Journal: PLoS Biology

Article Title: GSK3β-Dzip1-Rab8 Cascade Regulates Ciliogenesis after Mitosis

doi: 10.1371/journal.pbio.1002129

Figure Lengend Snippet: (A and B) Dzip1 interacts with GSK3β. Endogenous GSK3β was immunoprecipitated by Dzip1 but not IgG (A), and endogenous Dzip1 was immunoprecipitated with GFP-GSK3β in HEK 293T cells (B). (C and D) Dzip1 is co-localized with GSK3β at the basal body. G0-phase NIH 3T3 cells expressing GFP-GSK3β were immunostained for Dzip1 and AcTub (C), or cells expressing BFP-Centrin2 were immunostained with GSK3β and Dzip1 (D). Scale bar: 5 μm. (E) GSK3β binds Dzip1 in a kinase-substrate interaction manner. Wild-type (WT) GFP-GSK3β and the mutants S9A, K85R, and R96A were each co-expressed with Myc-Dzip1 in G0-phase HEK 293T cells, and treated with the CK1 inhibitor D4476 or the CK2 inhibitor CX4945. Note that treatment with CX4945 but not D4476 led to a significant decrease in the extent of the up-shifted Dzip1 bands, although the binding of Dzip1 to the GFP-GSK3β variants showed no difference. The extent of the up-shifting of the Dzip1 bands was decreased in K85R-expressing cells. (F) Phosphorylation of Dzip1 is coordinated with GSK3β activation. The kinase activity of GSK3β was negatively correlated with serum stimulation in NIH 3T3 cells. Note that the up-shifted bands (arrowheads) of Dzip1 became evident after serum depletion for 24–48 h, and disappeared after serum restimulation. γ-Tubulin was set as a loading control. (G) GSK3β phosphorylates Dzip1 in vivo. In resting mouse embryo fibroblast (MEFs) treated versus not treated with GSK3 and CK2 inhibitors, the Dzip1 bands were up-shifted less in GSK3- and CK2-inhibited cells. The protein levels of total β-Catenin and GSK3β were steady, but the phosphorylated (S33/37/T41) β-Catenin specifically disappeared from GSK3-inhibited cells. α-Tubulin was set as a loading control. (H) GSK3β phosphorylates Dzip1 in vitro. Auto-phosphorylation of GSK3β (55 kD), and the phosphorylated bands of the middle (28 kD), C-terminus (36 kD), and N- terminus (50 kD) of Dzip1 are shown (left panel). Coomassie blue staining of the gel shows the loaded amounts of Dzip1 fragments (right panel). Note that the S520A mutation resulted in decreased phosphorylation of Dzip1 by GSK3β. (I) Inhibition of GSK3 by BIO causes loss of phospho-S520 in Dzip1.

Article Snippet: The CK1 inhibitor D4476 (Tocris Bioscience, 2902), rabbit anti-GSK3β (Santa Cruz Biotechnology, sc-9166), β-Catenin (BD, 610154), phosphorylated GSK3β (S9, Cell Signaling Technology, #93365), and phosphorylated β-Catenin (N3, Cell Signaling Technology, #9561) were kind gifts from Dr. Wei Wu (Tsinghua University).

Techniques: Immunoprecipitation, Expressing, Binding Assay, Phospho-proteomics, Activation Assay, Activity Assay, Serum Depletion, Control, In Vivo, In Vitro, Staining, Mutagenesis, Inhibition

Journal: Nature Communications

Article Title: NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

doi: 10.1038/s41467-021-26142-w

Figure Lengend Snippet: a BMDMs transfected with the indicated siRNAs were treated with LPS followed by nigericin. Cell death was monitored by PI incorporation over time (for 140 min following nigericin treatment) and quantified by high content microscopy. Means of area under the curve normalized to non-targeting siRNA control and 1 SD are represented. b IL-1β and TNF secretions were measured by ELISA. c , d BMDMs transfected with Csnk1a1 siRNA were treated with LPS (6 h) followed by nigericin. IL-18 and TNF secretions were measured by ELISA ( c ). ASC specks were visualized and counted by immunofluorescence ( d ). Arrow, ASC specks. Scale bar, 20 μm. Quantification of >10 cells per replicates is shown (total cells n = 571 NT, 158 Csnk1a1 ). Means and 1 SD are represented. e GST-NLRP3 was incubated with GST-CSNK1A1, GST-CSNK2A1/GST-CSNK2B, or 6-His-GST-CAMK2B. In vitro phosphorylation was revealed by SDS-PAGE and autoradiography. Coomassie stainings serve as controls. f NLRP3 was ectopically expressed with HA-CSNK1A1 in 293T. HA-CSNK1A1 immunoprecipitates were analyzed for NLRP3. g BMDMs were treated with LPS (6 h). CSNK1A1 immunoprecipitates were analyzed for NLRP3. Lysate of Nlrp3 +/+ BMDMs incubated with isotype control and A/G-beads ( Nlrp3 +/+ +IgG) and lysates of Nlrp3 −/− BMDMs were used as negative controls. h NLRP3 WT or S806A mutant were ectopically expressed with HA-CSNK1A1 in 293 T. NLRP3 immunoprecipitates were analyzed for phospho-Ser by WB. i NLRP3-deficient U937 cells reconstituted with NLRP3 were treated with PMA and doxycycline for 16 h followed by D4476 and LPS (4 h). NLRP3 immunoprecipitates were analyzed for phospho-Ser by WB. Data are biological quadruplicates representative of two independent experiment ( a , c ), biological triplicates representative of four independent experiment ( b ), 11 technical replicates representative of two independent experiments ( d ), one representative of two ( e , h , i ) and three ( f , g ) independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple comparisons ( a ), ordinary two-way ANOVA with Tukey’s multiple comparisons ( c , d ) to corresponding non-targeting siRNA control; * p -value < 0.05; ** p -value < 0.01; *** p -value <0.001; **** p -value < 0.0001. Molecular weights are indicated in kDa ( e – i ). AUC area under the curve, NT non-targeting siRNA, Lys lysates, IP immunoprecipitates.

