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ). (E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). (F,G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH.
3,4-Dimethoxychalcone induces autophagy through activation of transcription factors TFE3 and TFEB (2019)
Figure 4

Figure 4. 3,4-DC induced TFEB-dependent lysosomal biogenesis and autophagy. (A,B) U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (A). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in (B). Scale bar equals 10 µm. (C, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm. (E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page. (G-I) MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels. (J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC) (M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr). Samples for immunoblots were run together in one gel (E, F) or in several parallel gels (I, J, M), then blots were cut into horizontal stripes and probed separately. Blots probed for P-p70 were stripped and reprobed to detect p70 independently from its phosphorylation status.