Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.

I.Introduction2II.Why Functional Proteomics?3 A. The Complementarity of Genomics and Proteomics3 B. Genotypes and Protein Phenotypes3 C. New Views of Biological Function5 D. The Role of Mass Spectrometry6 E. Current General Areas of Proteome Research7  1. Mapping of Total Cellular Proteins7  2. Subcellular Complexes and Organelles10  3. Protein Phenotypes and Function13 F. The Need for and Utility of Functional Proteomics15  1. Definition of Functional Proteomics.15  2. A New Biology—Networks, Fluxes, and Dynamics at the Molecular Level16  3. The Importance of Assigning Gene Functions in Context16  4. Practical Advantages of Functional Proteomics17III.Recent Applications of Functional Proteomics and Mass Spectrometry17 A. Changes in Cellular Environment17 B. Direct Observation of Growth Factor Signal Transduction19 C. Protein Synthesis Following Directed Cellular Perturbation22 D. Directed Measurement of Functional Classes of Proteins24 E. Global Detection of Specific Types of Functional Activities in Cells25 F. Investigating the Function of Individual Proteins26 G. Summary29IV.Technical Advances for Functional Proteomics29 A. “Total” Proteins29 B. Display Methodologies31 C. Quantification Methods33  1. Chemical Staining33  2. Antibody‐Based Detection Methods33  3. New Multi‐Photon Detection Methods for Detection of Radioactive Labels34 D. Implications for Automation34V.Perspectives36 A. Directions for Functional Proteomics36  1. Studies of Cells in Culture36 2. Single Cells36  3. Tissue Samples37  4. Medical and Pharmaceutical Themes37  5. Comparative Proteomics37 B. Perturbation Methods—The Importance of Time Scales, Kinetics, and Fluxes38 C. The “Virtual Proteome”39 D. Constructing the “Virtual Proteome”: Community‐Based Proteomics42  1. Standards for Display Maps of the Proteome43  2. Standards for Mass Spectrometric Identification of Proteins43  3. Databases44 E. Implications for Mass Spectrometry45  1. Sensitivity and Functional Proteomics45  2. Phenotypic Characterization of Proteins46  3. Automated Analysis of MS Data in Proteomics47 F. Conclusion48Acknowledgments48References48

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We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important for this interaction (Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, A. V., Ullrich, A., and Bö hmer,