Article Snippet: The following reagents were used: MG132 (Sigma-Aldrich), E-64d (Enzo Life Science), VX765 (Invivogen), G5 (Merck), doxycycline (Sigma-Aldrich), LPS (O111:B4, Sigma-Aldrich), Pam3CSK4 (Invivogen), nigericin (Invivogen), ATP (Sigma-Aldrich), H-Leu-Leu-OMe (LLOMe, Santa Cruz Biotechnology), MSU (Invivogen), silica (U.S.Silica), poly(dA:dT) (Invivogen), flagellin (FLA-ST Invivogen), oridonin (Euromedex), and D4476 (Sigma-Aldrich), SP600125 (Selleck Chemical).

Techniques: Transfection, Microscopy, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Incubation, In Vitro, Phospho-proteomics, SDS Page, Autoradiography, Mutagenesis

Journal: Scientific Reports

Article Title: Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic

doi: 10.1038/s41598-020-73248-0

Figure Lengend Snippet: Inhibition profiling of phosphorylation of Lyn-S13 in the presence of D4476, CX4945, IC261, or staurosporine. Lyn WT proteins synthesized by using the TnT T7 Insect Cell Extract Protein Expression System in the presence of D4476, CX4945, IC261, or staurosporine at concentrations of 62.5–500 µM were analyzed by Phos-tag SDS-PAGE (20 μM Zn 2+ –Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-Lyn antibody. Open arrowhead: nonphosphorylated species; closed arrowheads: phosphospecies containing a phosphorylated Ser-13 residue. The raw image is shown in Supplementary Figure .

Article Snippet: KN-93, X4945, and D4476 were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA).

Techniques: Inhibition, Phospho-proteomics, Synthesized, Expressing, SDS Page, Western Blot, Residue

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) Linear peptide docking sites for enzymes that regulate p53 function. The N-terminus is composed of three transactivation motifs,TAD1, TAD2, and Proline-repeat domain (PRD). A key regulatory domain in the C-terminus (REG) contains the acetylation motifs and phosphorylation site and flanks the Tetramerization domain (TET). The overlapping, but distinct, linear polypeptide docking motifs for p53 regulators include the acetyltransferase p300, the E3 ubiquitin ligase MDM2, iASPP, and the protein kinases including CDK, CK2, CK1, and CHK2 are highlighted. ( B ) Conservation of key phospho-acceptor sites between urochordate and human. The panel highlights the conservation of amino acids and phospho-acceptor sites in the BOX-I transactivation domain of p53 (TAD1 in Figure ) between human and urochordate ( Ciona intestinalis ). The ATM phospho-acceptor site at Ser15 and the Calcium Calmodulin kinase/CK1 phospho-acceptors sites at Thr18 and Ser20 are highlighted as indicated.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Ubiquitin Proteomics

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: p53 is activated by distinct stresses, some of which include as indicated, ionising radiation, viral infection, metabolic stress induced by an altered AMP/ATP ratio, and oncogene activation. The X-ray-induced Ser20 site kinase is ATM-dependent, but its identity is unknown (highlighted by "?"). CK1 is the DNA virus HHV-6B-induced p53 Ser20 kinase, but the upstream sensor is currently undefined (highlighted by "?"). The Ser20 site kinase induced by an elevated AMP/ATP ratio is AMPK, and LKB is the likely upstream sensor. DAPK-1 is the p53 Ser20 kinase induced by inappropriate oncogene activation, and ERK or ARF are the likely upstream sensors. These data support the formation of a model suggesting that the phosphorylation of p53 at Ser20 is triggered by distinct stress-responsive signaling cascades. Future analysis will be required to determine the identity of all the enzymes that mediate stress-induced phosphorylation at this site and "integrate" the p53 response and developing disease models that deregulate these signaling cascades.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Infection, Activation Assay, Virus, Phospho-proteomics

