active human pkcα  (Millipore)

Journal: Cell Death and Differentiation

Article Title: Blockage of PPARγ T166 phosphorylation enhances the inducibility of beige adipocytes and improves metabolic dysfunctions

doi: 10.1038/s41418-022-01077-x

Figure Lengend Snippet: a Differential HDX mass spectrometry data for PPARγ (PDB:3e00) with rosiglitazone (RSG) and CDDO. The ribbon diagram is colored according to HDX stabilization/destabilization. Percentages of deuterium difference are color-coded according to the color gradient key. b Sequence alignment of the PKCα catalytic consensus site in PPARγ, which is conserved in mammals. c Schematic representation of the PPARγ DBD revealing that T166 is located between two zinc finger structures. d Detection of PPARγ phosphorylation at T166 in HEK293T cells with overexpressing WT, TA mutant, or TD mutant PPARγ, or without overexpression (Mock). Experiments were repeated three times. e Annotation of MS/MS spectra of the peptides of PPARγ phosphorylated at T166. f Treatment of HEK293T cells overexpressing WT PPARγ with RSG or CDDO followed by the detection of p-T166. Experiments were repeated three times. g Co-immunoprecipitation of WT PPARγ and exogenous PKCα from HEK293T cells treated with PMA (100 nM) and Ro (2 μM), RSG (1 μM), or CDDO (100 nM). Experiments were repeated three times. Data were analyzed by one-way ANOVA followed by Tukey’s test ( d , f , g ). Data are presented as mean ± SEM. * P < 0.05. ** P < 0.01, *** P < 0.001.

Article Snippet: Human PPARγ was purchased from Cayman Chemical (Item No. 61700) and active human PKCα was purchased from Millipore (14−484).

Techniques: Mass Spectrometry, Sequencing, Phospho-proteomics, Mutagenesis, Over Expression, Tandem Mass Spectroscopy, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Blockage of PPARγ T166 phosphorylation enhances the inducibility of beige adipocytes and improves metabolic dysfunctions

doi: 10.1038/s41418-022-01077-x

Figure Lengend Snippet: a Isolated subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT), and brown adipose tissue (BATs) were homogenized. The whole tissue protein was analyzed by western blotting to evaluate the levels of p-PKCα and PPARγ p-T166 ( n = 4). b Body weight curve of 6-week-old C57/B6J mice treated with vehicle, RSG (5 mg/kg), or CDDO (3 mg/kg) for 14 days. c The content of adipose fat pads (percentage of body weight) from mice in the three groups. d The pure SAT adipocytes were isolated by using collagenase digestion method. The levels of PPARγ p-T166, PPARγ, p-PKCα, and PKCα in adipocytes were detected by western blotting. Each lane contains total protein from two mice. e H&E staining of SAT. 100× magnification, scale bar, 100 μm; 200× magnification, scale bar, 50 μm. f Relative mRNA levels of browning-related genes in SAT. Gene expression is normalized to the 36B4 endogenous control. g Detection of the level of UCP1 in SAT. Each lane contains total protein from two mice. β-Actin was the endogenous control. Biologically independent samples: ( b – g ), there were 6 mice in each group ( n = 6). Data are expressed as the mean ± S.E.M. Data were analyzed by two-way ANOVA followed by Bonferroni’s test ( b ) or one-way ANOVA followed by Tukey’s test ( a , c , d, f, g ). * P < 0.05. ** P < 0.01, *** P < 0.001.

Article Snippet: Human PPARγ was purchased from Cayman Chemical (Item No. 61700) and active human PKCα was purchased from Millipore (14−484).

Techniques: Isolation, Western Blot, Staining, Gene Expression, Control

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase Cα–mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it

doi: 10.1074/jbc.RA118.005921

Figure Lengend Snippet: Constitutively active PKCα (PKCαCAT) increases Twist1 protein expression independent of RNA. Human EOC cell clone R182 were transfected with PKCαCAT or empty vector control. A and B, expression of endogenous Twist1 and overexpressed PKCαCAT proteins were determined by Western blotting (A) and quantified by densitometry (B) reported as fold changes compared with empty vector control. *, p = 0.0008. C, effect on mRNA level was analyzed by qPCR, normalized to GAPDH, and reported as fold changes compared with empty vector control 24 h after transfection. ns, not significant (p > 0.05). D, different amounts of plasmid carrying WT Twist1 were transfected in HEK293T cells in the presence or absence of different amounts of plasmid carrying PKCαCAT, and the effect on Twist1 protein determined by Western blotting. Empty vector was used to control for total amount of plasmids transfected. E, HEK293T cells (panels i and ii) and EOC cells clone R182 (panels iii and iv) were transfected with WT Twist1 in the presence of different activation status of PKCα or empty vector control as indicated, and the effect on Twist1 protein was determined by Western blotting analysis, and the resulting bands were quantified by densitometry. CAT, constitutively active; DN, dominant negative; WT, full-length inactive WT. Densitometry readings are reported as fold changes compared with empty vector control. **, p = 0.0017; ****, p = 0.0001. Note that the truncated PKCαCAT (48 kDa) can be distinguished from the full-length DN and WT proteins (80 kDa). F, PKCα was knocked out in OCSC1-F2 ovarian cancer cells using CRISPR/Cas9, and the levels of PKCα and endogenous Twist1 were determined by Western blotting analysis. GAPDH was used as loading control for all Western blots. Each experiment was performed at least three independent times. Representative data are shown.

Article Snippet: Briefly, the kinase reaction consisted of 5 ng of recombinant human active full-length PKCα protein (Millipore, catalog no. 14-484), 200 or 300 ng recombinant human Twist1 protein (Abcam, catalog no. ab132349), PKC lipid activator (0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 4 m m MOPS, pH 7.2, 5 m m β-glycerol phosphate, 0.2 m m sodium orthovanadate, 0.2 m m DTT, 0.2 m m CaCl 2 ), 20 m m HEPES, pH 7.4, 20 m m MgCl 2 , 0.1 mg/ml BSA, 0.1 m m CaCl 2 , and 100 μ m ATP.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Activation Assay, Dominant Negative Mutation, CRISPR

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase Cα–mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it

doi: 10.1074/jbc.RA118.005921

Figure Lengend Snippet: PKCα induces Twist1 phosphorylation. A, Twist1 was transfected in HEK293T cells in the presence or absence of constitutively active PKCαCAT. Empty vector was used as control. Twist1 was subsequently immunoprecipitated, and the levels of phosphorylated Twist1 were determined by Western blotting using anti–phospho-serine/tyrosine/threonine antibody. GAPDH was used as a loading control for the input. B, in vitro kinase assay was performed as described under “Experimental procedures” with recombinant PKCα in the presence of increasing concentration of recombinant Twist1 as substrate. The ability of PKCα to phosphorylate Twist1 was quantified by the amount of ADP produced and presented as percentages of increase in ADP. *, p = 0.0022; **, p = 0.0001, compared with kinase reaction in the absence of Twist1. The experiments were performed at least three independent times. Representative data are shown.

Article Snippet: Briefly, the kinase reaction consisted of 5 ng of recombinant human active full-length PKCα protein (Millipore, catalog no. 14-484), 200 or 300 ng recombinant human Twist1 protein (Abcam, catalog no. ab132349), PKC lipid activator (0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 4 m m MOPS, pH 7.2, 5 m m β-glycerol phosphate, 0.2 m m sodium orthovanadate, 0.2 m m DTT, 0.2 m m CaCl 2 ), 20 m m HEPES, pH 7.4, 20 m m MgCl 2 , 0.1 mg/ml BSA, 0.1 m m CaCl 2 , and 100 μ m ATP.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Recombinant, Concentration Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase Cα–mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it

doi: 10.1074/jbc.RA118.005921

Figure Lengend Snippet: The WR domain of Twist1 is required for interaction with PKCα. A, HEK239T cell were co-transfected with Twist1–c-Myc and HA-PKCαCAT and whole-cell lysates were subjected to immunoprecipitation with either anti-HA antibody to capture PKCαCAT or anti-c-Myc antibody to capture Twist1. The resulting IP complexes were immunoblotted for PKCα and Twist1. B, schematic illustration of Twist1 protein deletions used to identify the domain required for binding to PKCα. C and D, Twist1 N-terminal deletion mutants with FLAG tag (C) and C-terminal deletions with Myc tag (D) were co-transfected with PKCαCAT in HEK293T cells. Whole-cell lysates were used to immunoprecipitate the corresponding tag on Twist1, and IP complexes were probed to detect presence of PKCα. IgG was used as control for all IPs. The experiments were performed at least three independent times. Representative data are shown.

Article Snippet: Briefly, the kinase reaction consisted of 5 ng of recombinant human active full-length PKCα protein (Millipore, catalog no. 14-484), 200 or 300 ng recombinant human Twist1 protein (Abcam, catalog no. ab132349), PKC lipid activator (0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 4 m m MOPS, pH 7.2, 5 m m β-glycerol phosphate, 0.2 m m sodium orthovanadate, 0.2 m m DTT, 0.2 m m CaCl 2 ), 20 m m HEPES, pH 7.4, 20 m m MgCl 2 , 0.1 mg/ml BSA, 0.1 m m CaCl 2 , and 100 μ m ATP.

Techniques: Transfection, Immunoprecipitation, Binding Assay, FLAG-tag, Control

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase Cα–mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it

doi: 10.1074/jbc.RA118.005921

Figure Lengend Snippet: Phosphorylation on Ser-144 of Twist1 promotes its stability. A, schematic representation of predicted PKCα phosphorylation sites on Twist1 adjacent to predicted Twist1 ubiquitination sites. B, location of phosphorylation sites included in this study (Thr-137, Ser-140, Ser-144, and Thr-148) on Twist1 3D structure (green) shown dimerized with another bHLH protein (gray). Note that the phosphorylation sites (yellow stars) are within the loop and accessible on the exterior of the dimer. WT Twist1 or phospho-mutants were transfected in HEK293T cells (C, panels i and ii) or EOC cells clone R182 (D, panels I and ii) as indicated; the levels of Twist1 protein were determined by Western blotting analysis; and the resulting bands were quantified by densitometry. Densitometry readings are reported as fold changes compared with empty vector control. *, p = 0.0126; **, p = 0.0021. E, WT Twist1 (panel i), phospho-deficient on 144 (144A, panel ii), or phosho-mimic on 144 (144D, panels iii) were transfected in HEK293T cells and treated with cyclohexamide (CHX, 20 μg/ml). The levels of Twist1 protein were determined by Western blotting, and the resulting bands were quantified by densitometry; densitometry readings are reported as fold changes compared with empty vector control. **, p = 0.0096. GAPDH was used as loading control.

Article Snippet: Briefly, the kinase reaction consisted of 5 ng of recombinant human active full-length PKCα protein (Millipore, catalog no. 14-484), 200 or 300 ng recombinant human Twist1 protein (Abcam, catalog no. ab132349), PKC lipid activator (0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 4 m m MOPS, pH 7.2, 5 m m β-glycerol phosphate, 0.2 m m sodium orthovanadate, 0.2 m m DTT, 0.2 m m CaCl 2 ), 20 m m HEPES, pH 7.4, 20 m m MgCl 2 , 0.1 mg/ml BSA, 0.1 m m CaCl 2 , and 100 μ m ATP.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Western Blot, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase Cα–mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it

doi: 10.1074/jbc.RA118.005921

Figure Lengend Snippet: PKCα-induced Ser-144 phosphorylation on Twist1 inhibits Twist1 ubiquitination and promotes stability. A, WT Twist1 or 144D phosphomimetic mutant were co-transfected in HEK293T cells with HA-tagged ubiquitin. Twist1 was subsequently immunoprecipitated, and the levels of ubiquitinated Twist1 were determined by Western blotting for HA. Ubiquitinated Twist1 appears as a smear on the HA blot. Note less ubiquitination in Ser-144 mutant compared with WT Twist1. Whole cell lysates were used as input control and GAPDH as loading control. B, phospho-deficient 144A, or phosphomimetic 144D were co-transfected in HEK293T cells with constitutively active PKCαCAT or empty vector to control for total amount of plasmids transfected. The levels of Twist1 protein detected by Western blotting. C, quantified by densitometry reported as fold changes compared with 144A. *, p = 0.04. D, WT Twist1 or 144D phosphomimetic mutant were co-transfected in HEK293T cells with PKCαCAT. Twist1 was subsequently immunoprecipitated, and phosphorylated Twist1 was detected by Western blotting using anti–phospho-(Ser/Thr)Phe antibody. Whole cell lysates were used as input control. The experiments were performed at least three independent times. Representative data are shown.

