Journal: Neuropharmacology

Article Title: Ciliary neurotrophic factor signaling in the rat orbitofrontal cortex ameliorates stress-induced deficits in reversal learning.

doi: 10.1016/j.neuropharm.2019.107791

Figure Lengend Snippet: Fig. 4. CNTF elicits a p38-dependent protein translation pathway. Rats were either left undisturbed in their home cages (No Stress) or were subjected to CIC stress (CIC), and injected with saline (VEH) or 50 nM CNTF (CNTF) 30 min before collection of OFC tissue. Male rats: CNTF increased pJAK2, pT-Akt, pS9-GSK3β and peIF4E in both non-stressed and stressed animals; CNTF increased pS6K only in non-stressed rats (A, B, C, D, E, *p < 0.05 and **p < 0.01 vs. respective vehicles, n = 4–6). Female rats: CNTF increased pJAK2, pT-Akt, pS9-GSK3β and peIF4E in both non-stressed and stressed animals; CNTF increased pS6K only in non-stressed rats (G, H, I, J, K, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. respective vehicles, n = 4–6). Panels F and L show images of representative western blots. CNTF induces p38 (M) and Mnk1 (P) and this effect is not sensitive to the PI3K inhibitor LY294002 (*p < 0.05, and ***p < 0.001 vs. VEH, n = 4–5). CNTF increases p- p38 and pMnk1 in both non-stressed and stressed male (N, Q) and female rats (O, R, *p < 0.05, **p < 0.01 vs. respective vehicles, n = 4–6). S. Proposed model of CNTF actions. CNTF (50 nM) binding to gp130/LIFR/CNTFRα activates JAK2, which in turns activates PI3K/Akt. Akt phosphorylates and inhibits GSK3β. Akt also inhibits TSC, an inhibitor of mTOR, thereby activating this pathway. mTOR phosphorylates S6K, a regulator of global protein synthesis. This branch is induced by CNTF in non-stressed rats, but it does not seem to be activated in stressed rats. In parallel to regulation of Akt, CNTF also induces the p38/Mnk1/eIF4E cascade that controls specialized mRNA translation. Finally, at 50 nM, CNTF does not seem to activate ERK or STAT3 in the OFC (dotted arrows).

Article Snippet: Subsequently, membranes were stripped with Restore Plus (Fisher Scientific) and re-probed with antibodies against un-phosphorylated STAT3 (mouse mAb 1:2,000, Santa Cruz Biotechnology, sc-8019), JAK2 (rabbit mAb, 1:2,000, CST 3230), Akt (mouse mAb, 1:1,000, CST 2920), GSK3β (mouse mAb, 1:1,000, CST 9832), S6K (rabbit pAb, 1:1,000, CST 9202), eIF4E (rabbit pAb, 1:1,000, CST 9742), ERK (rabbit pAb, 1:5,000, Santa Cruz Biotechnology, sc-94), p38 MAPK (rabbit mAb, 1:2,000, CST 8690), and Mnk1 (mouse mAb, 1:1,000, Novus Biologicals NBP211526), or GAPDH (CST; 1:10,000).

Techniques: Injection, Saline, Western Blot, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: MNK1/2 inhibition limits oncogenicity and metastasis of KIT -mutant melanoma

doi: 10.1172/JCI91258

Figure Lengend Snippet: (A) Western blot analysis of phospho-MNK1 (p-MNK1), MNK1, phospho-eIF4E (p-eIF4E), and eIF4E in a panel of melanoma cell lines. (B) Cell proliferation was assessed by SRB staining, 72 hours after vehicle (DMSO) or 10 nM dasatinib treatment in HBL, MM61, MM111, and M230 melanoma cell lines. (C) Western blot analysis of phospho–C-KIT (p-C-KIT), C-KIT, p-eIF4E, eIF4E, p-MNK1, and MNK1 in HBL, MM111, MM61, and M230 melanoma cell lines, following a 24-hour dasatinib treatment. (D) Cell proliferation was assessed by SRB staining, 96 hours after transfection with KIT siRNAs. (E) Western blot analysis of p-C-KIT, C-KIT, p-eIF4E, eIF4E, p-MNK1, and MNK1 in HBL, MM111, and M230 cell lines transfected with KIT siRNAs, at the indicated time points. (B and D) Data represent the mean ± SD, n = 3. **P < 0.01 by 2-way ANOVA. (A, C, and E) GAPDH used as loading control.

