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 degrees 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 x 10(-12) mol/liter) to increase the level 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 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).(ABSTRACT TRUNCATED AT 250 WORDS)
Thomas
A
A
Department of Medicine, Loma Linda University, California, USA.
Hall
S L
SL
Nicolas
V
V
Lau
K H
KH
Farley
J R
JR
D000469:Alkaline Phosphatase; D002116:Calcitonin / Q000494:pharmacology*; D002454:Cell Differentiation; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D012516:Osteosarcoma; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D011506:Proteins; D014443:Tyrosine; C028815:salmon calcitonin; D002116:Calcitonin; D000469:Alkaline Phosphatase