Journal: Journal of Cell Science

Article Title: Phosphorylation of axin within biomolecular condensates counteracts its tankyrase-mediated degradation

doi: 10.1242/jcs.261214

Figure Lengend Snippet: Axin phosphorylation increases its stability. (A-L) Representative western blots showing axin in hypotonic cell extracts. Red arrows indicate the phospho-axin variant. (A) HEK293T cells expressing GFP-tagged rat axin (GFP-axin) were left untreated (0) or were treated with 100 µM cycloheximide (CHX) for 8, 11 or 14 h. (B) GFP-axin or Flag-tagged rat axin (Flag-axin) expressed in SW480 and U2OS cells. (C) Flag-axin expressed in SW480 cells. Extracts were left untreated (–) or incubated at 37°C for 1 h without (37°C) or with calf intestinal phosphatase (CIP). (D) Endogenous human axin in SW480 (left) and U2OS cells (right). Extracts were untreated (–) or CIP-treated. (E) Flag-axin expressed in SW480 cells. Extracts were left untreated (–) or incubated at 37°C for 1 h without or with sodium fluoride (NaF, 10 mM). (F) Flag-axin expressed in SW480 cells. Extracts were supplemented with okadaic acid (OA, 1 µM) and/or incubated at 37°C, as indicated. (G) U2OS cells expressing Flag-axin were left untreated (0) or treated for 6 h with the indicated OA concentrations. (H) GFP-axin expressed alone (–) or together with Flag-tagged human PP1 (Flag-PP1) in U2OS cells. (I) U2OS cells expressing GFP-axin were left untreated (–) or treated for 6 h with CK1 inhibitors D4476 or IC261, or the GSK3 inhibitor BIO, as indicated. (J) Flag-axin expressed alone (–) or together with GFP-tagged human CK1α (GFP-CK1α) in U2OS cells. (K) GFP-axin transfected in U2OS cells either together with a control siRNA (–) or with siRNAs targeting human CK1α, CK1δ or CK1ε as indicated (siCK1α, siCK1δ, siCK1ε, respectively). (L) Endogenous axin in HEK293T and U2OS cells that were left untreated (0) or treated for 2 or 4 h with 100 µM D4476. α-Tubulin serves as loading control. All experiments were replicated at least three times.

Article Snippet: The CK1 inhibitors D4476 (ab120220) and IC261 (ab145189) were obtained from Abcam (Cambridge, UK) the GSK3 inhibitor BIO (B1686), the PP1 and PP2A inhibitor okadaic acid (O7885), the tankyrase inhibitor G007-LK (5.04907) and the protein synthesis inhibitor cycloheximide (239765) from Sigma-Aldrich (Merck, St. Louis, MO).

Techniques: Western Blot, Variant Assay, Expressing, Incubation, Transfection

Journal: Journal of Cell Science

Article Title: Phosphorylation of axin within biomolecular condensates counteracts its tankyrase-mediated degradation

doi: 10.1242/jcs.261214

Figure Lengend Snippet: Axin phosphorylation in condensates counteracts tankyrase-mediated degradation. (A) Western blotting for endogenous axin in lysates of U2OS (left) and HEK293T cells (right). Cells had been pre-treated overnight with the tankyrase inhibitor G007-LK (500 nM) (+) or not (−), and were then treated with 100 µM CK1 inhibitor D4476 for 2 h or 4 h, or left untreated (0). α-Tubulin serves as loading control. Numbers below the blots show 2D densitometry quantification of axin bands normalized to α-tubulin. To compare the effect of D4476 in cells pre-treated with G007-LK or not, the initial amount of axin in untreated cells (0 h D4476) was set to 100% for either pre-treatment, i.e. the amount of axin in D4476-treated cells is presented relative to the respective initial amount. (B) Quantification of the experiments shown in A ( n =4). Results are the mean±s.e.m.; ** P <0.01, *** P <0.001 (two-tailed, paired Student's t -test). (C) Schematic depicting how phosphorylation of axin prevents its degradation through tankyrase. Within condensates (light red background): CK1α (CK1) phosphorylates axin at its N-terminal consensus motif, thereby sterically hindering tankyrase binding and increasing axin stability. Outside condensates (light blue background): PP1 dephosphorylates axin (1), thereby allowing tankyrase (TNKS) binding (2) and promoting tankyrase-mediated degradation of axin (3).

Article Snippet: The CK1 inhibitors D4476 (ab120220) and IC261 (ab145189) were obtained from Abcam (Cambridge, UK) the GSK3 inhibitor BIO (B1686), the PP1 and PP2A inhibitor okadaic acid (O7885), the tankyrase inhibitor G007-LK (5.04907) and the protein synthesis inhibitor cycloheximide (239765) from Sigma-Aldrich (Merck, St. Louis, MO).

Techniques: Western Blot, Two Tailed Test, Binding Assay

Journal: Molecular Cell

Article Title: Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubiquitylation

doi: 10.1016/j.molcel.2021.07.013

Figure Lengend Snippet:

Article Snippet: CK1 inhibitor D4476 , Sigma-Aldrich , Cat# 218696.

Techniques: Recombinant, Protease Inhibitor, Silver Staining, Staining, Plasmid Preparation, Mutagenesis, Software, Fluorescence

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: Early phosphorylation of p53 in S20 induced by Resv is necessary for p53-stability in MCF-7 R cells. ( A ) MCF-7 R cells were treated with CDDP (6 μM) with or without Resv (100 μM) and ( B ) MCF-7 cells were treated with Resv (100 μM); both cell cultures were treated for 6 h with specific p53-pS20 site kinase inhibitors: CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM). Total and phospho-p53 contents are assessed by Western blot using antibodies directed against total p53 (DO-1) or against the specific phosphorylated residue on S20, as indicated. ( C ) MCF-7 and MCF-7 R cells were treated with a DMSO–ethanol vehicle as control or CDDP (6 μM) with resveratrol (100 μM) and cultured in combination with CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM) inhibitors for 48 h and were double-stained with Annexin V and propidium iodide (PI) followed by flow cytometry analysis to determine apoptotic cells. The viable cells are located in the lower left quadrant (double negative with Annexin V–/PI–). Apoptotic cells (Annexin V+/PI–) appear in the lower right (early apoptosis) and upper right (late apoptosis) quadrant of data plots. Data are presented as a percentage of the cell population. ( D ) The combined results of three independent cytometry analyses depicting the mean levels of total apoptotic cells are shown. Results are presented as the means ± SD. *** p < 0.001 by one-way ANOVA followed by Turkey’s Multiple Comparison test.

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Residue, Control, Cell Culture, Staining, Flow Cytometry, Cytometry, Comparison

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: In the MCF-7 resistant cell variant (MCF-7 R ), Resv attenuates phosphorylation in S15 and S46 of p53 by dephosphorylation and deactivation of ATM. However, it activates kinases CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20 (which is required to activate p53 in order to upregulate BAX and PUMA genes) and modifies the ratio between BCL-2/BAX expression. The BAX protein was increased while BCL-2 protein was decreased, restoring apoptosis and overcoming chemoresistance. On the other hand, the overexpression of BCL-2 in MCF-7 R cells after CDDP treatment maintains the chemoresistance and blocks apoptosis despite the phosphorylation of p53 in S15 and S46 and the upregulation of NOXA and PUMA .

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Variant Assay, Phospho-proteomics, De-Phosphorylation Assay, Expressing, Over Expression