Article Snippet: IHC analysis for phospho-eIF4E (Ser209, Abcam catalog ab76256), phospho-MNK1 (Thr197/202, Cell Signaling Technology catalog 2111), and MNK1 (Sigma-Aldrich, catalog SAB4503423) was conducted on formalin-fixed, paraffin-embedded tumor sections at 1:50 dilution, followed by a standard avidin-biotin detection protocol.

Techniques: Western Blot, Staining, Transfection

Journal: The Journal of Clinical Investigation

Article Title: MNK1/2 inhibition limits oncogenicity and metastasis of KIT -mutant melanoma

doi: 10.1172/JCI91258

Figure Lengend Snippet: (A) Western blot analysis of MNK1, p-eIF4E, and eIF4E in HBL or MM111 cells expressing shCTL and shMKNK1+2 (left). RT-qPCR was performed to examine the expression level of MKNK2 mRNA in HBL and MM111 cells expressing shCTL and shMKNK1+2 (right). (B) Cell migration was assessed by Transwell assay in shCTL versus shMKNK1+2 HBL and MM111 cells after 48 hours. Representative images are shown. Scale bars: 200 μm; original magnification, ×10. (A and B) Data represent the mean ± SD, n = 3. **P < 0.01 by 2-tailed Student’s t test. (C) Western blot analysis of MNK1, p-eIF4E, eIF4E, cyclin E1, and SNAIL in HBL and MM111 shCTL and shMKNK1+2 cell lines. (A and C) GAPDH is used as loading control.

Article Snippet: IHC analysis for phospho-eIF4E (Ser209, Abcam catalog ab76256), phospho-MNK1 (Thr197/202, Cell Signaling Technology catalog 2111), and MNK1 (Sigma-Aldrich, catalog SAB4503423) was conducted on formalin-fixed, paraffin-embedded tumor sections at 1:50 dilution, followed by a standard avidin-biotin detection protocol.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Migration, Transwell Assay

Journal: The Journal of Clinical Investigation

Article Title: MNK1/2 inhibition limits oncogenicity and metastasis of KIT -mutant melanoma

doi: 10.1172/JCI91258

Figure Lengend Snippet: (A) Representative images of p-MNK1 and p-eIF4E IHC staining. Bar graphs of p-MNK1 and p-eIF4E IHC scores in melanoma patients are shown in the right panel. χ2 test, P values shown in the figure. For p-MNK1 and p-eIF4E top panels, scale bars: 200 μm; original magnification, ×4. For p-MNK1 and p-eIF4E bottom panels, scale bars: 40 μm; original magnification: ×20. (B) The correlation between p-eIF4E and p-MNK1 IHC scores in melanoma patients. Pearson correlation test, r and P values are shown.

Article Snippet: IHC analysis for phospho-eIF4E (Ser209, Abcam catalog ab76256), phospho-MNK1 (Thr197/202, Cell Signaling Technology catalog 2111), and MNK1 (Sigma-Aldrich, catalog SAB4503423) was conducted on formalin-fixed, paraffin-embedded tumor sections at 1:50 dilution, followed by a standard avidin-biotin detection protocol.

Techniques: Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: MNK1/2 inhibition limits oncogenicity and metastasis of KIT -mutant melanoma

doi: 10.1172/JCI91258

Figure Lengend Snippet: (A) Chemical synthesis procedure of SEL201. (B) The in vitro ADP-Glo assay was performed by incubation of SEL201 with recombinant MNK1 and MNK2, peptide substrates, and ATP. After the kinase reaction, luminescence intensity generated by the remaining ADP was measured. (C) Kinome selectivity of SEL201 was assessed using KINOMEscan (DiscoverX) panel, which consists of 450 kinases. Circles represent targets that interacted with SEL201 at the concentration of 1 μM. (D) Body weight kinetics of tumor-bearing mice (6 animals per group) was assessed throughout the study (endpoint on day 37). Vehicle or SEL201 was administered p.o. to animals at the dosage of 50 mg/kg twice daily (100 mg/kg/d; mean ± SD). (E) Assessment of blood biochemistry in mice (3 animals per group) given SEL201 at the dosage of 50 mg/kg twice daily (100 mg/kg/d) was performed at the study endpoint (day 37). AST, aspartate aminotransferase; ALT, alanine transaminase; ALP, alkaline phosphatase.

Article Snippet: IHC analysis for phospho-eIF4E (Ser209, Abcam catalog ab76256), phospho-MNK1 (Thr197/202, Cell Signaling Technology catalog 2111), and MNK1 (Sigma-Aldrich, catalog SAB4503423) was conducted on formalin-fixed, paraffin-embedded tumor sections at 1:50 dilution, followed by a standard avidin-biotin detection protocol.

Techniques: In Vitro, Glo Assay, Incubation, Recombinant, Generated, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E *

doi: 10.1074/jbc.C116.740498

Figure Lengend Snippet: MNK1a-Ser353 is a novel AMPK target. A, upper panel, evolutionary conservation of putative MNK1a phosphorylation site. Sequence alignments of MNK1a proteins of human (Q9BUB5), mouse (O08605), rat (Q4G050), bovine (Q58D94), pig (B8XSK1), and Western clawed frog (Q66JF3) (www.uniprot.org) are shown. Lower panel, identification of MNK1a phospho-peptide (aa 351–369; Swiss-Prot: Q9BUB5-2). Intensities of phospho-peptide MS spectra in the presence or absence of ATP (±ATP) are shown. Probabilities for each phosphorylatable residue are indicated between brackets in the aa sequence. B, IVK assays. Purified GST-MNK1a protein was incubated with or without active AMPK in the presence of [γ-32P]ATP. Protease-mediated removal of the GST tag was done as indicated. Upper panel, pMNK1a autoradiograph (autorad). Lower panel, CBB staining image (used as loading control). C, IVK assays as in B, using “cold” ATP instead. Protease-mediated removal of the GST tag was performed as indicated in the figure. Immunoblot (IB) detection of phosphorylated MNK1a using phosphorylated AMPK substrate (AMPK-sub) antiserum or antiserum against total Mnk1 protein (tMNK1) is shown. D, as in C. IVK analyses include: active (WT), kinase-dead (KD), or no AMPK. IB detection was done using the indicated antisera. E, IVK. Purified recombinant GST-MNK1aWT and mutant GST-MNK1aS353A or GST-MNK1aS352A proteins were incubated with active AMPK and [γ-32P]ATP. Upper panel, pMNK1a autoradiograph. Lower panel, CBB image (used as loading control). F, as in C. IVK analyses were performed with cold ATP. IB detection was done using the indicated antisera. G, MNK1a and AMPK protein levels in HeLa cervical adenocarcinoma, U2OS, MEF, TIG3 human primary fibroblasts, HEK293T human embryonic kidney, and HL-1 immortal murine cardiomyocytes. IB analyses of total cell extracts using the indicated antisera were performed. H, U2OS cells expressing 2PY-MNK1aWT or MNK1aS353A (control: empty vector (ev)) were treated as indicated (c: control conditions; ss: serum-starved; AI: ss+AICAR). MNK1a was IP using PY antiserum; IB analysis of immunoprecipitation and input material was performed using the indicated antisera. I, as in C. IVK analyses using purified recombinant GST-MNK1aWT protein, active AMPK, and cold ATP were performed. Protease-mediated removal of the GST tag was performed as indicated in the figure. IB of phosphorylated MNK1a were performed using the indicated antisera. J, as in H. IVK analyses using purified GST-MNK1aWT or GST-MNK1aS353A with active AMPK and cold ATP were performed. IB was done using the indicated antisera. K, U2OS cells expressing 2PY-MNK1aWT, 2PY-MNK1aS353A, or transduced with empty vector (ev) were treated as indicated (serum-starved (ss) or ss+AICAR (AI)).

Article Snippet: Antibodies used are: total MNK1a (tMNK1a) (#2195), pMNK1 (Thr 209 /Thr 214 , #2111), AMPK-Thr(P) 172 (#2535), AMPK (#2532), eIF4E-Ser(P) 209 (#9741), and pERK (#9101; Cell Signaling Technology); pACC (#07-303; Merck Millipore); β-actin (bACT, #0869100; MP Biomedicals); and eIF4E (#610269; BD Biosciences).

Techniques: Sequencing, Western Blot, Purification, Incubation, Autoradiography, Staining, Recombinant, Mutagenesis, Expressing, Plasmid Preparation, Immunoprecipitation, Transduction

Journal: The Journal of Biological Chemistry

Article Title: AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E *

doi: 10.1074/jbc.C116.740498

Figure Lengend Snippet: Metabolic stress-induced MNK1a-Ser353 phosphorylation enhances MNK1a kinase activity toward eIF4E. A, AMPKWT and AMPKdKO MEFs expressing 2PY-MNK1aWT were treated with AICAR or A769662 as indicated. IB analyses of total cell extracts using the indicated antisera were performed. B, U2OS cells expressing 2PY-MNK1aWT (control: empty vector, ev), were treated as indicated: c, control conditions, serum-starved (ss), ss+AICAR (AI) (cf. Fig. 1G), or glucose-deprived (GD)). IB analyses of immunoprecipitation and input material using the indicated antisera were performed; bACT, loading control. C, U2OS cells expressing 2PY-MNK1aWT were glucose-deprived (GD, indicated in hours). #, cells were glucose-deprived, and glucose was re-administered for 1 h. D, lower panel, U2OS cells expressing 2PY-MNK1aWT, 2PY-MNK1aS353A, or 2PY-MNK1aS353D (control: empty vector, ev). IB analyses were performed using the indicated antisera. bACT, loading control. Upper panel, quantitation of peIF4E (FC: -fold change) in U2OS cells expressing 2PY-MNK1aWT or 2PY-MNK1aS353D (n = 5, *, p < 0.005). Error bars indicate means ± S.E. E, upper panels, U2OS cells expressing 2PY-tagged MNK1aWT, MNK1aS353A, MNK1aS353D, MNK1aT2→A2 (a double T209A/T214A mutant), or MNK1aK→M (a kinase-dead K78M mutant) were treated as indicated: AICAR, TPA, or combination treatment (all preceded by serum starvation (ss)). AI, ss+AICAR. 2PY-MNK1a was IP using PY antiserum (middle panels). IB analyses of IP and input material were performed using the indicated antisera. Immunoprecipitations were used in IVKs with recombinant GST-eIF4E and [γ-32P]ATP. White bars between sections indicate separate blots. Lower panels, peIF4E autoradiograph (autorad); CBB image (used as loading control). F, a functional role for Ser(P)353 in MNK1a activation based on a previously suggested conformational change model (21): inactive MNK1a is maintained in a “closed” conformation (upper schematic). ERK and P38 (M/SAPK) binding of the MNK1a C-terminal binding domain (C-term) and subsequent M/SAPK-mediated phosphorylation (encircled P) at MNK1a-Thr209/Thr214 (bold TT) renders MNK1a receptive to AMPK-dependent phosphorylation at Ser353 (bold S). In the context of metabolic stress, AMPK-mediated MNK1a-Ser353 phosphorylation stabilizes an active “open” conformation. Replacement of Thr209/Thr214 by two non-phosphorylatable alanine residues (bold AA) desensitizes MNK1a to M/SAPK signaling (“locked” state).

Article Snippet: Antibodies used are: total MNK1a (tMNK1a) (#2195), pMNK1 (Thr 209 /Thr 214 , #2111), AMPK-Thr(P) 172 (#2535), AMPK (#2532), eIF4E-Ser(P) 209 (#9741), and pERK (#9101; Cell Signaling Technology); pACC (#07-303; Merck Millipore); β-actin (bACT, #0869100; MP Biomedicals); and eIF4E (#610269; BD Biosciences).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Quantitation Assay, Mutagenesis, Recombinant, Autoradiography, Functional Assay, Activation Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Phospholipase Cγ-Erk Axis in Vascular Endothelial Growth Factor-induced Eukaryotic Initiation Factor 4E Phosphorylation and Protein Synthesis in Renal Epithelial Cells

