Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 2. Recombinant GST-HIF-1 is phosphorylated in vitro by p42 MAPK. A, recombinant GST-HIF-1 was incubated without (lanes 1 and 3) or with (lanes 2 and 4) 100 units of recombinant p42 MAPK kinase for 1 h at 30 °C in phosphorylation buffer without (lanes 1 and 2) or with [-32P]ATP (lanes 3 and 4) and analyzed by 10% SDS-PAGE followed by silver staining (lanes 1 and 2) or autoradiography (lanes 3 and 4). The slower migrating GST-HIF-1 band in lane 2 coincides with the radioactive band in lane 4. Numbers indicate the positions of molecular mass markers (in kDa). B, lanes 1 and 2 same as in A but analyzed by 8% SDS-PAGE followed by Western blot with anti-HIF1.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Recombinant, In Vitro, Incubation, Phospho-proteomics, SDS Page, Silver Staining, Autoradiography, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 3. Distinct domains of HIF-1 are targeted by p42 MAPK and another HeLa nuclear kinase. A, relative phosphorylation level of GST-HIF-1 and its deletion mutants used as substrates for phosphoryla- tion by either p42 MAPK or HeLa nuclear extracts and analyzed as shown in C and D. Values are means of three independent experiments. Schematic representation of the mutant inserts is shown on the right. B, Coomassie BluestainingfollowingSDS-PAGEoftheindicatedGST-taggedHIF-1fragments.Theamountsofrecombinant protein analyzed in the gel were also used for the phosphorylation assays shown in C and D. C and D, autora- diography following SDS-PAGE of the indicated HIF-1 forms phosphorylated by 50 units of recombinant p42 MAPK (C) or 2 g of HeLa nuclear protein extract (D) for 30 min at 30 °C. Dots indicate the position of the recombinant GST-tagged HIF-1 fragments.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Mutagenesis, SDS Page, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 4. Ser-641 and Ser-643 of HIF-1 are p42 MAPK phosphorylation sites. A, MS/MS spectrum of the (M 2H)2 747.4 m/z ion, corresponding to phosphorylated ILIASPSPTHIHK peptide. Fragment 698.4 represents loss of H3PO4. B, schematic representation of HIF-1 showing the position of the peptide sequence identified as a phosphopeptide by mass spectrometry in A and alignment of corresponding amino acid sequences from vertebrate HIF-1 homo- logues. Arrows indicate the mutation of the candidate phosphoacceptor serine residues into alanines. C, GST-HIF-1 or its point mutants (2 g each) as indicated were subjected to in vitro phosphorylation by either 50 units of recombinant p42 MAPK (left panels) or 2 g of HeLa nuclear protein extracts (right panels) and analyzed by SDS-PAGE followed by Coomassie Blue staining (upper panels) or autoradiography (lower panels). D, same as in C but the nuclear protein extracts used were prepared from cells treated with 50 M PD98059 for 5 h or left untreated as indicated. Only the relevant parts of the gels are shown. The numbers under each lane represent relative phosphorylation levels measured as described under “Experimental Procedures” and are means of three independent experiments.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Mass Spectrometry, Mutagenesis, In Vitro, Recombinant, SDS Page, Staining, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 5. Mutations in the MAPK phosphorylation sites of HIF-1 render itinactiveinHeLacells.A,Westernblotanalysisofimmunoprecipitatesfrom HeLa cells transfected with GFP alone (lane 1), wild-type GFP-HIF-1 (lanes 2 and 3), or its phosphorylation-deficient mutants (lanes 4–6) using anti-GFP antibodies for immunoprecipitation and anti-HIF-1 or anti-GFP antibodies forblottingasindicated.Panelsshowonlytherelevantpartsoftheblot:upper two panels for GFP-HIF-1; lower panel for GFP. In all cases cells were treated with MG132 and in one case (lane 3) PD98059 was also added. B, HIF-1 tran- scriptional activity determined 24 h after transfection of HeLa cells with the pGL3–5HRE-VEGF reporter plasmid, CMV-lacZ, and pEGFP-C1 plasmids carry- ing the indicated inserts. Values determined as a ratio of firefly luciferase activity over -galactosidase activity are expressed in relation to the results obtained from cells expressing GFP alone and represent the mean of four different experiments performed in triplicate (S.E.). In one case (HIF-1 PD) cells were treated with PD98059. C, Western blotting analysis of cytoplas- mic (cyt) and nuclear (nuc) extracts prepared from HeLa cells treated on not with PD98059 under the identical conditions as in B, using an antibody against MAPK (upper panel) or against its phosphorylated active form (lower panel). Only the relevant parts of the blots are shown.

