Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells

In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10^-13 to 10^-9 mol/liter, for 0-60 minutes at 37°C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with ¹²⁵I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and ¹²⁵I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3×10^-12 mol/liter) to increase the level of tyrosyl-phosphorylation 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 exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins 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 calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses-time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P<0.05 for each). Together, these studies demonstrate that sCt can have direct effects to modulate the level of tyrosyl-protein phosphorylation in human osteosarcoma cells, presumably by activation of tyrosyl-kinase activity and/or inhibition of phospho-tyrosyl-protein phosphatase activity.