doi: 10.1074/jbc.m504861200

Figure Lengend Snippet: FIG. 6. VEGF induces phosphoryla- tion of Mnk1 and promotes its shift from cytoplasm into nucleus. A, se- rum-deprived MCT cells were stimulated with VEGF 20 ng/ml. The lysates were fractionated by SDS-PAGE and immuno- blotted with phosphospecific antibodies for Mnk1. Probing with Mnk1 antibody (lower panel) was performed to assess loading. A representative blot from three experiments is shown. B, quiescent MCT cells were immunostained with anti-phos- pho-Mnk1 antibody with or without incu- bation with VEGF (20 ng/ml), for various time durations. Immunofluorescent stain- ing was performed as described under “Experimental Procedures,” and the im- ages were analyzed by confocal micros- copy. Control cells showed little staining for phosphorylated Mnk1. After incuba- tion with VEGF for 2 min, intense stain- ing for phospho-Mnk1 in the cytoplasm is seen followed by perinuclear staining at 5 min. The nuclear staining is maximal at 15 min and gets dispersed by about 30 min. The graph represents the percent of cells that showed nuclear localization of phospho-Mnk1 at individual time points following VEGF treatment. Cells at time 0 served as control. Equal numbers of cells were plated in 8-chambered slides; after treatment with VEGF, cells were viewed at uniform magnification of 40 and counted for phospho-Mnk1 nuclear localization. Composite data from three experiments are shown in a graph (p 0.01 time 0 versus 5 min and 15 min, determined by ANOVA).

Article Snippet: Antibody against Mnk1 and anti v-Src antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA) and Oncogene Research Products (San Diego, CA), respectively.

Techniques: SDS Page, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Phospholipase Cγ-Erk Axis in Vascular Endothelial Growth Factor-induced Eukaryotic Initiation Factor 4E Phosphorylation and Protein Synthesis in Renal Epithelial Cells

doi: 10.1074/jbc.m504861200

Figure Lengend Snippet: FIG. 7. VEGF-induced Mnk1 phosphorylation is dependent on PLC, c-Src, and Erk. Cells were serum-starved overnight and subjected to pretreatment with or without U73122 (5 M), a PLC inhibitor (A), PP2 (10 M), a Src inhibitor (B), and U0126 (25 M), a MEK inhibitor (C), prior to incubation with or without VEGF 20 ng/ml for 10 min. Equal amounts of cell lysates were run on SDS-PAGE and immunoblotted with a phosphospecific antibody for Mnk1. The lower panels of A, B, and C show the blots probed with antibody against Mnk1 to assess loading. Representative blots from three experiments with each kinase inhibitor are shown.

Article Snippet: Antibody against Mnk1 and anti v-Src antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA) and Oncogene Research Products (San Diego, CA), respectively.

Techniques: Phospho-proteomics, Incubation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Phospholipase Cγ-Erk Axis in Vascular Endothelial Growth Factor-induced Eukaryotic Initiation Factor 4E Phosphorylation and Protein Synthesis in Renal Epithelial Cells

doi: 10.1074/jbc.m504861200

Figure Lengend Snippet: FIG. 9. Mnk1 mediates eIF4E phosphorylation and is needed for VEGF-induced protein synthesis. A, MCT cells were transfected with a plasmid carrying dominant negative (DN)-Mnk1 (pEBG-T2/A2) or empty vector and allowed to grow for 48 h. Phosphorylation of eIF4E was assessed by immunoblotting as described in the legend to Fig. 8. A representative blot from three experiments is shown. B, MCT cells were transfected with dominant negative Mnk1 or empty vector as described in A, treated with or without VEGF prior to addition of [35S]methionine, and incubated for a total of 2 h. Equal amounts of protein from the lysates were taken for the estimation of 35S label incorporation into trichloroacetic acid-precipitable protein and expressed as percentage of control (Mean S.E.). Data from three experiments are shown in a graph. *, p 0.01 for VEGF versus vector-transfected control; **, p 0.01 for VEGF versus VEGF-treated dominant negative Mnk1-expressing cells, determined by ANOVA.

