Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 1. Ras-GRF1 is expressed at a higher level compared with Sos in rat forebrain slices. Lysates of COS-7 cells expressing HA13- Ras-GRF1 and HA13-Sos1 were subjected to Western blotting with the anti-HA1 monoclonal antibody (left panels) and standardized by dilu- tion such that equivalent levels of the expressed proteins were present. These amounts were then run in parallel with lysates of rat brain slices (right panels) and subjected to Western blotting with polyclonal anti- bodies to either Ras-GRF1 (upper right panel) or Sos (lower right panel). In all cases, a graded scale of four 3-fold dilutions of the lysates was loaded to ensure that the signals could be measured in a range where the signals were not saturated. Data shown are representative of four independent experiments and were collected digitally using a Fuji Film LAS-1000 Plus imaging system. Quantification was performed with ImageGauge Version 3.3 software.

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Expressing, Western Blot, Imaging, Software

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 2. Cyanogen bromide digests of Ras-GRF1 reveal the phosphopeptide that reflects Ser898 phosphorylation in forsko- lin-treated COS-7 cells and rat forebrain slices. COS-7 cells trans- fected with pMT3gluGRF1 (8) and rat forebrain slices were labeled with 32Pi and then treated with dimethyl sulfoxide vehicle (–) or 100 M isobutylmethylxanthine (IBMX) plus 20 M forskolin (FSK) (). The Ras-GRF1 proteins were immunoprecipitated and digested with cyan- ogen bromide, and the fragments were separated by SDS-PAGE using a Tricine cathode buffer to sharpen resolution of the phosphopeptides (26). The results shown are autoradiographs of a region of the dried gels, with molecular mass markers (in kilodaltons) shown to the left. Exposure times were overnight for the COS-7 cell-derived samples and 2 weeks with intensifying screens for the brain-derived samples. The arrow indicates a phosphopeptide fragment that was stimulated by the activation of PKA and represents phosphorylation of Ser898 (26). The result is representative of three independent experiments.

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Labeling, Immunoprecipitation, SDS Page, Derivative Assay, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 3. Phosphorylation of Ras- GRF1 at Ser916 promotes Ras-depend- ent outgrowth of neurites in PC12 cells. PC12 cells were transfected by elec- troporation and then grown for 48 h be- fore processing for indirect confocal im- munofluorescence using red detection for the Myc epitope tags at the N termini of the wild-type H-Ras constructs and green detection for the HA1 epitope tags at the N termini of the Ras-GRF1 constructs. Results shown in A–F are representative red fluorescence results (except for E, where green fluorescence is shown) from transfections with GRF1N plus H-Ras (A), GRF1900 plus H-Ras (B), GRF1- 900(S916A) (GRF1900,916A) plus H- Ras (C), GRF1976 plus H-Ras (D), GRF1N alone (E), and H-Ras alone (F). Scale bar 20 m. Neurite extension was quantified in the red fluorescence channel by measuring the projections that were longer than the diameter of the cell body. In all cases where cotransfections were performed, clear signals in both the red and green channels were required for a cell to be scored. Four independent exper- iments were performed, with 25 positive cells measured from each trial. Results for the longest neurite produced and for the sum of all neurites are presented in G and H as means S.D. Mutation of Ser916

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Transfection, Construct, Fluorescence, Produced, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 4. Antibody 2152 selectively recognizes Ras-GRF1 Ser(P)916. Recombinant GST fusion proteins of the indicated regions of Ras-GRF1 were incubated with () or without (–) PKA in the presence of ATP/Mg2 and then separated by SDS-PAGE on two gels run in parallel. A, one gel was transferred to nitrocellulose and subjected to Western blotting with antibody 2152. Note that reactivity was seen only when the GST-Ras-GRF1 constructs were phosphorylated by PKA and was not seen when Ser916 was mutated to alanine (916A). B, the other gel was stained with Coomassie Blue to confirm the relative loading of the recombinant proteins. C, experiments were repeated with graded loading of the unphosphorylated (open symbols) and phosphorylated (closed symbols) substrates to establish the degree to which antibody 2152 recognizes Ras-GRF1 only when it is phosphorylated at Ser916. Data shown were collected digitally from Western blots using the Fuji Film LAS-1000 Plus imaging system and analyzed with ImageGauge Version 3.3. software. wt, wild-type.

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Construct, Staining, Imaging, Software

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 5. Regulated phosphorylation of Ras-GRF1 at Ser916/898

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 6. Regulated phosphorylation of Ras-GRF1 at Ser916 in PC12 cells as revealed by Western blotting. PC12 cells were co- transfected with pKH3Ras-GRF1 plus expression vectors for human muscarinic receptor subtypes 1 and 2. The cells were deprived of serum overnight and then stimulated with 100 M carbachol, with 100 M IBMX plus 20 M forskolin, or with 10 ng/ml NGF. Whole cell extracts were prepared for Western blotting (32) with antibody 2152 (raised against Ras-GRF1 Ser(P)916 (GRF1(phospho916)); right panel) and with antibody sc-863 (raised against total Ras-GRF1; left panel). Data shown are representative of two independent experiments.

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Western Blot, Transfection, Expressing

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 7. Regulated phosphorylation of Ras-GRF1 at Ser916 in PC12 cells as revealed by indirect confocal immunofluores- cence. PC12 cells were cotransfected with pKH3Ras-GRF1 (left panels) or pKH3Ras-GRF1(S916A) (right panels) plus expression vectors for human muscarinic receptor subtypes 1 and 2. The cells were deprived of serum overnight and then stimulated with 100 M carbachol, with 100 M IBMX plus 20 M forskolin, or with 10 ng/ml NGF. The cells were fixed and processed for indirect confocal immunofluorescence using red detection for the HA1 epitope tags at the N termini of the Ras-GRF1 constructs and green detection for antibody 2152 (raised against Ras- GRF1 Ser(P)916 (Ras-GRF1phospho916)). The fluorescence images are presented overlaid with a phase-contrast image of the cells that reveals the presence of untransfected control cells. Note that green fluorescence due to antibody 2152 reactivity was found only when wild-type Ras- GRF1 (Ras-GRF1,wt) was present (thus colocalized with red fluores- cence and appears yellow) and only in cells that had been stimulated with agonists that induce Ser916 phosphorylation. Data shown are representative of five independent experiments. Ras-GRF1,916A, Ras-GRF1(S916A).

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Expressing, Immunofluorescence, Construct, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 8. Regulated phosphorylation of endogenous Ras-GRF1 at Ser898 in rat forebrain slices as revealed by Western blotting. Rat forebrain slices were stimulated with 100 M carbachol, with 100 M IBMX plus 20 M forskolin, or with 10 ng/ml NGF for 20 min at room temperature. Lysates were prepared, with 2.5% reserved for direct analysis. Ras-GRF1 was immunoprecipitated from 97.5% of the lysate using antibody sc-224; the immunoprecipitates were then split, with 10% subjected to Western blotting for total Ras-GRF1 using antibody sc-863 (left panel) and 90% subjected to Western blotting with antibody 2152 (raised against Ras-GRF1 Ser(P)898 (GRF1(phospho898)); right panel). Negative control immunoprecipitations ((-ve) i.p.) were per- formed with rabbit anti-mouse immunoglobulin (Jackson Laboratories). Data shown are representative of four independent experiments.

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex

doi: 10.1074/jbc.m209805200

Figure Lengend Snippet: FIG. 9. Regulated phosphorylation of endogenous Ras-GRF1 at Ser898 in rat cerebral cortex as revealed by indirect confocal immunofluorescence. Forebrain slices of postnatal day 15 rat brains were fixed and processed for indirect confocal immunofluorescence. The primary antibodies used were as follows: antibody 2152, raised against Ras-GRF1 Ser(P)898 (A, the yellow arrows indicate apical dendrites of pyramidal cells in the medial prefrontal cerebral cortex, and the inset shows a lower magnification view); antibody sc-224, raised against total Ras-GRF1 (B1, the yellow arrows indicate apical dendrites of pyramidal cells in the medial prefrontal cerebral cortex, and the red arrows indi- cate cell bodies); and antibody sc-863, which recognizes Ras-GRF1 only in Western blot protocols, but not in immunofluorescence and so was used as a staining control (B2).

