Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: (A) GNAQQ209L transfected alone or combined with PKC δ or ε into 293FT cells increased Ras-GTP levels. 293FT cells were transfected with indicated cDNA plasmids for 24 hr. Ras-GTP pull-down was performed and detected by western blot with a pan-Ras antibody. TPA stimulation was used a positive control. Expression levels of GNAQQ209L were monitored with Glu-Glu (EE) tag. PKC δ and ε were detected via HA tags.

Article Snippet: cDNA: HA-PKC α (catalytic domain) , addgene , 21234.

Techniques: Transfection, Western Blot, Positive Control, Expressing

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: cDNA: HA-PKC α (catalytic domain) , addgene , 21234.

Techniques: Recombinant, Activation Assay, Microarray, Fractionation, Expressing, Cell Culture

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: pHACE-PKC-α-CAT (PKCαCAT) (Addgene, catalog no. 21234), pHACE-PKC-α-DN (PKCαDN) (Addgene, catalog no. 21235) and pHACE-PKC-α-WT (PKCαWT) (Addgene, catalog no. 21232) plasmids were gifts from Bernard Weinstein ( 25 ).

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: pHACE-PKC-α-CAT (PKCαCAT) (Addgene, catalog no. 21234), pHACE-PKC-α-DN (PKCαDN) (Addgene, catalog no. 21235) and pHACE-PKC-α-WT (PKCαWT) (Addgene, catalog no. 21232) plasmids were gifts from Bernard Weinstein ( 25 ).

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: pHACE-PKC-α-CAT (PKCαCAT) (Addgene, catalog no. 21234), pHACE-PKC-α-DN (PKCαDN) (Addgene, catalog no. 21235) and pHACE-PKC-α-WT (PKCαWT) (Addgene, catalog no. 21232) plasmids were gifts from Bernard Weinstein ( 25 ).

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: 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: pHACE-PKC-α-CAT (PKCαCAT) (Addgene, catalog no. 21234), pHACE-PKC-α-DN (PKCαDN) (Addgene, catalog no. 21235) and pHACE-PKC-α-WT (PKCαWT) (Addgene, catalog no. 21232) plasmids were gifts from Bernard Weinstein ( 25 ).

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