Article Snippet: Antibody against Mnk1 and anti v-Src antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA) and Oncogene Research Products (San Diego, CA), respectively.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Incubation, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: β-Arrestin-mediated Signaling Regulates Protein Synthesis

doi: 10.1074/jbc.m710515200

Figure Lengend Snippet: FIGURE 1. Sensitivity of AngII-stimulated pMnk1 activation to kinase inhibitors. A, HEK-293 cells stably expressing HA-AT1AR and Mnk1 were serum- starved for 16 h, pretreated with vehicle Me2SO (DMSO) or the indicated kinase inhibitor for 30 min, and then stimulated with 100 nM AngII for the indicated times. Western blots for phospho-Mnk1 and tMnk1 were performed. The percentage of pMnk1/tMnk1 was calculated for each data point and plotted as a percentage of maximal activity (Me2SO-treated cells at 30 min) S.E. These data represent the average S.E. of seven independent experiments. B, repre- sentative pMnk1 Western blots. C, means of individual data points (as a percent of maximal stimulation) up to 30 min from experiments performed in A. D, calculations based on addition or subtraction of means from A as shown in the legend. Solid lines in C represent actual data; dashed lines in D represent mathematically calculated values.

Article Snippet: Pooled human Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39106).

Techniques: Activation Assay, Stable Transfection, Expressing, Western Blot, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: β-Arrestin-mediated Signaling Regulates Protein Synthesis

doi: 10.1074/jbc.m710515200

Figure Lengend Snippet: FIGURE 2. Effect of -arrestin siRNAs on AngII-stimulated pMnk1 and peIF4E activation. A, HEK-293 cells stably expressing HA-AT1AR and Mnk1 were transfected with either control siRNA (CTL) or -arrestin1-specific or -arrestin-2-specific siRNAs (-arr1, -arr2). 72 h post-transfection, cells were serum-starved for 16 h and stimulated with 100 nM AngII for the indicated times. Western blots for phospho-Mnk1 and total Mnk1 were performed. The percentage of pMnk1/tMnk1 was calculated for each data point and plotted as a percentage of maximal activity (CTL-transfected cells at 30 min). These data represent the average of six independent exper- iments S.E. Representative pMnk1- and -arrestin-silencing Western blots are shown. B, cells were treated as in A and stimulated with 100 nM AngII for 30 min, and Western blots were performed for phospho-eIF4E and total eIF4E. Data in graph represent the means S.E. of five experiments; statistical significance was calculated by one way ANOVA with post-test comparison of AngII-stimulated populations (*, p 0.05). Representative Western blot for p-eIF4E is shown.

Article Snippet: Pooled human Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39106).

Techniques: Activation Assay, Stable Transfection, Expressing, Transfection, Control, Western Blot, Activity Assay, Comparison

Journal: Journal of Biological Chemistry

Article Title: β-Arrestin-mediated Signaling Regulates Protein Synthesis

doi: 10.1074/jbc.m710515200

Figure Lengend Snippet: FIGURE3.-ArrestinsphysicallyinteractwithMnk1invivoandinvitro.A,HeLacellswerelysedandimmunoprecipitatedwitha-arrestin-specificantibody (A1CT) or pre-immune serum from the same rabbit. Western blots were performed with antibodies to detect human Mnk1 (upper panel) or a monoclonal antibody to detect immunoprecipitated -arrestins (lower panel). B, spleens were harvested from C57Black6 mice and immunoprecipitated with A1CT or pre-immune serum. Western blots were performed with an antibody for murine Mnk1 or with a monoclonal -arrestin antibody. C, Mnk1 and -arrestins interact in transfected cells. HEK-293 cells stably expressing Mnk1 were transfected with empty vector or plasmids expressing -arrestin1-FLAG or -arrestin2- FLAG and immunoprecipitated with a FLAG-M2 antibody. Western blots were performed for Mnk1. D, reciprocal IP. Empty vector or Mnk1-FLAG expression plasmid was cotransfected with a -arrestin2-HA expression plasmid. Lysates were immunoprecipitated with a FLAG-M2 antibody, and HA Western blots were performed to detect -arrestin2. All results shown are representative of three similar experiments. E, agonist effect on IP. Similar methods as in C, except cells were serum-starved and stimulated for 30 min with AngII or SII. Results of six experiments S.E. are shown in the graph. NB, no bait control, NS, nonstimulated. NB versus NS (p 0.05), NS versus AngII (*, p 0.05) or SII (*, p 0.05).

