Alpha-thrombin-induced tyrosine phosphorylation of 43,000- and 41,000-Mr proteins is independent of cytoplasmic alkalinization in quiescent fibroblasts.
Incubation of quiescent Chinese-hamster fibroblasts (CCL39) with alpha-thrombin, a potent mitogen for the cells, was found to stimulate the rapid phosphorylation of two 43,000-Mr and two 41,000-Mr proteins at tyrosine, threonine and/or serine, and two 63,000-Mr proteins at serine. Insulin, 12-O-tetradecanoylphorbol 13-acetate (TPA) and epidermal growth factor (EGF) are weak mitogens for cells; insulin and TPA did not stimulate the phosphorylation of those proteins significantly, whereas EGF stimulated their phosphorylation to the same extent as did alpha-thrombin. We analysed alpha-thrombin-induced protein phosphorylation at different external pH values in CCL39 and in the mutant derivative PS120, which lacks Na+/H+-antiport activity. We showed that cytoplasmic alkalinization, a common and early response to mitogens, is not required to trigger phosphorylation of 63,000-, 43,000- and 41,000-Mr proteins, either at tyrosine or serine and threonine residues. This finding contrasts with the phosphorylation of ribosomal protein S6, which takes place only at permissive pH for reinitiation of DNA synthesis. These results, demonstrating that phosphorylation of 63,000-, 43,000- and 41,000-Mr proteins and cytoplasmic alkalinization are not coupled, reinforce the idea that the site of action of intracellular pH controlling the commitment of G0/G1-phase-arrested cells to DNA synthesis might be restricted to mitogen-stimulated S6 phosphorylation.
D000468:Alkalies / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*
D000468:Alkalies; D011506:Proteins; D014443:Tyrosine; D013917:Thrombin