prk5 flag sestrin2  (Addgene inc)

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle

doi: 10.1152/ajpendo.00522.2018

Figure Lengend Snippet: Western blot analysis of Sestrin1, 2, and 3 protein expression in liver, gastrocnemius, tibialis anterior, heart, kidney, and brain. A: representative Western blot analysis of Sestrin and GAPDH expression. Membranes were stained with Pierce Reversible Stain for PVDF and then destained before Western blot analysis; a representative membrane is shown. Sestrin1, 2, and 3 antibodies were validated using HEK293T cells lacking all three Sestrin proteins [kindly provided by Dr. David Sabatini (21)]. B: FLAG-tagged Sestrin1, 2, and 3 were individually expressed in and purified from HEK293T cells as described under materials and methods. Purified proteins (2 µg) were resolved by SDS-PAGE, and the gel was stained with SimplyBlue SafeStain. Ratio, band intensity relative to Sestrin1. C: Western blot analysis of Sestrin1 and GAPDH expression in liver (n = 6) and various amounts of purified FLAG-Sestrin1. Membranes were stained for total protein loading before Western blot analysis as described above for A. D: representative standard curve for FLAG-Sestrin1. E: quantification of Western blot analysis of Sestrin1, 2, and 3 expression in various tissues. Values represent means ± SE (n = 6). Method used to quantify Sestrin1 shown in C and D was also used to quantify Sestrins 2 and 3 using the respective purified proteins (B). ND, Sestrin2 expression in gastrocnemius was too low to be accurately quantitated, and therefore is not included on the graph. For each tissue except gastrocnemius, values not sharing the same letter are significantly different by one-way ANOVA, P < 0.001. For gastrocnemius, P < 0.0005 by t-test. Gastroc, gastrocnemius; Tib. Ant., tibialis anterior.

Article Snippet: In a separate experiment, FLAG-Sestrin2 (pRK5-FLAG-Sestrin2; cat. no. RC-201386, Origene, Rockville, MD) was transfected into the left leg of 12 rats.

Techniques: Western Blot, Expressing, Staining, Purification, SDS Page

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle

doi: 10.1152/ajpendo.00522.2018

Figure Lengend Snippet: Leucine-induced dissociation of GATOR2 from Sestrin1, but not Sestrin2 or 3, in tibialis anterior muscle. A and B: a plasmid expressing FLAG-tagged Sestrin1 was transfected into the tibialis anterior muscle in one leg of a rat, and a plasmid expressing FLAG-tagged Sestrin3 was transfected into the contralateral muscle as described under materials and methods. In a second set of rats, a plasmid expression FLAG-metap2 was transfected into the tibialis anterior muscle in one leg of a rat, and a plasmid expressing GFP was transfected into the contralateral muscle. In a third set of rats, a plasmid expressing FLAG-Sestrin2 was transfected into the tibialis anterior muscle. Four days later, the rats were fasted for ~18 h and were then gavaged with a suspension containing 54 g/l leucine (2.5 ml/100 g body weight) or an equivalent volume of saline. FLAG-tagged proteins were immunoprecipitated from muscle homogenates using anti-FLAG beads and then subjected to Western blot analysis. Representative blots are shown in B. Muscles transfected with a plasmid expressing GFP were processed for immunofluorescence analysis as described under materials and methods. A representative image is shown in A. C: quantitation of Western blots from FLAG-immunoprecipitates from tibialis anterior muscle. Because WDR24, Mios, and Sec13 are subunits of GATOR2, and leucine-induced changes in their association with the Sestrins were qualitatively similar, values for WDR24, Mios, and Sec13 were combined to provide an estimate for GATOR2 associated with Sestrin. Values represent the means ± SE (n = 6–7 rats/group). *P < 0.05 vs. Leu. GATOR2, GAP activity toward Rags 2; GFP, green fluorescent protein; Leu, leucine; Mios, meiosis regulator for oocyte development; p70S6K1, ribosomal protein S6 protein kinase; p70S6K1(p-T389), phosphorylated p70S6K1; WDR24, WD repeat-domain-containing protein 24.

Article Snippet: In a separate experiment, FLAG-Sestrin2 (pRK5-FLAG-Sestrin2; cat. no. RC-201386, Origene, Rockville, MD) was transfected into the left leg of 12 rats.

