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¹ Laboratory of Tumor Phosphoproteomics, Division of Medical Sciences, National Cancer Centre, Singapore; ² Institute of Molecular and Cell Biology, National University of Singapore, Singapore; ³ Oregon Cancer Institute, Oregon Health Sciences University, Portland, OR; and ⁴ Department of Medicine, University of Hong Kong, Hong Kong, China

Requests for Reprints:Yoon-Pin Lim, Laboratory of Tumor Phosphoproteomics, Division of Medical Sciences, National Cancer Centre, 11 Hospital Drive, Singapore 169610. Fax: (65) 6226 5694. E-mail: dmslyp{at}nccs.com.sg

Many proteins regulating cancer cell growth are tyrosine phosphorylated. Using antiphosphotyrosine affinity chromatography, thiourea protein solubilization, two-dimensional PAGE, and mass spectrometry, we report here the characterization of the epidermal growth factor (EGF)-induced phosphoproteome in A431 human epidermoid carcinoma cells. Using this approach, more than 50 distinct tyrosine phosphoproteins are identifiable within five main clusters—cytoskeletal proteins, signaling enzymes, SH2-containing adaptors, chaperones, and focal adhesion proteins. Comparison of the phosphoproteomes induced in vitro by transforming growth factor- and platelet-derived growth factor demonstrates the pathway- and cell-specific nature of the phosphoproteomes induced. Elimination of both basal and ligand-dependent phosphoproteins by cell exposure to the EGF receptor catalytic inhibitor gefitinib (Iressa, ZD1839) suggests either an autocrine growth loop or the presence of a second inhibited kinase in A431 cells. By identifying distinct patterns of phosphorylation involving novel signaling substrates, and by clarifying the mechanism of action of anticancer drugs, these findings illustrate the potential of immunoaffinity-based phosphoproteomics for guiding the discovery of new drug targets and the rational utilization of pathway-specific chemotherapies.

Received 7/25/03; revised 10/ 2/03; accepted 10/ 7/03.

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