Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Effect of H 2 O 2 and antioxidant treatment on autophosphorylation of FLT3 in intact cells and in vitro (A, B) RS4-11 cells were serum-starved and treated with H 2 O 2 at the indicated concentrations at 37 °C for 30 min. Positive control was FL stimulation with 100 ng/ml for 10 min. Endogenous FLT3 was immunoprecipitated and autophosphorylation was detected with anti-pY591 FLT3 antibodies (denoted pFLT3); blots were stripped and reprobed with pan-specific anti-FLT3 antibodies (FLT3, C20). (A) Example experiment, (B) quantification of pY-FLT3/FLT3 ratios for multiple experiments (n = 5, significance tested with one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001 for comparison with control) (C, D) FLAG-tagged FLT3 was transiently expressed in HEK293 cells and enriched by anti-FLAG immunoprecipitation and elution with FLAG peptide under anaerobic conditions. Samples were treated with H 2 O 2 for 10 min, and autokinase reactions were performed in absence or presence of 5 μM ATP and 5 mM MnCl 2 as indicated. Samples were analyzed by SDS-PAGE and immunoblotting with anti-pY591 FLT3 antibodies, and blots were reprobed with anti-FLT3 (S18). (C) Representative experiment, (D) quantification of pY-FLT3/FLT3 ratios for multiple experiments (n = 7). Box plot illustrating medians within boxes from first quartile (25th percentile) to the third quartile (75th percentile) and whiskers ranging from the 10th to the 90th percentiles. Differences did not reach significance by testing with one-way ANOVA. (E) MV4-11 cells were treated for 8 h with diphenyleneiodonium (DPI, concentrations indicated) or N -acetylcysteine (NAC, 10 mM), or the FLT3 kinase inhibitor AC220 (20 nM). Receptor autophosphorylation, and activation of the downstream mediators of cell transformation STAT5 and ERK1/2 were detected by immunoblotting using phosphospecific antibodies (pFLT3, pSTAT5, pERK1/2) and reblotting with corresponding pan-specific antibodies (FLT3 (S18), STAT5, ERK1/2). (F) Quantification of 6 experiments (treatment time 6–12 h). Values were calculated as arbitrary units (a.u.) relative to untreated cells and are given as means ± SEM. *p < 0.05, **p < 0.01,***p < 0.001 by one-way ANOVA compared to control.

Article Snippet: C-terminally FLAG-tagged hFLT3 in pCMV2 (FLT3-FLAG, HG10445-CF) was purchased from Sino Biological, Beijing, China.

Techniques: In Vitro, Positive Control, Immunoprecipitation, SDS Page, Western Blot, Activation Assay, Transformation Assay

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: DCP-Bio1 labeling reveals sulfenic acid modification of FLT3ITD cysteine residues. (A, B) MV4-11 cells (A) or RS4-11 cells (B), expressing endogenously FLT3ITD or wild-type FLT3, respectively, were serum-starved and treated as indicated with diphenyleneiodonium (DPI, 10 μM, 4 h), H 2 O 2 (100 μM, 30 min), or FL (100 ng/ml, 10 min). Cells were lysed in presence (or for control absence) of 1 mM DCP-Bio1, and subjected to pulldown with streptavidine (SA) or corresponding control (ctrl) beads. Pulldowns and corresponding lysate aliquots were analyzed for FLT3 (pan-FLT3 S18) by immunoblotting. pFLT3 was also assessed (pY591 phosphorylation) to detect FL stimulation. (C) HEK293 cells were transiently transfected with the indicated plasmids (EV, empty vector). Two days after transfection cells were processed as in A, and B. Note that all parts of the blot in C are from the same membrane and exposure, but have been rearranged for better clarity. Shown results are representative of three experiments with consistent results. (D) DCP-Bio1 labeling of FLT3ITD in HEK293 cells. Cells were pre-treated with DPI at the indicated concentrations or 10 mM N -acetylcysteine (NAC) for 4 h and then processed for DCP-Bio1 labeling as in (A) and (B). Lower panel shows the quantification of 4 independent experiments. *p < 0.05 for comparing with control by t -test.

Article Snippet: C-terminally FLAG-tagged hFLT3 in pCMV2 (FLT3-FLAG, HG10445-CF) was purchased from Sino Biological, Beijing, China.

Techniques: Labeling, Modification, Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Positions and indirect assessment of oxidation of cysteine residues in the FLT3 cytoplasmic domain. (A) Alignment of class III RTKs reveals conserved (highlighted yellow) and non-conserved unpaired cysteines in the FLT3 cytoplasmic domain. (B) Position of cysteine residues in the cytoplasmic domain of FLT3. The structure of FLT3 (PDB 1RJB ) is shown in ribbon representation. The main chain is colored in blue and the Cys residues are highlighted in yellow and their positions in the full-length protein are indicated by black numbers. Several functional motifs of the protein are also indicated: Juxtamembrane domain Tyr572-Trp603 in dark grey, catalytic loop His809-Asn816 in pink, activation loop Asp829-Ala856 in light green. (C) HEK293 cells were transiently transfected with the indicated plasmids expressing FLT3ITD or corresponding Cys-Ser mutants. Two days after transfection, cells were processed as described in and pulldowns with streptavidine (SA) beads were assessed for presence of FLT3 (S18 antibody). For FLT3ITD, two forms are detectable: The larger (ca. 170 kDa), mature, complex glycosylated (CG) form, and the smaller (ca. 150 kDa) major immature, high-mannose (HM) form. (D) Multiple experiments as in (C) were quantified and the ratio of FLT3 signals in pulldowns and whole cell lysate (WCL) aliquots was calculated. Data are depicted as medians within boxes from first quartile (25th percentile) to the third quartile (75th percentile) and whiskers ranging from the 10th to the 90th percentiles of independent experiments. (n = 6, *p < 0.05 by one-way ANOVA for comparison with control). Results for Cys828Ser, Cys925Ser, and Cys945Ser were very variable and not included in the statistical analysis.

Article Snippet: C-terminally FLAG-tagged hFLT3 in pCMV2 (FLT3-FLAG, HG10445-CF) was purchased from Sino Biological, Beijing, China.

Techniques: Functional Assay, Activation Assay, Transfection, Expressing

Journal: Redox Biology

Article Title: Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation

doi: 10.1016/j.redox.2019.101325

Figure Lengend Snippet: Signaling phenotypes of FLT3ITD cysteine mutants are not caused by altered dimerization. HEK293 cells were transiently transfected with the indicated FLT3 expression constructs. Dimers were stabilized by crosslinking with BS3, controls were performed in absence of crosslinker (−). Samples were analyzed by SDS-PAGE with 5.4% gels and immunoblotting. (A) Effect of FLT3 ligand (FL) on dimerization of wild-type (WT) FLT3. Cells were starved from serum 4 h before ligand stimulation. FLT3 was enriched by wheat-germ agglutinin (WGA) pulldown before analysis. The positions of FLT3 monomers (Mon), which exist as immature high-mannose form (HM), or complex glycosylated (CG) mature form, and of FLT3 dimers (Dim) are indicated (detection with pan-FLT3 S18). (B) Spontaneous dimer formation of FLT3ITD, and of the efficiently dimerizing FLT3ITD Cys694S mutant. Analysis of whole cell lysates (WCL) (C) Comparison of dimerization of FLT3ITD and different Cys-Ser mutants as indicated. The blot was also probed with anti-pFLT3 (pY591) to detect the autophosphorylation activity of dimers and monomers.

Article Snippet: C-terminally FLAG-tagged hFLT3 in pCMV2 (FLT3-FLAG, HG10445-CF) was purchased from Sino Biological, Beijing, China.

Techniques: Transfection, Expressing, Construct, SDS Page, Western Blot, Mutagenesis, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds *

doi: 10.1074/jbc.M114.585901

Figure Lengend Snippet: Inhibition of TLR dimerization by iberin. A, effect of iberin on the binding of LPS to RAW264.7 cells. The cells were pretreated with iberin (0–20 μm) or polymyxin B (PB; 100 μg/ml) for 30 min and then treated with 300 ng/ml biotin-labeled LPS (Biotin-LPS) for 15 min. After incubation, the cells were analyzed by ELISA using HRP-conjugated avidin. Error bars represent S.D. B, the TLR/NF-κB signaling pathway. C, effect of iberin on NF-κB activation mediated by TLR2. The HEK293 cells were transiently co-transfected with the expression plasmid for TLR2 or empty vector and NF-κB reporter plasmid. After transfection, the cells were cultured in the absence or presence of iberin for 24 h. #, p < 0.05 versus control; *, p < 0.05 versus TLR2 overexpression (0 μm iberin). Error bars represent S.D. D, effect of iberin on NF-κB activation mediated by MyD88. HEK293 cells were transiently co-transfected with the expression plasmid for adaptor protein MyD88 or empty vector and NF-κB reporter plasmid. After transfection, the cells were cultured in the absence or presence of iberin for 24 h. Error bars represent S.D. E, effect of iberin on poly(I:C)-induced phosphorylation of TBK1 (p-TBK1). RAW264.7 cells were pretreated with iberin (10 μm) for 30 min and then treated with poly(I:C) (10 μg/ml) for 30 min. F, effect of iberin on co-immunoprecipitation of MyD88 with TLR4. The HEK293 cells were transfected with expression vectors encoding TLR4-His and/or MyD88 together with MD2. Twenty-four hours after transfection, the cells were pretreated with iberin (15 μm) for 30 min and then treated with LPS (500 ng/ml) for 20 min. After incubation, the cells were washed and lysed. The cell lysates were immunoprecipitated (IP) with the anti-His-tag antibody, and the immunoprecipitates were analyzed by immunoblotting using the anti-MyD88 and anti-His tag antibodies. G, immunoblot analysis of phosphorylation of IRAK4. The RAW 264.7 cells were pretreated with iberin for 60 min and then treated with LPS (100 ng/ml) for 30 min. H, inhibition of TLR4 dimerization by iberin. The HEK293 cells expressing TLR4-HA and/or TLR4-His together with MD2 were pretreated with iberin (20 μm) for 30 min and then treated with LPS (500 ng/ml) for 20 min. The cells were then subjected to immunoprecipitation with anti-His tag antibody and immunoblotted with the anti-HA tag (upper) or anti-His tag (lower) antibody. I, inhibition of TLR2/TLR6 heterodimerization by iberin. The HEK293 cells expressing TLR2-His and/or TLR6-HA were pretreated with iberin (20 μm) for 30 min and then treated with Pam2CSK4 (500 ng/ml) for 20 min. The cells were then subjected to immunoprecipitation with the anti-His tag antibody and immunoblotted with anti-HA tag (upper) or anti-His tag (lower) antibody.

Article Snippet: The pMetluc2-NF-κB reporter vector was obtained from Clontech. pCMV2-FLAG-mouse MyD88 was obtained from Addgene. pEFBOS-FLAGHis-mouse TLR4, pEFBOS-FLAGHis-mouse TLR2, and pEFBOS-FLAGHis-mouse MD2 were kind gifts from Dr. K. Miyake (University of Tokyo).

Techniques: Inhibition, Binding Assay, Labeling, Incubation, Enzyme-linked Immunosorbent Assay, Avidin-Biotin Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Over Expression, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds *

doi: 10.1074/jbc.M114.585901

Figure Lengend Snippet: Effect of iberin on NF-κB signaling pathway. A, immunoblot analysis of nuclear translocation of p65. The RAW 264.7 cells were pretreated with iberin for 30 min and then treated with LPS (100 ng/ml) for 4 h. After incubation, the nuclear fractions were prepared, and immunoblotting was performed with the anti-p65 and anti-lamin A antibodies. A representative immunoblot (top) and densitometric analysis of p65 protein normalized to lamin A (bottom) are shown. Error bars represent S.D. B, immunoblot analysis of IκB degradation. The cells were pretreated with iberin for 30 min and then treated with LPS (100 ng/ml) for 30 min. After incubation, immunoblotting was performed with the anti-IκB and anti-GAPDH antibodies. A representative immunoblot (top) and densitometric analysis of IκB protein normalized to GAPDH (bottom) are shown. Error bars represent S.D. C and D, immunoblot analysis of phosphorylation of mitogen-activated protein kinases. The RAW264.7 cells were pretreated with iberin for 30 min and then treated with LPS (100 ng/ml) for 30 min. A representative immunoblot (C) and densitometric analysis of phosphorylated (p-) MAPKs normalized to total MAPKs (D) are shown. The values of LPS-treated cells are shown as a control (100%). Error bars represent S.D. E, effect of quercetin and Q4′G on NF-κB activation mediated by MyD88. The HEK293 cells were transiently co-transfected with the expression plasmid for the adaptor protein MyD88 or empty vector (Mock) and NF-κB reporter plasmid. After transfection, the cells were treated with quercetin (QUE) or Q4′G for 24 h. #, p < 0.01 versus Mock; ***, p < 0.005 and **, p < 0.01 versus vehicle (MyD88 expression). Error bars represent S.D. F, effect of quercetin and Q4′G on poly(I:C)-induced phosphorylation of TBK1 (p-TBK1). RAW264.7 cells were pretreated with quercetin or Q4′G (10 μm) for 30 min and then treated with poly(I:C) (10 μg/ml) for 30 min.

Article Snippet: The pMetluc2-NF-κB reporter vector was obtained from Clontech. pCMV2-FLAG-mouse MyD88 was obtained from Addgene. pEFBOS-FLAGHis-mouse TLR4, pEFBOS-FLAGHis-mouse TLR2, and pEFBOS-FLAGHis-mouse MD2 were kind gifts from Dr. K. Miyake (University of Tokyo).

Techniques: Western Blot, Translocation Assay, Incubation, Activation Assay, Transfection, Expressing, Plasmid Preparation

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition requires the SCF βTrCP E3 ubiquitin ligase. ( A ) Time course of YAP decline after Rac1 inhibition. HPAF/CD18 cells were harvested at the indicated time point after the addition of EHT-1864 (50 μM). Levels of YAP and S127-phosphorylated YAP were quantified by immunoblotting. GAPDH was used as an internal standard. ( B ) MG132 blocks the degradation of YAP elicited by the inhibition of Rac1. After 12 h of exposure to EHT-1864 (50 μM; +) or vehicle control (−), HPAF/CD18 cells were exposed or not to MG132 (20 μg/mL) for 4 h prior to harvesting. ( C ) Schematic description of YAP structure showing the position and sequence of critical degrons and phosphorylation sites. Upper drawing shows the structure of YAP, including its heterodimerization domain (TEAD), WD40 domains (WW), transactivation domain (TAD), putative degrons (βTrCP1/2, FBXW7), and LATS1/2 phosphorylation sites (S61, S109, S127, S128, S131, S163, S164, and S381). Lower left panel: Sequence of the putative FBXW7 phosphodegron highlighting its consensus and its potentially required phosphoserine group (S351). Changes introduced by the S351A/P352A mutation are also shown. Lower right panel: Sequence of the βTrCP degron of YAP highlighting its consensus and its required CK1 (S384, S387) and LATS1/2 (S381) phosphorylation sites. Changes introduced by the D383A/S384A mutation are also shown. ( D ) Detection of the Flag-YAP proteins in retrovirally infected Panc1 cells. Panc1 cells infected with pLXSH viruses carrying no insert (Empty), Flag-tagged YAP (WT), or its various mutant versions (5SA, D383A/S384A, S351A/P352A) were analyzed for the presence of Flag-YAP. GAPDH was used as an internal control. ( E ) The βTrCP degron is needed for YAP degradation after Rac1 inhibition, but not the S381 LATS1/2 phosphorylation site. In duplicate, Panc1 cells expressing the different mutants of Flag-YAP were exposed to EHT-1864 (50 μM). Sixteen hours later, Flag-tagged proteins were quantified using western blotting. Again, GAPDH was used as an internal control. ( F ) The siRNA-mediated knockdown of Skp1 blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with skp1 siRNA or with a non-targeting siRNA. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. ( G ) The siRNA-mediated knockdown of the βTrCP1/2 proteins blocks YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against βTrCP1, βTrCP2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, Skp1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: PCR products encoding Flag-tagged YAP and its 5SA mutant were respectively amplified from vectors p2xFlag CMV2-YAP2 (a gift from Marius Sudol’s lab; purchased from Addgene cat# 19045) and pCMV2-flag YAP2 5SA (a gift from Kunliang Guan’s lab; purchased from Addgene cat# 27371) using the same forward (5′-GTACGC GTCGAC AGTGAACCGTCAGAATTGATCTA-3′; Sal1 site underlined) and reverse (5′-CATGGA AGATCT CTATAACCATGTAAGAAAGCTT-3′; Bgl2 site underlined) primers.

Techniques: Inhibition, Ubiquitin Proteomics, Western Blot, Control, Sequencing, Phospho-proteomics, Mutagenesis, Infection, Expressing, Knockdown, Transfection

Journal: Cancers

Article Title: Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases

doi: 10.3390/cancers16213605

Figure Lengend Snippet: YAP degradation after Rac1 inhibition is LATS1/2-independent but requires CK1. ( A ) The silencing of LATS1/2 fails to prevent YAP degradation after Rac1 inhibition. Panc1 cells were transfected with a non-targeting siRNA or with siRNA against LATS1, LATS2, or both proteins. Forty-eight hours later, transfected cells were treated with EHT-1864 (50 μM; +) or the vehicle control (−) for 16 h prior to western blot analysis. GAPDH was again used as an internal standard. ( B ) Detection of LATS1 and LATS2 in the LATS1/2-proficient and -deficient HeLa cells. Cells were probed with the indicated antibodies. ( C , D ) LATS1/2 are not needed for YAP degradation after Rac1 inhibition. LATS1/2-proficient and -deficient cells HeLa cells were exposed to 50 μM EHT-1864 ( C ) or the DMSO vehicle ( D ) for 16 h, after which YAP levels were measured. ( E , F ) Panc1 cells expressing the Flag-YAP protein ( E ) or its 5SA mutant ( F ) were exposed or not to CK1 inhibitor IC-261 (10 μM), after which YAP levels were measured. GAPDH was used as an internal standard. For all panels, densitometry readings for the indicated intensity ratios (pYAP/GAPDH, YAP/GAPDH, LATS1/GAPDH, and FLAG/GAPDH) are shown below the lanes of each western blot.

Article Snippet: PCR products encoding Flag-tagged YAP and its 5SA mutant were respectively amplified from vectors p2xFlag CMV2-YAP2 (a gift from Marius Sudol’s lab; purchased from Addgene cat# 19045) and pCMV2-flag YAP2 5SA (a gift from Kunliang Guan’s lab; purchased from Addgene cat# 27371) using the same forward (5′-GTACGC GTCGAC AGTGAACCGTCAGAATTGATCTA-3′; Sal1 site underlined) and reverse (5′-CATGGA AGATCT CTATAACCATGTAAGAAAGCTT-3′; Bgl2 site underlined) primers.

Techniques: Inhibition, Transfection, Control, Western Blot, Expressing, Mutagenesis