Article Snippet: Pooled human Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39106).

Techniques: Western Blot, Immunoprecipitation, Transfection, Stable Transfection, Expressing, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: β-Arrestin-mediated Signaling Regulates Protein Synthesis

doi: 10.1074/jbc.m710515200

Figure Lengend Snippet: FIGURE 4. Selective -arrestin pathway stimulation via SI4I8 AngII or AT1ARDRY/AAY results in Mnk1 acti- vation. A, HEK-293 cells stably expressing HA-AT1AR and Mnk1 were serum-starved and pretreated with Me2SO(DMSO),U0126,orRo31–8425for30min,followedbystimulationwitheither100nMAngIIor10MSI4I8 AngII for 30 min. Western blots for phospho-Mnk1 and total Mnk1 were performed. The percentage of pMnk1/ tMnk1 was calculated and plotted as a percentage of maximal AngII-mediated activity. Results are represent- ative of six independent experiments S.E. Post test comparisons: NS versus AngII (**, p 0.001), NS versus SII (*, p 0.01), Ro31 NS versus Ro31 SII (**, p 0.01), SII versus U0126 SII (p 0.01), U0126 NS versus U0126 SII (not significant). NS, nonstimulated. B, HEK-293 cells were transiently transfected with a plasmid encoding the HA-AT1ARDRY/AAY mutant (which is uncoupled from G protein signaling) and Mnk1. Cells were serum-starved and stimulated with 100 nM AngII for the indicated times. Western blots for phospho-Mnk1 were performed and quantitated as in A. Results are representative of the mean of three independent experiments S.E.

Article Snippet: Pooled human Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39106).

Techniques: Stable Transfection, Expressing, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: β-Arrestin-mediated Signaling Regulates Protein Synthesis

doi: 10.1074/jbc.m710515200

Figure Lengend Snippet: FIGURE 6. -Arrestin2 signaling stimulates Mnk1 activation and protein synthesis in vascular smooth muscle cells. A, rVSMCs were serum-starved and pretreated with vehicle Me2SO (DMSO), the PKC inhibitor Ro31–8425, or the MEK inhibitor U0126 for 30 min. Cells were stimulated with either 100 nM AngII (A) or 10 M SII-AngII (S) for an additional 30 min. Western blots for pMnk1 were performed and quantitated. Results depicted are representative of four experiments S.E. Statistical significance was calculated using a one-way ANOVA with post test. Me2SO-nonstimulated was compared with the following, Me2SO AngII (**, p 0.01), Me2SO SII (*, p 0.05), Ro31 AngII (***, p 0.001), and Ro31 SII (**, p 0.01). No other conditions are significant with respect to the Me2SO-nonstimulatedgroup.B,rVSMCsweretransfectedwithsiRNAfor-arr2andsubjectedtopMnk1analysisasinAat96hpost-transfection.Graphdepicts results from six experiments S.E. Statistical significance was calculated using a one-way ANOVA with post test. CTL-nonstimulated was compared with CTL AngII (***, p 0.001) and CTL SII (**, p 0.01). CTL AngII was compared with arr2 AngII (**, p 0.01). No other comparisons are significant. C, rat vascular smooth muscle cells (rVSMC) were serum-starved and stimulated with AngII or SII in the presence of [3H]leucine for 24 h. Radiolabeled proteins were quantitated as described under “Experimental Procedures.” Data are normalized such that nonstimulated cells are set to 100%. Results depicted are repre- sentative of four experiments (means S.E.). Statistical significance was calculated using a one-way ANOVA with post test: N.S. versus AngII was *, p 0.01 and N.S. versus SII was **, p 0.05. D, rVSMC were transfected with siRNAs for -arrestin2 or Mnk1 and subjected to protein synthesis analysis as in C at 96 h post-transfection. Results depicted are representative of six experiments (means S.E.). Statistical significance was calculated using a one-way ANOVA with post test. CTL N.S. compared with CTL AngII was *, p 0.05. CTL AngII compared with arr2 AngII and Mnk1 AngII were both **, p 0.01.