Article Snippet: The production of the anti-Ras-GRF1 Ser(P)916 antibody was carried out by Cell Signaling Technology, Inc.

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Staining, Control

Journal: Disease Markers

Article Title: Evodiamine as the Active Compound of Evodiae fructus to Inhibit Proliferation and Migration of Prostate Cancer through PI3K/AKT/NF- κ B Signaling Pathway

doi: 10.1155/2022/4399334

Figure Lengend Snippet: Evodiamine induces G 2 /M cell cycle arrest in DU145 cells. (a) After treatment with evodiamine (0, 1.25, 2.5, and 5.0 μ M) for 24 h, the cell cycle distributions were analyzed by flow cytometry. The cell population in the G 2 /M phase significantly augmented in a dose-dependent manner. (b) The cell populations were quantified using Prism. Each column represents the cell population in different phases ( n = 3). (c) DU145 cells were treated with evodiamine (0, 1.25, 2.5, and 5.0 μ M) for 24 h. The protein expression levels of CDK1, p-CDK1 Thr14 , cyclin B1, and p-Cdc25C Ser216 were detected by Western blot. β -Actin was used as the loading control. Evodiamine-induced G 2 /M cell cycle arrest is associated with upregulation of p-CDK1 Thr14 /CDK1 and cyclin B1 and downregulation of p-Cdc25C Ser216 . (d) Quantitative analysis of the relative protein expression. Data are presented as the mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus the control group.

Article Snippet: The primary antibodies against CDK1, p-CDK1 Thr14 , cyclin B1, p-Cdc25C Ser216 , Bax, Bcl-2, AKT, β -actin, p-AKT Ser473 , NF- κ B, PARP, and PI3K were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Flow Cytometry, Expressing, Western Blot, Control

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: Strains used in this study

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques:

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: mcs1 delays the initiation of mitosis. (A) mcs1-77 is synthetically lethal with cdc25-22. Wild-type, mcs1-77, cdc25-22, and mcs1-77 cdc25-22 cells were grown on YEPD at 28°C and then streaked onto YEPD plates at 28, 31, or 36°C, and the growth of the strains was monitored after 3 d at these temperatures. (B) mcs1-77 cdc25-22 arrested cells are highly elongated. mcs1-77 cells were crossed to cdc25-22 cells, and the resulting asci were subjected to tetrad dissection on YEPD plates at 31°C. Colonies from a tetratype that were genotypically mcs1-77 (left), cdc25-22 (middle), or mcs1-77 cdc25-22 (right) were visualized after 2 d of growth at 31°C.

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques: Dissection

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: mcs1-77 encodes a point mutant in the DNA-binding domain of Res2. (A) Localization of inactivating transposon insertions in a genomic clone containing Res2. Simultaneous localization and sequencing of complementing regions was performed by transposon mutagenesis of the pURB1-Res2 genomic clone. The structure of the Res2 protein is shown with the N-terminal DNA-binding domain (black bar), central ankyrin repeats (gray bar), and the C-terminal Cdc10 interaction domain (hatched bar). Arrows indicate the location of transposon insertions, and the ability of the transposed plasmid to rescue mcs1-77 cdc25-22 cells at 31°C is shown as either rescue (+) or no rescue (−) after 3 d of growth on Edinburgh minimal medium (EMM) plates lacking uracil. (B) Sequence of the DNA-binding domain of Res2 from wild-type and mcs1-77 cells. Alignment of amino acids 28–89 of Schizosaccharomyces pombe (S.p.) Res2 protein with homologous regions of S.p. Res1 and Saccharomyces cerevisiae (S.c.) Mbp1 and S.c. Swi4 transcription factors. The arrow indicates the change of glycine 68 to a serine residue in mcs1-77 cells.

