Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
FIG. 3

FIG. 3. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

Phosphorylation of Y1105 by c-Src in vitro. (A) Phosphotryptic peptide map of the GST-middle domain (p190 residues 380 to 1180) phosphorylated in vitro with [γ-32P]ATP by purified c-Src (UBI); ∼1,000 cpm was loaded. (B) Rechromatography of the isolated peptide A depicted in panel A. (C) Phosphotryptic peptide map of full-length p190, metabolically labeled with 32P; ∼1,000 cpm was loaded. (D) Mix of samples depicted in panels B (∼600 cpm) and C (∼1,000 cpm). The comigrating phosphopeptides are labeled A and 3, respectively. Maps represent one experiment of three, all giving identical results.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.
2.
FIG. 6

FIG. 6. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

Binding of p120 RasGAP to GST fusion proteins containing p190 middle domain variants. GST fusion proteins containing a variant (wt, Y1105F, or Y1087F) of the p190 middle domain (MD; residues 380 to 1180) or GST alone attached to glutathione-agarose beads were either mock phosphorylated (−) or phosphorylated (+) by c-Src in vitro, washed, and incubated with detergent lysates of Neo cells for 3 h at room temperature. Beads were pelleted and washed, and precipitated proteins were subjected to SDS-PAGE and Western immunoblotting with 6F2 anti-p120 RasGAP (A) or 4G10 anti-p-Tyr (B) MAb and 125I-labeled goat anti-mouse Ig.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.
3.
FIG. 2

FIG. 2. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

CAD mass spectra of unlabeled phosphorylated p190 peptides. (A) The sequence of the p190 tryptic peptide spanning amino acids 1099 to 1115 and the predicted m/z ratios of ion fragments of this peptide are shown above the CAD spectrum of this tryptic peptide from endogenous p190 from K+ c-Src overexpressers. Ion fragments originating from the amino terminus are listed as b1 to b17. Ions originating from the carboxy terminus are listed as y1 to y17. y8 to y16 are listed in both the singly and doubly charged forms due to the contribution of a positive charge by the His1109 residue. Underlined ions and m/z ratios represent values detected in the spectrum. (B) CAD spectrum of the synthetic peptide 1099-1115 phosphorylated at Tyr1105. (C) CAD spectrum of the synthetic peptide 1099-1115 phosphorylated at Ser1106.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.
4.
FIG. 4

FIG. 4. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

Y1105 is a preferred in vivo c-Src phosphorylation site. Indicated cell lines were brought to near confluence, serum starved and labeled with 32P for 18 h, and either left unstimulated or stimulated for 30 min with EGF (100 ng/ml). P190 was immunoprecipitated and processed to generate phosphotryptic peptides. Phosphopeptides were analyzed by electrophoresis in the first dimension and chromatography in the second dimension on TLC plates. Amounts (in counts per minute) of sample loaded: (A) 2,025; (B) 2,450; (C) 2,360; (D) 1,796; (E) 2,212; (F) 2,254; (G) 2,117; (H) 1,834; (I) 1,389; (J) 760. Times of exposure on BioMax film were 20 (A to F), 24 (G and H), and 72 (I and J) h.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.
5.
FIG. 7

FIG. 7. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

In vivo binding of p120 RasGAP to ectopically expressed p190 variants. The mammalian expression plasmid pRc/CMV, encoding full-length, HA-tagged wt p190 (lanes 2 and 4) or variant Y1105F (lane 5), Y1087F (lane 6), or Y1105F/Y1087F (lane 7), was transiently transfected into COS-7 cells with (lanes 3 to 7) or without (lanes 1 and 2) a pcDNA3 plasmid encoding wt c-Src. pRc/CMV plasmid lacking the p190 coding sequences was transfected to control for nonspecific protein interactions (lanes 1 and 3). Ectopically expressed p190 was immunoprecipitated with anti-HA MAb 12CA5, immunoprecipitates were divided into two equal parts, and each was subjected to SDS-PAGE. Western immunoblotting was then carried out, probing one membrane first with anti-p190 MAb 8C10 (top panel, lanes 3 to 7) or anti-HA MAb 12CA5 (lanes 1 and 2) and then with anti-p120 MAb 6F2 (middle panel). The second membrane was probed with anti-p-Tyr MAb 4G10 (bottom panel). Primary antibodies were visualized with 125I-labeled goat anti-mouse Ig. Results are representative of three independent experiments.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.
6.
FIG. 1

