QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01397

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Biskup, C. Articles by Böhmer, F.-D. Search for Related Content PubMed PubMed Citation Articles by Biskup, C. Articles by Böhmer, F.-D. Social Bookmarking                         What's this?

Visualization of SHP-1-target interaction

Christoph Biskup^1,*, Annette Böhmer^2,*, Rico Pusch^2,*, Laimonas Kelbauskas¹, Alexander Gorshokov³, Irina Majoul³, Jörg Lindenau⁴, Klaus Benndorf¹ and Frank-D. Böhmer^2,

¹ Institute of Physiology II, Medical Faculty, Friedrich Schiller University, Drackendorfer Str. 1, 07747 Jena, Germany
² Research Unit Molecular Cell Biology, Medical Faculty, Friedrich Schiller University, Drackendorfer Str. 1, 07747 Jena, Germany
³ Max-Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
⁴ Application Laboratory, Advanced Imaging Microscopy, Carl-Zeiss GmbH, Carl-Zeiss-Promenade 10, 07745 Jena, Germany

View larger version (34K): [in a new window]   Fig. 2. Structure and expression of TrkA-Ros and SHP-1 fluorescent fusion proteins. (A) Schematic presentation of fluorescent fusion protein constructs. Ros is used as a TrkA-Ros chimerical protein, which allows stimulation of the kinase by nerve growth factor (NGF). Protein domains and important amino acid residues are indicated. (B) Expression of TrkA-Ros-ECFP and EYFP-SHP1-WT in HEK293 cells. TL, transmitted light; bar, 10 µm. (C) Localization of TrkA-Ros-ECFP in the plasma membrane of HEK293 cells shown by immuno-electron microscopy. Membranes of HEK293 cells expressing TrkA-Ros-ECFP and wild-type SHP-1, or mock-transfected cells for control, were freeze-fractured and TrkA-Ros-ECFP was detected with anti-GFP antibodies, followed by immuno-gold labeled secondary antibody. The inner plasma membrane surface, identified by morphological criteria, is shown. Outer surfaces, nuclear membrane or mitochondria showed no labeling (not depicted).

View larger version (21K): [in a new window]   Fig. 1. High-affinity bindings sites for the N-terminal SH2 domain of SHP-1 in the C-terminal region of Ros receptor tyrosine kinase. (A) Synthetic phosphopeptides representing sequences around Ros pY2267 and pY2327, and Epo pY429 were analyzed for binding of the N-terminal SH2 domain of SHP-1 by surface plasmon resonance. Representative experiments are shown (left panel). The data were fitted to determine the kinetic constants kass and kdiss depicted in Table 1. Signals at equilibrium were also determined and plotted against concentrations (right panel). Dissociation constants determined by this procedure are also given in Table 1. (B) The same set of peptides was used for activation experiments. Recombinant SHP-1 was incubated with different concentrations of the phosphopeptides and activity was then determined with pNPP as a substrate. (C) Wild-type EGFR and an EGFR with the sequence around Ros pY2267 (LNY2267MVL) inserted in place of the EGFR pY1173 (AEY1173LR) were overexpressed in HEK293 cells and cell lysates were subjected to pulldown with a GST-fusion protein of the SHP-1 N-terminal SH2 domain. Affinity-precipitated receptor is visualized by immunoblotting with anti-EGFR antibodies. (D) Phosphopeptides representing the sequence around pY2267 or activation loop phosphotyrosines pY2103, pY2107 and pY2108 were subjected to a phosphatase reaction with the catalytic domain of SHP-1. Phosphate release was determined with Malachit Green. Note that the more efficient dephosphorylation of the activation loop phosphopeptide is also obvious at matched concentrations of phosphotyrosine (100 µM activation loop phosphopeptide versus 300 µM pY2267 peptide).

View larger version (15K): [in a new window]   Fig. 3. Fluorescent TrkA-Ros fusion proteins are functional. (A) A fluorescent TrkA-Ros fusion protein signals in response to NGF. Stable NIH3T3 cell lines expressing TrkA-Ros-sgBFP were subjected to NGF stimulation as indicated and TrkA-Ros-sgBFP autophosphorylation and Erk1/2 activation were detected by immunoblotting with lysate aliquots (upper panels). TrkA-RossgBFP autophosphorylation was also measured with immunopreciptated receptor (lower panel). Experiments with the parental cells are shown for comparison. (B) A fluorescent TrkA-Ros fusion protein transduces biological signals. NIH3T3 cells, stably expressing TrkA-Ros-sgBFP, were stimulated with NGF as indicated, and focus formation (upper panel) or proliferation (lower panel) were assessed.

View larger version (26K): [in a new window]   Fig. 4. Constitutive association of TrkA-Ros and SHP-1 in intact cells. (A) Association of TrkA-Ros and SHP-1 in HEK293 cells demonstrated by streak camera measurements. TrkA-Ros-ECFP was coexpressed with different EYFP-SHP1 variants as indicated. Fixed cells were excited at 430 nm and emitted fluorescence was recorded by a combination of a spectrograph and a streak camera. Typical examples of fluorescence emission spectra (upper panel) and fluorescence decay curves in the wavelength range from 465-495 nm (lower panel) that were calculated from the recorded data (streak images) are shown. Cumulated data are presented in Table 2. (B) Detection of constitutive interaction between TrkA-Ros-ECFP and EYFP-SHP-1 by fluorescence spectroscopy. TrkA-Ros-ECFP was coexpressed with the EYFP-SHP1-SH2 or EYFP-SHP1-R32K fusion proteins, as indicated, in COS7 cells. Cells were suspended and spectra were recorded in a fluorescence spectrophotometer with excitation at optimal wavelength for ECFP (ex=424 nm). EYFP spectra (ex=494 nm) were recorded in the same samples and revealed that the amount of EYFP constructs in both sets of cells was identical (not shown).