Techniques: Plasmid Preparation, Expressing, Transfection, Immunoprecipitation, Western Blot, Immunofluorescence, Quantitation Assay, Activity Assay

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: Exogenous expression of a Sestrin2 phospho-mimetic variant results in repressed p70S6K1 phosphorylation and interaction with Mios in a leucine-independent manner. HEK293 cells were transiently transfected with either an empty vector (EV), a plasmid expressing wild-type Sestrin2 (WT Sesn2), or a plasmid expressing a triple phospho-mimetic Sestrin2 variant (Sesn2EDE). (A) Cells were incubated in medium lacking serum and leucine for 2 h (-SL) prior to stimulation with 760 μM leucine for 30 min (LAB). Phosphorylation of p70S6K1 on Thr389 (p-p70), FLAG-tagged Sestrin2 (FLAG-Sesn2), and GAPDH were evaluated by Western blot analysis. * indicates significantly different than LAB, # indicates significantly different than EV. Results represent the mean ± SEM of 5 independent experiments with 2-3 replicates performed in each experiment. (B) Cells were incubated in complete medium (CM) or deprived of serum and leucine for 2 h (-SL). FLAG-tagged Sestrin2 was immunoprecipitated from the supernatant fraction of cell lysates. Co-immunoprecipitation of Mios with FLAG-tagged Sestrin2 was assessed by Western blot analysis. Letters above the bars indicate significant differences. Results represent the mean ± SEM of 3 independent experiments with 2 replicates performed in each experiment.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Expressing, Variant Assay, Transfection, Plasmid Preparation, Incubation, Western Blot, Immunoprecipitation

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: Leucine alters the electrophoretic migration of Sestrin2. (A) HEK293 cells were incubated in complete medium (CM) or medium lacking leucine (-L) for 2 h prior to harvest and samples were resolved on a BioRad Criterion gel prior to Western blot analysis for p70S6K1 (phospho-Thr389 (p-p70) and total (p70)), Sestrin2 (Sesn2), and GAPDH. (B and C) HEK293 cells were incubated in complete medium, medium lacking leucine for 2 h prior to stimulation with 760 μM leucine (LAB) for 30 min prior to harvest and cell extracts were fractionated on 7.5% polyacrylamide gels with 0.19% bisacrylamide. (D) HeLa cells were incubated as in panels B and C. All samples were analyzed on the same blot, but not in contiguous lanes; a white line separates non-contiguous lanes. * indicates significantly different than CM, p <0.05. Results represent the mean ± SEM of 3 independent experiments with 2 replicates performed in each experiment.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Migration, Incubation, Western Blot

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: The altered migration of Sestrin2 is specific to changes in leucine content. (A and B) HEK293 cells were incubated in complete medium (CM) or medium lacking leucine, arginine, glutamine, glycine, and histidine (−5AA) for 2 h prior to stimulation with either 760 μM leucine (+L), 400 μM arginine (+R), 4 mM glutamine (+Q), or 200 μM histidine (+H) alone or in combination for 30 min prior to harvest. Samples were subjected to Western blot analysis for (A) total p70S6K1 (p70), p70S6K1 phosphorylated on Thr389 (p-p70), and GAPDH, or (B) Sestrin2. Letters above the bars indicate significant differences. Significance was set at p < 0.05 for all analysis. Results represent the mean ± SEM of 3 independent experiments with 1-2 replicates performed in each experiment.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Migration, Incubation, Western Blot

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: Time course and dose response analysis of leucine-induced changes in Sestrin2 electrophoretic migration. (A) HEK293 cells were incubated in medium lacking leucine (−L) for the indicated time prior to harvest. (B) HEK293 cells were incubated in complete medium (CM) or medium lacking leucine for 2 h prior to stimulation with 760 μM leucine (LAB) for the indicated time. (C and D) HEK293 cells were incubated in CM or medium with the indicated concentration of leucine for 2 h prior to harvest. Sestrin2 (Sesn2) and GAPDH protein content and phosphorylation of p70S6K1 on Thr389 (p-p70) were determined by Western blot analysis. * indicates significantly different than 0 μM leucine. Significance was set at p < 0.05 for all analyses. Results represent the mean ± SEM of 3 independent experiments with 2 replicates performed in each experiment.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Migration, Incubation, Concentration Assay, Western Blot