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Activity Assay, Plasmid Preparation, Luciferase, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 8. Treatment with LMB restores the transcriptional activity of the HIF-1 MAPK site mutants. Transcriptional activity of GFP- (A) or GAL4-DBD (B) fusion constructs of wild-type HIF-1 or its SDMA mutant determined 24 h after transfection of HeLa cells together with the corresponding reporter and control plas- mids and 4 h incubation in the absence or presence of 20 ng/ml LMB as indicated. Values (relative luciferase units, RLU), determined as a ratio of firefly over Renilla luciferase activity in abstract units, represent the mean of two independent experiments performed in triplicate (S.E.).

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Activity Assay, Construct, Mutagenesis, Transfection, Control, Incubation, Luciferase

Journal: Journal of Biological Chemistry

Article Title: Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

doi: 10.1074/jbc.m605058200

Figure Lengend Snippet: FIGURE 9. A proposed model for the regulation of the subcellular distri- butionandactivationofHIF-1byaMAPK-andCRM1-dependentmech- anism. HIF-1 that enters the cell nucleus after stabilization is rapidly exported back to the cytoplasm by a CRM1-dependent mechanism. Activa- tion of the MAPK pathway causes phosphorylation of HIF-1-Ser-641/643 by p44/22 MAPK and inhibition of its export. Thus, HIF-1 accumulates insides the nucleus, heterodimerizes with ARNT, binds to HRE sequences in the pro- moter/enhancer regions of its target genes, and stimulates transcription by interacting with co-activators such as CBP/p300. This interaction can be fur- ther enhanced by MAPK-dependent phosphorylation of CBP/p300 (21).

Article Snippet: SDS-PAGE and Western Blot—Proteins were resolved by 8% SDS-PAGE, and analyzed by Coomassie Blue or Western blotting using an anti-HIF-1 mouse monoclonal antibody (BD Transduction Laboratories), a rabbit polyclonal antiGFP serum generously provided by Dr. H. Boleti (Hellenic Pasteur Institute, Athens, Greece), or antibodies against p44/42 MAP kinase and phospho-p44/42 MAP kinase (Cell Signaling, Beverly, MA).

Techniques: Phospho-proteomics, Inhibition

Journal: iScience

Article Title: Mitochondria-targeted cancer analysis using survival and expression: Prioritizing mitochondrial targets that alleviate pancreatic cancer cell phenotypes

doi: 10.1016/j.isci.2024.110880

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Recombinant, Modification, Ligation, Transfection, Infection, Electron Microscopy, Protease Inhibitor, Negative Control, shRNA, Sequencing, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Regulation of MYC by CARD14 in human epithelium is a determinant of epidermal homeostasis and disease

doi: 10.1016/j.celrep.2024.114589

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ERK (rabbit) (WB (1:1000)) , Cell Signaling Technologies, Danvers, MA , Cat# 4695; RRID: AB_390779.

Techniques: FLAG-tag, Plasmid Preparation, Recombinant, Modification, Transfection, Staining, Protease Inhibitor, Blocking Assay, Western Blot, Stripping, XF Assay, Reporter Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Sequencing, Cloning, Mutagenesis, Software, Membrane

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-p38 antibody Clone D13E1 , Cell Signaling Technology , Cat# 8690s; RRID:AB_10999090.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Reverse Transcription, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Gene Expression, CRISPR, Software, Sequencing, Modification

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Boxplots of growth phenotypes for PC9-Cas9-mCherry cells expressing the indicated pgRNA compared to PC9-Cas9-GFP cells expressing a double-safe-targeting control pgRNA. Boxes indicate mean ± SEM of six biological replicates, which are shown as overlaid points. Growth phenotype is defined as the log 2 -scaled ratio of mCherry:GFP cell counts at the late time point compared to the day 1 mCherry:GFP cell counts. Expected DKO phenotypes are the sum of single KO growth phenotypes. The expected and observed DKO phenotypes were compared using a one-tailed t test. Data shown are for the time point with the most extreme difference between expected and observed DKO growth phenotypes, termed the late time point: CCNL1/CCNL2 (day 12), CDK4/CDK6 (day 7), MEK1/MEK2 (day 11), and OXSR1/STK39 (day 10). Full time course data are shown in . (B) Fluorescence microscopy images of competitive fitness assays on early (day 1) and late time points as indicated above for (A). Scale bar, 100 μM. (C) Western blot validation of single KO and DKO pgRNA-induced gene inactivation. For CCNL1, pie charts of percent mutant alleles based on next-generation sequencing are shown due to lack of a suitable CCNL1 antibody for western blotting. Additional genomic DNA-level validation data are presented in . See also and and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Expressing, Control, One-tailed Test, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Mutagenesis, Next-Generation Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Rank plot of target-level GI scores in HeLa cells. Table insert, top synthetic lethal paralogs based on GI score. (B) Volcano plot of target-level GI scores in HeLa cells. FDR indicates the multiple hypothesis-adjusted p values from a two-tailed t test . Blue, synthetic lethal paralog GIs with GI < −0.5 and FDR < 0.1; red, buffering paralog GIs with GI > 0.25 and FDR < 0.1. (C) Scatterplot of target-level GI scores for paralog pairs in PC9 versus HeLa cells. Blue, synthetic lethal paralog pairs with GI < −0.5 and FDR < 0.1 in either PC9 or HeLa cells; gray, all paralog pairs with GI ≥ −0.5 or FDR ≥ 0.1. (D) CRISPR scores for representative synthetic lethal paralog pairs identified in the PC9 and HeLa cell screens. Top row: data shown are the mean CRISPR score for each single KO or DKO target across three biological replicates with replicate data shown in overlaid points. Shared synthetic lethal paralogs (e.g., CCNL1/CCNL2 and MEK1/MEK2 ) have FDR < 0.1 in both cell lines; PC9-specific paralogs (e.g., CDK4/CDK6 and OXSR1/STK39 ) have FDR < 0.1 in PC9 only; and HeLa-specific paralogs (e.g., GFTP1/GFPT2 and SOS1/SOS2 ) have FDR < 0.1 in HeLa only. Dashed lines indicate CRISPR score < −0.5. Bottom row: paralog gene expression in PC9 and HeLa cells from RNA-seq analysis. Dashed lines indicate log 2 (TPM) = 1, the threshold for gene expression. (E) Boxplots comparing the effect of CRISPR-mediated KO of the indicated gene in DepMap cell lines with high (top quartile) compared to low (bottom quartile) copy number of its paralogous gene. For boxplots, the middle line, hinges, notches, and whiskers indicate the median, 25th/75th percentiles, 95% confidence interval, and data points within 1.5× the interquartile range from the hinge, respectively. p values were computed using a two-tailed Wilcoxon rank-sum test. CRISPR score and copy number data were obtained from DepMap. (F) As in (E), but for gene expression. (G) Bar plot indicating the p values (computed using a two-tailed Wilcoxon rank-sum test) obtained by comparing the effect of a single paralog KO to the copy number (as in E) or gene expression (as in F) of its pair across human cancer cell lines profiled by DepMap. Bar color indicates whether each pair was synthetic lethal in PC9 only, HeLa only, or both cell lines in the pgPEN screens. Dashed line indicates p = 0.05. See also and , , and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Two Tailed Test, CRISPR, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Software

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Example immunoblots and summary data of dose dependent decline in ET-1 induced S6K and RSK2 phosphorylation by ERK1/2 inhibition (SCH) in NRVMs. Concomitant ERK1/2 phosphorylation itself did not decline, whereas Akt phosphorylation rose with increasing SCH dose (0.01–10 μM). N = 6/group; **** P < 1 × 10 −4 , *** P ≤ 0.0006, ** P ≤ 0.006, * P = 0.01; Welch one-way ANOVA, Dunnett’s multiple comparisons test (MCT). (B) Inhibition of Akt does not significantly reduce p/t S6K stimulated by ET-1. N = 6/group; Kruskal–Wallis test P < 0.0007 for each; Dunn’s MCT: ** P = 0.002; * P < 0.026. (C) Expression of TSC2 S1364E (SE) reduces ET-1–stimulated S6K in a dose-dependent manner; ERK1/2 activity is unchanged, and Akt activity rises with higher SE expression. N = 4/group; one-way ANOVA ( P ≤ 3 × 10 −10 for each protein), Sidak’s MCT: **** P < 10 −5 ; *** P < 0.0002. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Western Blot, Inhibition, Expressing, Activity Assay

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Effect of ERK-1/2 inhibitor U0216 on ET-1–stimulated response. NRVMs were stimulated with ET-1 in presence or absence of ERK-1/2 inhibitor U0216. (n = 6/group). Summary analyzed by Welch one-way ANOVA (variance differences between groups) and multiple comparisons Dunnett’s test P -values displayed. (B) Effect of TSC2S S1364E (SE) expression on RSK2 activation. NRVMs infected with WT or SE form of TSC2, exposed to ET-1 ± ERK1/2 inhibitors. (n = 6/group). Summary data analyzed by one-way-ANOVA, Dunnett’s multiple comparisons test P -values shown.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Expressing, Activation Assay, Infection

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A, B) Example immunoblots and (B) summary data showing ET-1–stimulated S6K in NRVMs is augmented by expression of TSC2 S1364A (SA) without altering ERK1/2 phosphorylation. This stimulation is blocked by ERK1/2 (SCH) or mTOR (rapamycin, Rapa) inhibition. N = 4/group; P -values for two-way ANOVA for genotype and interaction of genotype and condition. **** P < 0.0001 by Sidak’s MCT. (C) Effect of ET-1 stimulation on S6K and ERK1/2 phosphorylation in NRMVs co-expressing TSC2 S1364A and PKG1α C42S and then exposed to vehicle (Veh), PDE5 (P5-i) (1 μM), or PDE9 (P9-i) (0.1 μM) inhibition, or soluble guanylate cyclase activation (GC-1) (0.1 μM). N = 6/group; Welch one-way ANOVA, Dunnett’s MCT; *** P ≤ 0.001; **** P = 0.00006. (D) Combined analysis of data from and bi-normalized to span from 1.0 with vehicle control to 4.0 for peak p/t S6K response. Data show strong correlation between p/t S6K and p/t ERK1/2 but not p/t Akt. P -values are for linear regression of each respective relation. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Western Blot, Expressing, Inhibition, Activation Assay

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Insulin-stimulated pS6K in MEFs is not altered by ERK1/2 inhibition but is by Akt inhibition; example immunoblot (left) and summary data (right) shown. N = 6/group; one-way ANOVA, Holm–Sidak MCT: ** P ≤ 0.004, *** P = 0.0002; ****P ≤ 2 × 10 −5 . (B) Example immunoblot and summary results for insulin stimulation in TSC2 KO MEFs infected with AdV expressing empty vector (KO), or WT, SA, or SE TSC2. N = 12/group; two-Way ANOVA, Sidak’s MCT; *P < 2 × 10 −7 versus Vehicle KO; †P < 3 × 10 −7 versus KO+ insulin; ‡P ≤ 6 × 10 −7 versus KO+ insulin. (C) Insulin-stimulated S6K is fully blocked by rapamycin (Rapa) or torkinib (Tork), contrasting to effect from SE mutant. N = 6/group; Welch one-way ANOVA, Dunnett’s test; * P < 0.0003 versus other groups. (D) PDGF stimulation potently activates Akt and S6K, and this response is not significantly altered by TSC2 mutants versus WT. N = 6/group; two-way ANOVA, P = 3 × 10 −12 for PDGF effect, 0.7 for genotype effect, and 0.8 for genotype–PDGF interaction. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Inhibition, Western Blot, Infection, Expressing, Plasmid Preparation, Mutagenesis

