Abstract
Protein phosphorylation of G0/G1-arrested Chinese hamster lung fibroblasts (CC139 line) has been analyzed following stimulation by fetal calf serum (FCS) or by a variety of growth factors. FCS stimulated the phosphorylation of three major polypeptides separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nuclear protein with a Mr of 62,000 daltons, the ribosomal protein S6, and a cytosoluble peptide of 27,000 daltons. These phosphorylations occurred rapidly after serum stimulation (1 min for the 27,000-dalton peptide, 5 min for S6 and the 62,000-dalton proteins) and were maximal after 30 min. In nonstimulated cells the 27,000-dalton phosphopeptide exists in two forms with isoelectric points of 5.7 and 6.0; serum increased the amount of the most acidic form. At low concentrations, the "commitment" growth factors, alpha-thrombin, eye-derived growth factor (EDGF), platelet-derived growth factor (PDGF), stimulated phosphorylation of the 27,000-dalton peptide. At higher concentrations, these factors alone reinitiated DNA synthesis and, like FCS, stimulated phosphorylation of the three major peptides. In contrast, and suggesting a different mechanism of action, "progression" factors such as insulin (1-10 micrograms/ml) and multiplication-stimulating activity (MSA) are unable to stimulate phosphorylation of the 27,000-dalton peptide. However, insulin or MSA which are known to potentiate the mitogenic action of alpha-thrombin, PDGF, EDGF, ... were also found to potentiate phosphorylation of the ribosomal protein S6. These results support the existence of two classes of growth factors and suggest that protein phosphorylation is an early event involved in the control of the cellular G0 leads to G1 transition.
Highlights
Protein phosphorylation of Go/G1-arrested Chinese rylation is a rapid and general response of cells to growth hamster lung fibroblas(CtsC139 line) has been analyzed factors ( 2 4, yet itis not known whether each growth factor following stimulation by fetal calf serum (FCS) or by a stimulates thephosphorylation of a specific or common set of variety of growth factors.FCS stimulated thephospho- proteins when added to Go-arrested cells
A chemically defined medium containing a set of purified growth factors can support growth of rg 3 0
The inhibition of a phosphatase medium is depletedof growth factors, growthstops reversibly activity has been implicated for the phosphorylationof S6 in in Go/GI [1, 12]
Summary
Jean-Claude Chambard$, Arlette Franchi$A,lphonse Le Cam@a, nd Jacques Pouyssegurt. From the $Centre dBe iochimie, Centre National dela Recherche Scientifique and 8Faculte dMe edecine, Uniuersite de Nice, P a r c Valrose, 06034 Nice Cedex, France. Another point of rylation of three major polypeptides separated on so- interest is the potentiating action observed between growth dium dodecyl sulfate-polyacrylamide gel electrophoresis: a nuclear protein with a M, of 62,000 daltons, the ribosomal protein S6, and a cytosoluble peptide of 27,000 daltons These phosphorylations occurred rapidly after serum stimulation (1 min for the 27,000-dalton factors [7,8], in particular between twodistinct classes (9-ll), suggesting dual modes of action. Serum-deprived cells were incubated with 100 pCi/nll of [23] modified by Studier [24].The resolving gel was a linear gradient ""I', during 45 min, and 10'r FCS was added( + I for the time indicated.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
