CALCITONIN ACUTELY INCREASES TYROSYL-PHOSPHORYLATION OF PROTEINS IN HUMAN OSTEOSARCOMA (SAOS-2) CELLS

In order to test the hypothesis that salmon calcitonin has direct effe cts to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal al kaline phosphatase) were exposed to calcitonin, in duplicate serum-fre e cultures, at concentrations ranging from 10(-13) to 10(-9) mol/ lite r, for 0-60 minutes at 37 degrees C. Phospho-tyrosyl proteins were ide ntified by autoradiography of Western blots after incubation with (125 )-I-labeled antiphosphotyrosine antibodies (Or with unlabeled antibodi es and (125)-I-labeled protein A) and quantitated by laser densitometr y. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3 x 10(-12) mol/liter) to increase the le vel of tyrosylphosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P < 0.05 for each ); and (2) dose-dependent effects of sCt (during 15 minutes of exposur e) to increase the level of tyrosyl-phosphorylation of at least 10 pro teins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P < 0.05 for each). A supplementary study of ca lcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkalin e activity revealed similar responses-time and dose-dependent increase s in the tyrosyl-phosphorylation of at least seven proteins with appar ent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P < 0.0 5 for each), Together, these studies demonstrate that sCt can have dir ect effects to modulate the level of tyrosyl-protein phosphorylation i n human osteosarcoma cells, presumably by activation of tyrosyl-kinase activity and/or inhibition of phospho-tyrosyl-protein phosphatase act ivity.