FIG. 1. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

Two-dimensional tryptic phosphopeptide analysis of metabolically 32P-labeled p190 and phosphoamino acid analysis of p-Tyr-containing peptides. (A and B) Two-dimensional tryptic phosphopeptide maps were generated from metabolically 32P-labeled p190, immunoprecipitated with MAb 8C10 from v-Src-transformed IV5 cells (A) or K+ c-Src cells (B) and processed as described in Materials and Methods. Peptides were spotted onto TLC plates at the origin (marked “o”), resolved by electrophoresis in the horizontal dimension and chromatography in the vertical dimension, and visualized by autoradiography. Peptides displaying the same migration pattern in both maps are numbered identically. In panel A, 1,150 sample cpm was applied, and plates were exposed for 5 days on Kodak BioMax film. In panel B, 2,200 cpm was applied, and plates were exposed for 48 h on the same film. (C and D) Two-dimensional phosphoamino acid analysis. Phosphopeptides 3 (C) and 8 (D) were isolated from peptide maps of p190 from K+ c-Src cells and hydrolyzed as described in Materials and Methods. Unlabeled p-Ser, p-Thr, and p-Tyr standards were added, and the samples were spotted onto cellulose plates and separated by electrophoresis in pH 1.9 buffer in the horizontal dimension and in pH 3.5 buffer in the vertical dimension. Standards were visualized with 0.25% ninhydrin; 400 cpm of peptide 3 and 1,500 cpm of peptide 8 were applied, and plates were exposed for 54 h on BioMax film.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.
7.

FIG. 5. From: Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation.

p-Tyr-dependent and -independent mechanisms of p190-p120 association. (A) p120 RasGAP was quantitatively immunoprecipitated from 500 μg of lysate derived from serum-deprived, nonstimulated Neo cells (lane 1), K+ c-Src cells (lane 3), or K c-Src cells (lane 5), using MAb 6F2. In lanes 2, 4, and 6, mouse IgG (Jackson ImmunoResearch Laboratories) was used as a negative antibody control. Precipitated proteins were resolved by SDS-PAGE, transferred to an Immobilon membrane, and immunoblotted with anti-p120 MAb 6F2 (bottom lanes) and anti-p190 MAb 8C10 (top lanes). The membrane slice containing p190 was stripped of 8C10 and reprobed with anti-p-Tyr MAb 4G10 (middle lanes). Localization of primary antibodies was revealed by binding of 125I-labeled rabbit anti-mouse IgG and autoradiography. (B) The amount of radioactivity in each lane of panel A was quantitated by PhosphorImager analysis, and the PIU for p190 protein and p-Tyr p190 were adjusted to the amount of p120 in each immunoprecipitate. Adjusted PIU for each antibody (anti-p-Tyr or anti-p190) were then compared across the cell lines, arbitrarily setting the normalized value for Neo control cells to 1.00 in each experiment for each antibody. Results from two independent experiments, one done in triplicate and one done in quadruplicate for each cell line and antibody, were pooled and expressed as the mean ± standard error. Results from both nonstimulated and EGF-stimulated (not shown in panel A) cells were included in the analysis. Differences in mean p-Tyr levels of the three cell lines are significantly different by Student’s paired t test (P < 0.005). Differences in mean p190 protein associated with p120 between the three cell lines are significant to a P value of <0.02. To assess the relative differences in p190 p-Tyr content versus p190 protein associated with p120 RasGAP between the different cell lines, the mean p-Tyr/p190 protein ratio was calculated for each cell line and is shown in the inset.

Richard W. Roof, et al. Mol Cell Biol. 1998 December;18(12):7052-7063.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk