Tyrosine phosphorylation of p145met mediated by EGFR and Src is required for serum-independent survival of human bladder carcinoma cells.
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 beta-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).
Yamamoto
Natsumi
N
Graduate School of Science and Technology, Kobe University, Nada, Kobe, Japan.
Mammadova
Gunay
G
Song
Robert X-D
RX
Fukami
Yasuo
Y
Sato
Ken-ichi
K
D000595:Amino Acid Sequence; D017209:Apoptosis; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D006801:Humans; D026121:Indole Alkaloids; D007211:Indoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology; D001749:Urinary Bladder Neoplasms; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
D002227:Carbazoles; D016895:Culture Media, Serum-Free; C066855:HGF protein, human; D026121:Indole Alkaloids; D007211:Indoles; D021122:Protein Subunits; C416927:SU 6656; D013449:Sulfonamides; D020032:Tyrphostins; C086599:tyrphostin A46; D014443:Tyrosine; D017228:Hepatocyte Growth Factor; C049985:staurosporine aglycone; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met; D019061:src-Family Kinases