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Tyrosine Phosphorylation of a M_r 63,000 Protein Induced by an Endogenous Mitogen in Human Colon Carcinoma Cells, but not in Normal Colonocytes¹

Gastroenterology Service and Laboratory of Gastrointestinal Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Transformation of normal human colonocytes makes them sensitive to new mitogenic signals. Long-chain diglycerides (LCDGs) found in the human colon are mitogens selective for colon tumor cells, inducing mitogenesis in premalignant cells from each of 13 adenomas and in malignant cells from two of four carcinomas, but having no mitogenic effects on normal colonocytes (E. Friedman, P. Isaksson, J. Rafter, B. Marian, S. Winawer, and H. Newmark, Cancer Res., 49:544–548, 1989). Parallel to this biological activity pattern, LCDGs induce protein phosphorylation only in adenomas and carcinomas. Immunoblotting with an anti-phosphotyrosine monoclonal antibody demonstrated that the LCDG dimyristin, at concentrations found within the body, induced a 6-fold increase of tyrosine phosphorylation of an M_r63,000 protein found in the particulate fraction of colon carcinoma cells. Tyrosine phosphorylation was maximal 0.5 min after addition of the LCDG, then fell, but remained elevated 40% over constitutive levels for at least 6 h. The M_r 63,000 tyrosine phosphoprotein was found in each of four colon carcinoma cell lines and an adenoma, but not in normal colonocytes, suggesting that the tyrosine kinase is activated only in tumor cells. Constitutive levels of the M_r63,000 substrate were enhanced 2-fold by incubation of cells for 20 h with sodium orthovanadate, a tyrosine phosphatase inhibitor. This result suggested that carcinoma cells continually phosphorylate and dephosphorylate this tyrosine kinase substrate during growth. Thus, the colon tumor cell mitogen, dimyristin, utilizes a signal transduction pathway, containing the M_r 63,000 tyrosine kinase substrate, which is already in use during cell growth, possibly by other mitogens or growth factors.

¹ Supported by American Cancer Society Grant BC613 to E. F. and partially supported by Erwin Schrodinger Auslandsstipendium Fellowship J0160M to B. M.

² Present address: Institute for Tumor Biology-Cancer Research, University of Vienna, Borschkegasse 8a, Vienna, Austria.

³ To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, Box 5614, 1275 York Avenue, New York, NY 10021.

Received 1/31/89. Revised 4/12/89. Accepted 5/ 3/89.

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