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: Leucine-induced changes in Sestrin2 electrophoretic mobility are due to phosphorylation. (A) HEK293 cells were transiently transfected with a control plasmid or one encoding FLAG-tagged Sestrin2 18 h prior to incubation in phosphate-free medium containing (CM) or lacking leucine (−L) for 1 h. [32P]orthophosphate was added to the medium and cells were harvested 30 min later. FLAG-tagged Sestrin2 was immunoprecipitated, subjected to SDS-PAGE, and the gel was dried and 32Pi incorporation was imaged on a Typhoon Imager. Incorporation of 32Pi was from a single experiment. An aliquot of the immunoprecipitate was also subjected to Western blot analysis for FLAG content. (B) HEK293 cells were incubated in medium containing or lacking leucine for 2 h prior to harvest. A portion of the cell extracts were incubated with lambda protein phosphatase prior to Western blot analysis for Sestrin2 (Sesn2) and 4E-BP1 as indicated in the figure.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, SDS Page, Western Blot

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: Identification of Sestrin2 phosphorylation sites by mass spectrometry. Sestrin2 was isolated from cells incubated in the presence or absence of leucine as described under “Methods” and sent to the Taplin Mass Spectrometry Facility for analysis. Three sets of samples were independently analyzed. Ser249 was identified as being phosphorylated in cells incubated in medium containing leucine and Thr232 and Ser279 were identified as being phosphorylated in cells deprived of leucine. (A) Alignment of Sestrin2 phosphorylation sites across species. Hs, Homo sapiens; Pt, Pan troglodytes; Bt, Bos Taurus; Mm, Mus musculus; Rn, Rattus norvegicus; Dr, Danio rerio. (B) Multisequence alignment of human Sestrin2 phosphorylation sites with Sestrin1 and Sestrin3.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Mass Spectrometry, Isolation, Incubation

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: ULK1 promotes Sestrin2 phosphorylation and interaction with GATOR2. (A) Wild type (ULK1+/+) and ULK1 knockout (ULK1−/−) MEF were incubated in medium lacking serum and leucine for 2 h (-SL) prior to stimulation with 760 μM leucine for 30 min (LAB). Phosphorylation of p70S6K1 on Thr389 (p-p70), total p70S6K1 (p70), ULK1, Sestrin2 (Sesn2), and GAPDH were evaluated by Western blot analysis. Results are representative of 2 independent experiments with 2-3 replicates performed in each experiment. Letters above the bars indicate significant differences. (B) HEK293 cells were transfected with the indicated plasmids, and the next day cell lysates were subjected to Western blot analysis for FLAG-Sestrin2 (FLAG-Sesn2) and HA-ULK1. Results are representative of 3 independent experiments with 2 replicates performed in each experiment. (C) HEK293 cells were transfected with an empty vector (EV) and/or plasmids expressing HA-p70S6K1 (HA-p70) and HA-ULK1. The next day cell lysates were immunoprecipitated using anti-HA beads and immunoprecipitates were subjected to Western blot analysis for HA and p70S6K1 phosphorylated on Thr389 (p-p70). Results represent 1 experiment with 2 replicates. (D) HEK293 cells were transfected with the plasmids indicated in the figure, and the next day FLAG-Sestrin2 was immunoprecipitated from the supernatant fraction of cell lysates. Co-immunoprecipitation of Mios with FLAG-tagged Sestrin2 was assessed by Western blot analysis. Results are representative of 2 independent experiments with 2 replicates performed in each experiment.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Knock-Out, Incubation, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation

Journal: Cellular signalling

Article Title: Leucine Induced Dephosphorylation of Sestrin2 Promotes mTORC1 Activation

doi: 10.1016/j.cellsig.2016.03.008

Figure Lengend Snippet: Working model for the mechanism whereby leucine availability acts to repress mTORC1 activity via phosphorylation of the Sestrin2 linker domain at Thr232, Ser249, and Ser279. CTD, C-terminal domain; NTD, N-terminal domain; linker, unstructured region between the C-terminal and N-terminal domains.

Article Snippet: Transient transfection and [ 32 P i ] incorporation HEK293 cells were transfected with plasmids encoding FLAG-tagged Sestrin2 with a Myc and FLAG-tag at the C-terminus (pCMV6-Sestrin2-myc-DDK, catalog #RC501386, Origene Technologies, Inc.) in Opti-MEM reduced serum medium (Life Technologies, Carlsbad, CA) at a reagent to DNA ratio (μl/μg) of 4:1 as previously described [ 19 ].

Techniques: Activity Assay