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) A CK1 inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by HHV-6B infection. MOLT-3 cells were infected with (even-numbered lanes) or without (odd-numbered lanes) HHV-6B for 48 hours in the presence of increasing concentrations (10-100μM) of the CK1 inhibitor D4476 (lanes 3-14) or a DMSO solvent control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( B ) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Infection, Solvent, Control, Western Blot, Cell Culture

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) An AMPK inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( B ) An AMPK inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( C ) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( D ) An ATM inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Solvent, Control, Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A-B) Bars present relative mRNA expression of CSNK1G isotypes. After treatment with 2 μM D4476 for 24 h, (A) MCF-7 and (B) MDA-MB-231 silenced with CSNK1G2 siRNA were analyzed for the mRNA expression of CSNK1G isotype genes such as CSNK1G1 , CSNK1G2 , and CSNK1G3 . (C-D) Bars denote relative percentage of % survival of (C) ER + or (D) ER - breast cancer cells treated with D4476 alone or in combination with TAM for 24 h. MCF-7 and MDA-MB-231 cells were treated with 1 to 10 μM of D4476. For combination treatment, 1 μM TAM was also used. Cellular toxicity was measured using the MTT assay (mean (SD), n = 5; ** P < 0.01 vs . each represented counterpart). (E-G) Western blotting analysis from the breast cells treated with vehicle or 2 μM D4476 together with 1 μM TAM for 24 h were performed. Immunoblots for (E) ERα and phospho-ERα (at Ser 118 or Ser 167 ) in MCF-7 cells and for PI3K/AKT/mTOR/S6K signaling-associated proteins in (F) MCF-7 and (G) MDA-MB-231 cells. NC siRNA or CSNK1G2 siRNA-transfected cells were treated with 1 μM of TAM together with vehicle or with 2 μM of D4476 for 24 h. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis (mean ± SEM, n = 4).

Article Snippet: D4476 (#74014) was purchased from Stem Cell Technologies Inc. (Cambridge, MA, USA).

Techniques: Expressing, MTT Assay, Western Blot, Transfection

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A) Cytotoxicity assay of either GFP-C1 or GFP-ERα-transfected ER - breast cancer cells. Bar denote relative percentage of % survival of transfected MDA-MB-231 cells after treatment with vehicle, 1 μM TAM, 2 μM D4476, and 1 μM TAM plus 2 μM D4476 for 24h. Cellular toxicity was measured using the MTT assay (mean (SD), n = 5; * P < 0.05, ** P < 0.01 vs . each represented counterpart). (B) Western blotting analysis from GFP-ERα-transfected ER - breast cells treated with vehicle or 2 μM D4476 together with 1 μM TAM for 24 h were performed. Immunoblots for PI3K/AKT/mTOR/S6K signaling-associated proteins in ER - MDA-MB-231 cells. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis (mean ± SEM, n = 4).

Article Snippet: D4476 (#74014) was purchased from Stem Cell Technologies Inc. (Cambridge, MA, USA).

Techniques: Cytotoxicity Assay, Transfection, MTT Assay, Western Blot, Expressing

Journal: PLoS Genetics

Article Title: Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds

doi: 10.1371/journal.pgen.1007850

Figure Lengend Snippet: (A) Lysates from NIH/3T3 stable cell lines expressing the dog, Myc-tagged wild-type (Wt) or mutant variant (Mut) DVL2, which is 23 aa shorter than wild-type exogenous DVL2, were analyzed by western blotting using an anti-c-Myc antibody. To assess the ability of the wild-type and mutant proteins to respond to WNT stimulation, cells were treated with WNT5A or WNT3A for 6 hours. Both treatments resulted in increased gel mobility shifts of the wild-type DVL2 protein, indicative of increased phosphorylation; this effect was reduced on the mutant DVL2 protein. (B) To confirm that the DVL2 gel mobility shifts observed in (A) were due to phosphorylation, cell lysates were subjected to mock treatment (30 min incubation at 37 C), or calf intestinal phosphatase (CIP) treatment (30 min incubation at 37 C in the presence of CIP) before separation by SDS-PAGE. The DVL2 gel mobility shifts above wild-type and mutant proteins were lost after CIP treatment, confirming that they are caused by phosphorylation. (C) To test whether the DVL2 gel mobility shifts observed in (A) were driven by casein kinase 1 (CK1), cells were treated with D4476, a CK1 inhibitor, for 1 hour prior to and concurrently during the Wnt stimulation for 6 hours. The DVL2 gel mobility shifts were lost after D4476 treatment, further indicating that they are caused by CK1-dependent phosphorylation. α-tubulin was used for loading controls. Cell lysates were normalized by BCA assays for total protein.

Article Snippet: For casein kinase 1 inhibitor treatment, cells were pre-treated with D4476 (APEXBIO catalog #A3342, 100nM final concentration) for 1h prior to WNT5A or WNT3A treatment.

Techniques: Stable Transfection, Expressing, Mutagenesis, Variant Assay, Western Blot, Phospho-proteomics, Incubation, SDS Page