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First published online 24 October 2006
doi: 10.1242/jcs.03236

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Tyrosine phosphorylation of p145^met mediated by EGFR and Src is required for serum-independent survival of human bladder carcinoma cells

¹ Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan
² Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA
³ Laboratory of Molecular Biology, Research Center for Environmental Genomics, Kobe University, Nada, Kobe 657-8501, Japan

^* Author for correspondence (e-mail: kksato{at}kobe-u.ac.jp)

Accepted 31 August 2006

Here we address the molecular mechanism of serum-independent survival and growth of human bladder carcinoma cell line 5637. Serum starvation promoted tyrosine phosphorylation of a 145-kDa protein and activation of the tyrosine kinase Src and the receptor for epidermal growth factor (EGFR) over a slow time course (>8 hours). The phosphorylated 145-kDa protein was identified as the ß-subunit of c-Met/hepatocyte growth factor (HGF) receptor, p145^met, in which tyrosine residues 1003, 1234, and 1235 were phosphorylated. Inhibitors of Src (PP2, SU6656) or EGFR (AG99), but not p145^met (K252a), effectively blocked tyrosine phosphorylation of p145^met and promoted cell death accompanied by activation of caspase-like proteases. Conditioned medium from the serum-starved 5637 cells or purified EGF readily promoted the activation of Src and EGFR, and tyrosine phosphorylation of p145^met in normally grown 5637 cells, suggesting that autocrine signaling of EGFR ligands is responsible for signal transduction events in serum-starved cells. Consistent with this idea, a monoclonal antibody against EGFR that would interfere with the ligand binding to EGFR blocked tyrosine phosphorylation events and promoted the caspase activation and cell death in serum-free conditions. Such apoptotic cell death was also induced by pretreatment of cells with a high concentration of HGF that downregulated endogenous p145^met. Nevertheless, Cu²⁺ ions, competitive inhibitors for HGF-binding to p145^met, did not show any effect on cellular functions in serum-free conditions. These results suggest that the serum-independent growth of 5637 cells involves the transmembrane signaling cascade via EGFR ligand(s) (but not HGF), EGFR, Src and p145^met.

Key words: EGFR, Src, Met, Cancer cells, Serum-independent growth, Signal transduction

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