Article Snippet: Briefly, the kinase reaction consisted of 5 ng of recombinant human active full-length PKCα protein (Millipore, catalog no. 14-484), 200 or 300 ng recombinant human Twist1 protein (Abcam, catalog no. ab132349), PKC lipid activator (0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 4 m m MOPS, pH 7.2, 5 m m β-glycerol phosphate, 0.2 m m sodium orthovanadate, 0.2 m m DTT, 0.2 m m CaCl 2 ), 20 m m HEPES, pH 7.4, 20 m m MgCl 2 , 0.1 mg/ml BSA, 0.1 m m CaCl 2 , and 100 μ m ATP.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase Cα–mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it

doi: 10.1074/jbc.RA118.005921

Figure Lengend Snippet: Proposed model of PKCα-dependent Twist1 stabilization. A, Twist1 is a target for ubiquitination around the loop region. Twist1 3D structure is shown in green dimerized with another bHLH protein shown in gray. B, active PKCα binds to Twist1 via Twist1's WR domain and phosphorylates Twist1 on Ser-144 located in the loop region. This phosphorylation precludes ubiquitination, leading to Twist1 stabilization.

Article Snippet: Briefly, the kinase reaction consisted of 5 ng of recombinant human active full-length PKCα protein (Millipore, catalog no. 14-484), 200 or 300 ng recombinant human Twist1 protein (Abcam, catalog no. ab132349), PKC lipid activator (0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 4 m m MOPS, pH 7.2, 5 m m β-glycerol phosphate, 0.2 m m sodium orthovanadate, 0.2 m m DTT, 0.2 m m CaCl 2 ), 20 m m HEPES, pH 7.4, 20 m m MgCl 2 , 0.1 mg/ml BSA, 0.1 m m CaCl 2 , and 100 μ m ATP.

Techniques: Ubiquitin Proteomics, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: PKC-mediated phosphorylation of nuclear lamins at a single serine residue regulates interphase nuclear size in Xenopus and mammalian cells

doi: 10.1091/mbc.E16-11-0786

Figure Lengend Snippet: Altering PKC activity in cultured mammalian cells influences nuclear size . Representative images for the cell lines, with visualization methods indicated to the left. HeLa cells stably express H2B-GFP, whereas the Ptk2 and MRC-5 nuclei were visualized either with Hoechst or H2B-mCherry introduced by cotransfection. Cross-sectional nuclear area was quantified for >150 nuclei per sample, averaged, and normalized to the relevant control (set at 1.0). (A) Nearly confluent cells grown on glass coverslips were treated with a PKC activator (6 nM PMA) or cPKC inhibitor (2 µm Gö 6976; inhibits PKC α and β) for 90 min. Cumulative data from four independent experiments. Gö 6976 was used for these experiments rather than chelerythrine, which is known to disrupt cell adhesion ( Zimmerman et al. , 2004 ; Tan et al. , 2011 ). (B) Cells were transiently cotransfected with plasmids expressing constitutively active cPKC α or βII and H2B-mCherry. Cells were fixed and imaged, using H2B-mCherry fluorescence to identify transfected cells. Cumulative data from three independent experiments. (C) Cells were transiently cotransfected with siRNA against the indicated PKC isoforms and a plasmid expressing H2B-mCherry. Cells were fixed and imaged, using H2B-mCherry fluorescence to identify transfected cells. The PKC ζ isoform was used as a negative control, as its expression is exclusively neuronal. Cumulative data from three independent experiments. These siRNA oligos designed against human PKC sequences were ineffective in Ptk2 rat-kangaroo cells. Bars, 10 µm. *** p < 0.005; ** p < 0.01; * p < 0.05; N/S, not significant. Errors bars represent SD.

Article Snippet: We used mammalian expression vectors (pHACE) expressing constitutively active human PKC α (21233; Addgene) and mouse PKC βII (16383; Addgene).

Techniques: Activity Assay, Cell Culture, Stable Transfection, Cotransfection, Control, Expressing, Fluorescence, Transfection, Plasmid Preparation, Negative Control