Protein phosphorylation in somatomedin-stimulated chick cartilage cells

Cell cultures obtained from chick embryo pelvic rudiments which respond to somatomedin C (SM) by increased incorporation of /sup 35/(SO/sub 4/) into proteoglycans were stimulated with SM in the presence of /sup 32/P(PO/sub 4/). Cells were harvested at zero time, 50 min, and 7 hrs following addition of the /sup 32/P and SM, homogenized, and dialyzed against phosphate buffered saline. After centrifugation at 100,000 xg, the pellet and the supernatant were incubated with DNase and RNase in the presence of benzamidine HCl, phenylmethylsulfonyl fluoride, 6-aminohexanoic acid and EDTA, and subjected to electrophoresis in a 5 to 15% polyacrylamide gel gradient in sodium dodecyl sulfate. In samples from cells stimulated for 50 min with SM, radioautography revealed two labeled bands at molecular weights of approximately 400K and 500K in the 100,000 xg pellet; no labeled bands were observed in the corresponding control cells. After 7 hr incubation extensive labeling was observed in both control and stimulated cells without discernible differences between control and SM-stimulated samples. These results suggest that an early event in SM stimulation of proteoglycan synthesis may be phosphorylation of proteins distinct from the SM receptor. It is interesting that one of the labeled bands corresponds approximately to themore » molecular weight of the proteoglycan core protein precursor.« less