View larger version (52K): [in a new window]   Fig. 5. Ligand-stimulated complex formation between SHP-1 and TrkA-Ros in COS7 cells and effect of binding site mutations. COS7 cells expressing TrkA-Ros-ECFP, or TrkA-Ros-ECFP mutants together with EYFP-SHP1-SH2 as indicated, were starved in serum-free medium for 6 hours and were either left untreated or were stimulated with 100 ng/ml NGF for 20 minutes. Thereafter, the cells were fixed and subjected to fluorescence lifetime imaging. (A-D) Left panels: fluorescence lifetime images. Amplitude weighted mean lifetimes (m) were color coded as indicated in the right panels. Right panels: distribution of mean lifetimes over the entire cell. The mean fluorescence lifetime was calculated by averaging over all pixels of the cell taking into account their relative intensity. (E) Bar graph of the mean of the fluorescence lifetimes () determined in 5-11 cells. White bars represent measurements in the absence, and grey bars in the presence of NGF. Error bars represent the standard error of the mean. Significant (Student's t-test, P<0.05) differences between the data sets are marked by brackets. Green brackets mark a significant decrease of mean lifetimes that can be attributed to NGF. Dark and light blue brackets indicate data sets that reflect significant changes in constitutive association.

View larger version (49K): [in a new window]   Fig. 6. Ligand-stimulated complex formation between SHP-1 and TrkA-Ros in living COS7 cells. (A) Left panel: fluorescence lifetime image of a COS7 cell coexpressing TrkA-Ros-ECFP and EYFP-SHP1-SH2. Amplitude weighted mean fluorescence lifetimes are encoded by color as specified in the right panels. Right panel: distribution of mean fluorescence lifetimes over the entire cell. (B) Lifetime image of the cell shown in A 40 minutes after stimulation with 100 ng/ml NGF. (C) Subsequent recording of the same cell after photobleaching of the acceptor (EYFP) with the Ar 514 nm line. ECFP fluorescence lifetimes recovered to control values. (D,E) COS7 cells expressing TrkA-Ros-ECFP and EYFPSHP1-C455S before (D) and after (E) treatment with 100 ng/ml NGF for 20 minutes. Images were recorded by confocal LSM in the EYFP channel. Note that a fraction of the SHP1 derivative is enriched at the plasma membrane. Similar translocations were seen in 8 of 10 analyzed cells, but not upon mock-treatment or in cells expressing SHP1 variants with inactivated N-terminal SH2 domain.

View larger version (79K): [in a new window]   Fig. 7. Detailed analysis of lifetime components in NGF-stimulated cells. COS7 cells expressing TrkA-Ros-ECFP and EYFP-SHP1-SH2 were stimulated with 100 ng/ml NGF for 20 minutes. Cells were fixed and confocal and fluorescence lifetime images were recorded. (A) Left panel: LSM image with superimposed ECFP and EYFP channel. Right panel: the quantitative distribution of ECFP (blue curve) and EYFP (yellow curve) along the line depicted in the left panel is shown. (B) Color-coded images of the amplitude weighted mean lifetime (m), the fast lifetime component (f) and its amplitude (Af). The color scale used is shown in the respective panels of C. (C) Distribution of the parameters visualized in B over the entire cell.

View larger version (20K): [in a new window]   Fig. 8. Effect of binding site mutations on TrkA-Ros association with SHP-1 and dephosphorylation as revealed by biochemical methods. (A) TrkA-Ros-ECFP-wt or the respective mutants were coexpressed with catalytically inactive SHP-1CS in HEK293 cells. TrkA-Ros-ECFP was immunoprecipitated from cell lysates with anti-GFP antibodies, and associated SHP-1CS was detected with anti-SHP-1 antibodies. Control: IgG isotype precipitation. Consistent results were obtained in corresponding experiments using untagged TrkARos. (B) Dephosphorylation of TrkA-Ros-ECFP or the respective mutants by coexpressed SHP-1-wt. TrkA-Ros-ECFP-wt or the YF mutants, as indicated, were expressed in HEK293 cells with or without different amounts of SHP-1 wild-type. Lysate aliquots were analyzed with anti-phosphotyrosine antibodies (4G10) for TrkA-Ros-ECFP phosphorylation. Expression levels of receptor and SHP-1 were comparable (not shown). The resulting bands for three independent experiments with a ratio of TrkA-Ros-ECFP:SHP-1 of approximately 1:2 were quantified, normalized to TrkA-Ros-ECFP expression levels, and relative ratios of tyrosine phosphorylation in presence or absence of SHP-1 were calculated (lower panel: means±s.e.m.; *significantly different from wild-type, P<0.05).

View larger version (25K): [in a new window]   Fig. 9. Proposed model of Ros-SHP-1 interaction. For a discussion of the different steps, see text.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter