Journal: Journal of Biological Chemistry

Article Title: Molecular Identification and Characterization of a Family of Kinases with Homology to Ca2+/Calmodulin-dependent Protein Kinases I/IV

doi: 10.1074/jbc.m513212200

Figure Lengend Snippet: FIGURE7.CL2-inducedsuppressionofCREBactivityismediatedbyaphosphor-Ser-171-TORC2-depend- ent mechanism. A, kinase activity of CL2 favored nuclear export/cytoplasmic retention of TORC2. Under our basal culture condition, TORC2-GFP was found to be largely concentrated in the nucleus of COS-7 cells. How- ever, co-expression of an active form of CL2 (Dx-CL2C) potently inhibited nuclear import of TORC2-GFP and increased its cytoplasmic content (arrowheads). Scale bar, 20 m. B, monitoring of total fluorescence values of TORC2-GFPrevealedasignificanttransferofTORC2proteinfromthenucleustothecytosolinaCL2-dependent manner, as measured by a shift in cytosol/nucleus ratio. **, p 0.01 (two-tailed Student’s t test). C, CL2-induced suppression of CREB activity was eliminated by a phosphorylation-deficient TORC2 mutant. An active (Dx- CL2C), but not a kinase-dead (Dx-CL2KD) CL2 is able to induce a significant reduction in CRE-dependent luc reporter activity in the presence of wild-type (WT) TORC2. However, such kinase-dependent inhibition of CRE-mediated transcription is abolished in the presence of a phosphorylation-deficient TORC2 mutant (TORC2-S171A). Remarkably, a kinase-active form of CaMK2, CaMK2C, can elicit inhibition of CRE-depend- ent transcription, irrespective of the TORC2 status, consistent with the idea that CaMK2-dependent inhibition may occur by a mechanism distinct from that mediated by CL2 and TORC2. Each data point on the graphs representsmeans S.E.calculatedfromtriplicates.Theresultsshownarerepresentativeofmultipleindepend- ent sets of experiments. ***, p 0.001 (one-way ANOVA with Tukey post hoc test).

Article Snippet: To construct TORC2-GFP, an open reading frame of human TORC2 (OriGene clone TC107319) was subcloned by PCR into the EcoRI/SalI sites of pEGFP-C1 (Clontech).

Techniques: Activity Assay, Expressing, Fluorescence, Two Tailed Test, Phospho-proteomics, Mutagenesis, Inhibition

Journal: Archives of biochemistry and biophysics

Article Title: Keap1/Nrf2 pathway activation leads to a repressed hepatic gluconeogenic and lipogenic program in mice on a high-fat diet

doi: 10.1016/j.abb.2015.11.040

Figure Lengend Snippet: Antibodies used in western blot.

Article Snippet: Anti-p-Torc2-Ser171 , BS-3415R , Bioss.

Techniques: Western Blot

Journal: Archives of biochemistry and biophysics

Article Title: Keap1/Nrf2 pathway activation leads to a repressed hepatic gluconeogenic and lipogenic program in mice on a high-fat diet

doi: 10.1016/j.abb.2015.11.040

Figure Lengend Snippet: A. Immunoblotting analysis of p-Ampkα-Thr172, total Ampkα, p-Ampkβ1-Ser108, total Ampkβ1 show that Ampk is activated in the Keap1-hypo mice after 90 days on HFD. Downstream targets of Ampk, Torc2 and Acc1 show increased phosphorylation at Ser171 and Ser79 correspondingly. Total Acc1 levels are lower in the Keap1-hypo mice.

Article Snippet: Anti-p-Torc2-Ser171 , BS-3415R , Bioss.

Techniques: Western Blot

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 2. GIP modulates CREB/TORC2 activity in human adipocytes. Human adipocytes were serum starved in DMEM/Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP or GLP-1 (100 nM) in the presence of insu- lin (1 nM). A: Effects of GIP on phosphorylation of CREB and nuclear localization of TORC2. Nuclear extracts were isolated and Western blot analyses performed with antibodies against phosphorylated Ser133-CREB, CREB, TORC2, and Histone H3. Cyto- plasmic extracts were immunoprecipitated (IP) with phospho-Serine/Threonine followed by immuno- blotting (IB) for TORC2. Input represents one-tenth of total cytoplasmic extracts used in assay. B: Confo- cal microscopy. Human adipocytes were treated as described above. Immunocytochemical staining was performed using antibodies against phospho-CREB (Ser133, green) or TORC2 (red) and nuclei were stained with DAPI (blue). The scale bar indicates 20 m and all imaging data were analyzed using the Northern Eclipse program (ver.6). Shown are repre- sentative of n = 3.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Activity Assay, Phospho-proteomics, Isolation, Western Blot, Immunoprecipitation, Microscopy, Staining, Imaging, Northern Blot

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 3. CREB/TORC2 are functionally involved in the regulation of LPL expression. A: Effects of different CREB constructs on GIP-responsiveness of the LPL promoter. GIPR-HEK-293 cells were cotransfected with an LPL promoter reporter construct (chr8+: 19840052 - 19841249; -992 to +206 of LPL gene, 2 µg), and various CREB constructs or control vector pCMV5 (1 µg). After transfection, cells were treated with insulin (1 nM) plus GIP (100 nM), and the reporter activities are shown as the relative luciferase activity normalized to protein concentration. All data represent three independent experiments, each carried out in triplicate. Signifi cance was tested using ANOVA with Newman-Keuls post hoc test, where ** represents P < 0.05 versus untreated Vector Control, ## represents P < 0.05 versus respective untreated Control. B, C: RNAi-mediated suppression of CREB and TORC2 reduces GIP-stimulated LPL expression. B: Effects of CREB and TORC2 siRNAs on GIP-stimulated LPL protein expression. Human adipocytes were transfected with a pool of three siRNAs for CREB and TORC2 and incubated for 72 h. At 48 h after transfection, cells were treated with in- sulin (1 nM) plus GIP (100 nM) for 24 h and Western blot analyses were performed as described in Materials and Methods, using antibody against phospho-CREB (Serine-133), CREB, TORC2, LPL, Histone H3, and -actin. C. Effects of CREB and TORC2 siRNAs on GIP-stimulated LPL mRNA expression. Human adipo- cytes were treated as described above and real-time RT-PCR performed to quantify LPL mRNA levels; shown as the fold difference versus control normalized to 18S rRNA expression levels. Signifi cance was tested using ANOVA with Newman-Keul’s post hoc test where ** represents P < 0.05 versus Control siRNA, ## represents P < 0.05 versus GIP.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Expressing, Construct, Control, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Protein Concentration, Incubation, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 4. GIP regulates CREB/TORC2 through a pathway involving PI3-K/PKB/AMPK. Human adipocytes were serum starved in DMEM/ Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM). A: Effect of PI3-K inhibition on GIP-mediated decreases in phospho-AMPK Thr172 and nuclear CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PI3-K inhibitors, LY 294002 (40 M) or wortman- nin (400 nM). B: Effect of AMPK modulation on CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of AMPK inhibitor (Compound C, 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]3-pyridin-4-yl- pyrrazolo[1,5-a]pyrimidine, 40 M) or AMPK activator (5-(3-(4-(2-(4-Fluorophenyl)ethoxy)phenyl)propyl)furan-2-carboxylic acid, FPPF, 50 M). AMPK inhibitor or activator was added to cells during 1 h preincubation as well as GIP stimulation. Nuclear/cytoplasmic extracts were isolated from each sample and Western blot analyses were performed. TORC2 and histone H3 blots are from nuclear extracts and phospho-AMPK Thr172 and AMPK blots are from cytoplasmic extracts. C: Effect of AMPK modulation on LPL activity. Human adipocytes were treated as described above and LPL activity was determined as described in Materials and Methods. D: Effect of GIP or GLP-1 on cAMP accumulation. Human adipocytes were treated as described above and incubated with GIP or GLP-1 (100 nM) in the presence of insulin

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Inhibition, Activity Assay, Isolation, Western Blot, Incubation

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 6. GIP increases protein-protein interaction between phospho-CREB and TORC2 in the nucleus. A: Coimmunoprecipitation. Human adipocytes were serum-starved in DMEM/Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM) and in the presence or absence of PI3-K inhibitors, LY 294002 (40 M), wortmannin (400 nM) or AMPK activator (50 M). Nuclear extracts were isolated from each sample and immunoprecipitated (IP) with phospho-CREB (Ser133) followed by immunoblotting (IB) for TORC2. Input represents one-tenth of total nuclear extract used in the coimmunoprecipitation assay. B: Confocal microscopy. Human adipocytes were treated as described above and fi xed following stimulation with GIP (100 nM) in the presence or absence of PI3-K inhibitors or AMPK activator. Immunocytochemical staining was performed using antibodies against phospho-CREB (Ser133, green) or TORC2 (red) and nuclei were stained with DAPI (blue). The scale bar indicates 20 m and all imaging data were analyzed using the Northern Eclipse program (ver.6). Shown are representative of n = 3.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Isolation, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Staining, Imaging, Northern Blot

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 7. Phospho-CREB/TORC2 binds CRE-II se- quence of human LPL promoter. A: Identifi cation of putative CRE sequences in the promoters of several adipocyte-specifi c genes. Potential CREs present in these promoters are indicated by the box-enclosed regions and consensus CRE sequences shown at the top of the fi gure. B, C: Representative gel of a ChIP assay for the binding of phospho-CREB (B) and TORC-2 (C) in the human LPL promoter. Human adipocytes were serum-starved in DMEM/Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the pres- ence of insulin (1 nM). Phospho-CREB and TORC2 were respectively immunoprecipitated from intact chromatin isolated from human adipocytes using anti-phospho-CREB (Ser 133) and anti-TORC2 anti- body. Precipitated DNA fragments were analyzed by PCR using primers fl anking the CRE-I and CRE-II sites in the LPL promoter. An isotype-matched IgG was used as negative control and 1% Input (PCR product of one-hundredth of the total isolated DNA used in the ChIP assay) as positive control, respec- tively. Shown are representative of n = 3.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Binding Assay, Immunoprecipitation, Isolation, Negative Control, Positive Control

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 8. Proposed pathway by which GIP increases LPL activity in adipocytes. GIP receptor interaction results in PI3-K activation, increased phosphorylation of Ser473 and Thr308 in PKB, and reduced phosphorylation of Ser428 in LKB1 and Thr172 in AMPK. An associated de- crease in AMPK phosphorylation leads to dephosphorylation of TORC2, thus allowing increased translocation from the cytoplasm and into the nucleus. PKB also increases phospho-CREB Ser133 levels in the nucleus. TORC2 complexes with phospho-CREB Ser133 in the nucleus and binds to CRE-II of the human LPL promoter, thus turning on the transcriptional machinery for up-regulation of LPL .

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, De-Phosphorylation Assay, Translocation Assay

Journal: Journal of Cell Communication and Signaling

Article Title: The MAPK and PI3K pathways mediate CNTF-induced neuronal survival and process outgrowth in hypothalamic organotypic cultures

doi: 10.1007/s12079-015-0268-8

Figure Lengend Snippet: Pharmacological inhibitors used in this study

Article Snippet: Torin-1 (500 nM) , mTORC1 and mTORC2 , Tocris (R&D Systems) #4247.

Techniques: Concentration Assay, Negative Control