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Example immunoblot of MEFs exposed to thrombin showing balanced activation of ERK1/2 and Akt. (B) Example immunoblot and summary data for TSC2 KO MEFs and cells with WT TSC2 re-expressed, exposed to thrombin ± Akt inhibition (MK2206). N = 4/group TSC2-KO; N = 11/group WT. P -values displayed are for Kruskal–Wallis test, **** P = .00009, ** P = 0.009 by Dunn’s MCT. (C) Example immunoblot and summary data for the same experiment but ± ERK1/2 inhibitor (SCH). N = 6/group; KW test P -values displayed; Dunn’s MCT: † P = 0.1, ** P = 0.007. (D) Example immunoblot for KO MEFs re-expressing either TSC2-WT or TSC2 S1364E , stimulated with thrombin ± Akt inhibitor (MK2206). (E) Percent rise in p/t S6K due to thrombin in WT versus SE TSC2 expressing KO MEFs. N = 7–8/group; P -value displayed Mann–Whitney U test. (F) Effect of Akt inhibition on p/t S6K response to thrombin in TSC2-KO MEFs expressing WT or SE TSC2. N = 8/group; two-way ANOVA, Sidak’s MCT— P -values displayed. (G) Effect of ERK1/2 inhibition by SCH772984 on p/t S6K in TSC2-KO MEFs expressing WT or TSC2 S1364E . two-way ANOVA with Sidak’s MCT P -values shown. † P = 0.04 versus WT; * P = 0.022 versus SE with vehicle controls. (H) Differential effect of ET1 or insulin stimulation on TSC2 phosphorylation at S1364 N = 6/group; one-way ANOVA, Sidak’s MCT. * P = 0.012; ** P = 0.001; **** P = 0.00008. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Western Blot, Activation Assay, Inhibition, Expressing, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: A G q,11 – GPCR typified by ET-1 receptor and tyrosine kinase receptor typified by insulin receptor, and mixed receptor (e.g., thrombin) are depicted with their downstream signaling. ET-1 prominently activates PLCβ-PKC-ERK1/2 and Ras-ERK1/2 pathways leading TSC2 phosphorylation reducing its suppression of mTORC1 to increase p/t S6K. Insulin more prominently activates PI3K-Akt, phosphorylating TSC2 at different sites but also leading to mTORC1 activation. Thrombin engages both cascades. TSC2 S1364—modified by mutagenesis to block (S1364A, SA) or mimic (S1364E, SE) its phosphorylation—alters co-modulation of mTORC1 due to ERK1/2 but not to Akt signaling via TSC2. In vivo examples of this bias is found by marked impact of S1364 modulation on cardiac pressure overload as reported , but negligible effect on high-fat diet obesity and metabolic syndrome observed in the current study.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Activation Assay, Modification, Mutagenesis, Blocking Assay, In Vivo

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: A A schematic diagram of the Hog1 MAPK signaling pathway. B Analyses of Hog1 phosphorylation by immunoblotting with anti‐phospho‐p38 (Hog1‐P) and anti‐Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207, and KY594‐1. C Analyses of Hog1 phosphorylation by Phos‐tag band‐shift assay. Yeast strain KY594‐1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 (Hog1‐P [%]) were calculated as explained in Materials and Methods and are shown beneath the panel. D Analyses of Hog1 phosphorylation by immunoblotting with anti‐phospho‐p38 (Hog1‐P) and anti‐Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single‐copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild‐type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (−) 1 M NaCl for 5 min. E–H Analyses of Hog1 phosphorylation by Phos‐tag band‐shift assay. Yeast strains (E) KY603‐3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. I Comparison of the NaCl dose–responses of Hog1 activation by various strains. Phos‐tag band‐shift assays shown in (C and E–H) were independently repeated three times, and average values were plotted. Data information: (C and E–H) Representative results from three independent experiments. (I) Error bars are SEM ( n = 3). Source data are available online for this figure.

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Electrophoretic Mobility Shift Assay, Transformation Assay, Mutagenesis, Plasmid Preparation, Incubation, Comparison, Activation Assay

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: A Alignment of the amino acid sequences of the CD (green) and L16 (pink) domains of yeast Hog1 and mammalian p38α. The alpha helix αL16 forms the core of the L16 domain (Wang et al , ). B Schematic diagrams of Hog1‐WT and its deletion constructs used in this study. C Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416‐FLAG‐Hog1 (WT) or its indicated deletion derivatives. FLAG‐Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti‐phospho‐p38 (for Hog1‐P; upper panel) or anti‐FLAG (for total FLAG‐Hog1; lower panel) D–G Phos‐tag band‐shift assay of Hog1 phosphorylation. Yeast strain (D and E) KT235 or (F and G) KT290 carrying the single‐copy expression plasmid YCplac22I’‐Pbs2 S514D/T518D was transformed with either pRS416‐Hog1 (WT) or its indicated mutant derivatives and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarize the averages of three independent experiments. H Phos‐tag band‐shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single‐copy expression plasmid pRS416‐Hog1 (WT) or pRS416‐Hog1‐ΔL16 and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: (E, G, and H) Error bars are SEM ( n = 3). Source data are available online for this figure.

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Construct, Western Blot, Phospho-proteomics, Transformation Assay, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Expressing, Plasmid Preparation, Mutagenesis

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: KT299 ( MAT a hkr1 Δ msb2 Δ ssk2 / 22 Δ) was exposed to the indicated concentrations of α‐factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting. KT299 ( MAT a ssk2 / 22 Δ hkr1 Δ msb2 Δ) was exposed to the indicated concentrations of α‐factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average values of four independent experiments from (B) were plotted. HM06‐1 ( MAT a ΔS/O/H/M ssk2 / 22 Δ) was exposed to the indicated concentrations (log scale) of α‐factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average values of three or more independent experiments were plotted. KT306 ( MAT a hkr1 Δ msb2 Δ ssk2 / 22 Δ hog1 Δ) was transformed with pRS416‐Hog1 (WT) or pRS416‐Hog1‐N149H D162G (N/H D/G) and was exposed to 10 μM α‐factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average of three independent experiments is plotted. A schematic model showing that the lack of osmotic enhancement of the Pbs2‐Hog1 reaction prevents the pheromone‐to‐Hog1 crosstalk. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy‐p38 (Tomida et al , ) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in . Distribution of p38 activity in individual cells from sets (a)–(d) in (G). Data information: (B) Representative results from three independent experiments. (C–E) Error bars are SEM: (C) n = 4; (D), n = 3 or more; and (E) n = 3. (G) Scale bars: 20 μm. (H) Statistics, Student's two‐tailed t ‐test. Source data are available online for this figure.

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Electrophoretic Mobility Shift Assay, Transformation Assay, Activation Assay, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: Alignment of the amino acid sequences around the DFG motif of various MAP kinases. The positions of N149 and D162 in Hog1, the β7 and β8 strands, the DFG motif, the activation loop, and activating phosphorylation sites (the TXY motif) are indicated. The sequences of the mouse p38α and yeast Hog1 are highly conserved in this segment (23 residues out of 33 are identical, and the other residues show mostly conservative changes). Sc, Saccharomyces cerevisiae ; Mm, Mus musculus (mouse). The 3D structure of the mouse p38α MAPK (left), and an annotated enlargement of the relevant segment (residues 151–183; right). The corresponding amino acid sequence is shown in (A). Side chains of N155, D168, and activating phosphorylation sites T180 and Y182 are also shown. N155 and D168 correspond to, respectively, the yeast Hog1 residues N149 and D162, whose mutations created the constitutively enhanced phenotype. The coordinate data were from PDB (ID 5UOJ) (Wang et al , ) and were visualized using the MOLMOL program (Koradi et al , ). The 3D structure of the mouse p38α MAPK showing the spatial relationship between the L16 domain and the DGF motif. Four side views of the mouse p38α are shown, each of which was rotated 90° from the previous one around the vertical axis. Following segments are highlighted by coloring: the DFG motif (brown), the CD domain (green), and the L16 domain (pink).

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Activation Assay, Phospho-proteomics, Sequencing

Journal: Cell reports

Article Title: EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR-activated lung cancer

doi: 10.1016/j.celrep.2022.111827

Figure Lengend Snippet: (A) Phosphorylation levels altered by GDH1 and RSK2 knockdown. Human phosphorylation pathway profiling array results were obtained using 55 antibodiesdetecting AKT, JAK/STAT, MAPK, NF-κB, and TGF-β signaling in A549 lysates. (B) A549 and H157 cells with GDH1 and RSK2 knockdown were cultured under attached or detached conditions and assayed for CREB, AKT, and ERK1/2 phosphorylation by immunoblotting. GDH1, RSK2, and β-actin blots were obtained from attached conditions, and similar stable knockdown efficacy was observed in detached conditions. (C) Effect of RSK2 and GDH1 knockdown on CREB activity was assessed by CREB transcription factor assay. Nuclear extracts from the detached A549 and H157 cells were incubated with a specific CRE consensus sequence, and the activated CREB-CRE complex was quantified by phospho-CREB S133 ELISA. (D) RNA levels of CREB transcription targets PTK6, ING3, and Fascin-1 in RSK2 and GDH1 knockdown cells were determined by quantitative RT-PCR. (E) Effect of CREB phosphorylation-mimetic mutant S133D (SD) or -deficient mutant S133A (SA) expression on cell invasion and anoikis resistance in GDH1 and RSK2 knockdown cells. GDH1 and RSK2 double knockdown cells were overexpressed with myc-tagged CREB SD or SA mutants, and invasive and anoikis resistant potentials were determined by Matrigel cell invasion assay and annexin V staining. (F) Effect of p38 or CREB inhibitors on p38 and CREB activity. A549 cells were treated with 5 μM BIRB 796 (p38 inhibitor) or 100 nM 666–15 (CREB inhibitor) for 24 h. The activities of p38 and CREB were assessed by p38 T180/Y182 and CREB S133 phosphorylation. (G and H) Effect of CREB S133D overexpression or 10 μM of p38 activator U-46619 on cell invasion and anoikis resistance in fmk- and R162-treated cells. A549 cells were treated with CREB S133D and/or U-46619 for 24 h, and invasive and anti-anoikis potentials were determined as in (E). Western blot results shown are representative of four (B) and two (F) independent biological experiments. Error bars represent ±SD from two replicates for (A) and three replicates for the others. p values were obtained by one-way ANOVA (ns, not significant; *0.01 < p < 0.05; **p < 0.01). See also – .

Article Snippet: Rabbit polyclonal anti-p38 antibody , Cell Signaling Technology , Cat#9212; RRID: AB_330713.

Techniques: Phospho-proteomics, Knockdown, Cell Culture, Western Blot, Activity Assay, Transcription Factor Assay, Incubation, Sequencing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Mutagenesis, Expressing, Invasion Assay, Staining, Over Expression

Journal: Cell reports

Article Title: EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR-activated lung cancer

doi: 10.1016/j.celrep.2022.111827

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-p38 antibody , Cell Signaling Technology , Cat#9212; RRID: AB_330713.

Techniques: Microarray, Recombinant, Purification, Membrane, SYBR Green Assay, Viability Assay, Phospho-proteomics, Kinase Assay, Reverse Transcription, Transcription Factor Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Extraction, Isolation, shRNA, Sequencing, Plasmid Preparation, Software