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques: Mutagenesis, Binding Assay, Sequencing, Plasmid Preparation, Residue

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: Mutation of MBF components causes a G2 delay

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques: Mutagenesis

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: Mitotic delay is not dependent on Rad3 kinase

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques:

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: Cdc13 and Cut2 are unstable in mcs1-77 cells in G2 phase. (A) Stability of cyclin B and Cut2 proteins in mcs1-77 cdc25-22 cells. Log-phase cultures of either cdc25-22 cut2-364::cut2+-HA-LEU2 or mcs1-77 cdc25-22 cut2-364::cut2+-HA-LEU2 cells growing in YEPD at 28°C were shifted to 35.5°C for 3 h and then incubated in the same medium containing 100 μg/ml cycloheximide at 35.5°C for the times indicated. Cell extracts were prepared and probed for the presence of either Cdc13, with the use of affinity-purified anti-Cdc13 polyclonal antibodies, or Cut2, with the use of anti-hemagglutinin mAbs. (B) Quantification of cyclin B and Cut2 stability from A. The abundance of Cdc13 (squares) and Cut2 (circles) was expressed as a percentage of the initial level after the addition of cycloheximide to either cdc25-22 cut2-364::cut2+-HA-LEU2 (○, □) or mcs1-77 cdc25-22 cut2-364::cut2+-HA-LEU2 cells (●, ▪). (C) Aberrant chromosome segregation in mcs1-77 cdc25-22 cells. Log-phase cultures of either cdc25-22 or mcs1-77 cdc25-22 cells growing in YEPD at 25°C were shifted to 36°C for 3 h. Cells were then fixed and stained with DAPI to visualize chromatin.

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques: Incubation, Affinity Purification, Staining

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: Instability of Cdc13 in mcs1-77 cells is due to the activity of APCSte9 (A) Deletion of Ste9/Srw1 rescues the cell cycle delay in mcs1-77 cells. mcs1-77 cdc25-22 or ste9::ura4 mcs1-77 cdc25-22 cells were grown on YEPD at 25°C and then streaked onto YEPD plates at either 25 or 31°C, and the growth of the strains was monitored after 3 d at these temperatures. (B) Deletion of Ste9 stabilizes Cdc13 cyclin B in mcs1-77 cells. Log-phase cultures of either mcs1-77 cdc25-22 or ste9::ura4 mcs1-77 cdc25-22 cells growing in YEPD at 28°C were shifted to 35.5°C for 3 h and then incubated in the same medium containing 100 μg/ml cycloheximide at 35.5°C for the times indicated. Cell extracts were prepared and probed for the presence of Cdc13 with the use of affinity-purified anti-Cdc13 polyclonal antibodies.

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques: Activity Assay, Incubation, Affinity Purification

Journal:

Article Title: A Role for the START Gene-specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

doi:

Figure Lengend Snippet: MBF complex controls periodic transcription of ste9/srw1. (A) Transcription of ste9/srw1 is cell cycle regulated. A log-phase culture of cdc25-22 cells growing in YES was arrested in G2 by incubation at 36.5°C for 4 h. Cells were shifted to 25°C and collected for Northern blot analysis and examined microscopically for the appearance of septa at the times indicated. Total RNA was extracted, and equal quantities were separated by electrophoresis and probed with the use of DNA specific to the ste9/srw1 and cdc18 genes. Reprobing with cdc2-specific probes verified approximately equal loading of RNA. (B) Res1 and Res2 control transcription of ste9/srw1. Log-phase cultures of wild-type, res1::ura4, or res2::ura4 cells growing in YES were incubated in the presence of 11 mM hydroxyurea for 4 h and then washed and resuspended in fresh YES lacking hydroxyurea for the times indicated. Cells were harvested, and total RNA was extracted. Equal quantities of RNA were separated by electrophoresis and probed with the use of DNA specific to the ste9/srw1 and cdc18 genes. Reprobing with cdc2-specific probes verified approximately equal loading of RNA.

Article Snippet: [ PMC free article ] [ PubMed ] Millar JB, McGowan CH, Lenaers G, Jones R, Russell P. p80 cdc25 mitotic inducer is the tyrosine phosphatase that activates p34 cdc2 kinase in fission yeast.

Techniques: Incubation, Northern Blot, Electrophoresis, Control