Article Snippet: Pooled human Mnk1 siRNA was purchased from Santa Cruz Biotechnology (sc-39106).

Techniques: Activation Assay, Western Blot, Transfection

Journal: Molecular cancer research : MCR

Article Title: Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

doi: 10.1158/1541-7786.MCR-17-0397

Figure Lengend Snippet: MNK1 is required for ATO-induced eIF4E phosphorylation in GBM and arsenic binds to MNK1 and activates kinase activity. A and B, U87 (A) and LN229 (B) cells were transfected with control siRNA or siRNA targeting MNK1, MNK2, or combination. Cells were treated with or without ATO (2 μmol/L) for 90 minutes. Whole cell lysates were subjected to immunoblotting with antibodies against MNK1, phospho-eIF4E (Ser209), or eIF4E. C and D, Cells from A and B were collected and RNA was extracted. Expression of MKNK1 and MKNK2 genes was determined by qRT-PCR normalizing to GAPDH. Data represent means ± SEM of two independent experiments. E, LN229 (MNK1 expressing) or LN18 (MNK1 undetectable) cells were treated with or without ATO (5 μmol/L) for 90 minutes. Whole cell lysates were subjected to SDS-PAGE followed by transfer to PVDF membranes. Membranes were incubated with antibodies against phospho-eIF4E (Ser209), eIF4E, or MNK1. F, LN229, U87, or recombinant MNK1-GST protein were treated with DMSO or the biotinylated arsenic compound, As-Biotin (AsB), for 2 hours. Whole cell lysates or recombinant protein were incubated with streptavidin beads overnight. Streptavidin pull-down and input control were subjected to immunoblotting with an antibody against MNK1. G, Recombinant MNK1-GST protein was treated with increasing concentrations of ATO for 30 minutes in the presence of the MNK1 peptide substrate, EIF2S. Kinase activity was assessed using the ADP-Glo Kinase Assay. Data represent means ± SEM of three independent experiments. Unpaired, two-tailed t test, **, P ≤ 0.01.

Article Snippet: Recombinant, active MNK1 protein (SignalChem) was used for the in vitro kinase assay.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, SDS Page, Incubation, Recombinant, Kinase Assay, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: Differential Response of Glioma Stem Cells to Arsenic Trioxide Therapy Is Regulated by MNK1 and mRNA Translation

doi: 10.1158/1541-7786.MCR-17-0397

Figure Lengend Snippet: Expression of MES GSC markers correlate with MKNK1 and MKNK1 expression predicts poor survival in GBM patients. A and B, Multiple correlation analysis of MES and PN GSC markers in TCGA patients. Pearson’s correlation analysis,****, P ≤ 0.0001. C, Heatmap showing expression of MKNK1, MES GSC markers (ALDH1A3, CD44) and PN GSC markers (OLIG2, SOX2) in different tumor regions: microvascular proliferation (MP), infiltrating tumor (IT), leading edge (LE), and cellular tumor (CT). Bar graphs depict summarized data from heatmap. Comparisons between MKNK1 and SOX2 means in different tumor regions is shown with Tukey’s honest significant difference test,*, P ≤ 0.05; **, P ≤ 0.01;***, P ≤ 0.001. D, Expression of MKNK1 in GBM and low-grade gliomas: oligodendroglioma (OG), oligoastrocytoma (OA), and astrocytoma (AS). One-way ANOVA,*, P ≤ 0.05;****, ≤ 0.0001. E and F, Overall survival (OS) analysis of low-grade glioma (E), all GBM (F), and MGMT promoter unmethylated; non-G-CIMP GBM (G) patients with high and low expression of MKNK1 (MNK1). Log-rank (Mantel-Cox) test P-values are shown. Expression data downloaded from GlioVis (http://gliovis.bioinfo.cnio.es; ref. 57). Heatmap generated using the IVY Glioblastoma Project. ©2015 Allen Institute for Brain Science. Ivy Glioblastoma Atlas Project. Available from: glioblastoma.alleninstitute.org.

Article Snippet: Recombinant, active MNK1 protein (SignalChem) was used for the in vitro kinase assay.

Techniques: Expressing, Generated