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Ligand-induced Phosphorylation of a Murine Tumor Surface Protein (TSP-180) Associated with Metastatic Phenotype¹

Laboratorio di Oncogenesi Molecolare Istituto Regina Elena per lo studio e la cura dei tumori, Roma, Italia [A. S., R. F., G. P., M. A. G.]; Istituto di Oncologia Sperimentale e Clinica, Facoltà di Medicina, Catanzaro, Italia [N. P.]; and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 [S. J. K.]

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to M_r 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (M_r 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate ³²PO₄, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins.

Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the M_r 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine.

Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.

¹ Partially supported by Consiglio Nazionale delle Ricerche Progetto Finalizato ‘Oncologia’ Grant 870158344, by Associazione Italiana per la Ricerca sul Cancro, and by Oak Ridge National Laboratory.

² To whom requests for reprints should be addressed, at Laboratorio di Oncogenesi Molecolare, Istituto Regina Elena per lo studio e la cura dei tumori, Viale Regina Elena, 291-00161 Rome, Italy.

Received 6/10/88. Revised 11/29/88. Accepted 1/25/89.

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