Cyclic AMP-dependent phosphorylation of high molecular mass proteins in pig thyroid cells

Abstract

Four high molecular mass (H M r) proteins were found to be phosphorylated in a cyclic AMP-dependent manner in both partially purified pig thyroid membrane fractions and in pig thyroid cells in culture. These phosphoproteins did not correspond to major cellular proteins; they were found in both soluble and particulate subfractions of homogenates from cultured thyroid cells. The molecular mass of the 4 proteins named H M r1 to H M r1 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate was about 310000 for H M r1, 250000 for H M r2, 240000 for H M r3 and 220000 for H M r4. H M r1 comigrated with brain MAPi 1 whereas H M r3 and H M r4 had the same mobility as α-and β-spectrins, respectively. The 4 high molecular mass phosphoproteins are substrates of cyclic AMP-dependent protein kinase(s) since (a) their phosphorylation was increased by cyclic AMP and not by cyclic GMP or calcium alone or calcium in the presence of calmodulin or phospholipid; (b) the effect of cyclic AMP was prevented by the thermostable inhibitor of cyclic AMP-dependent protein kinases; (c) the purified catalytic subunit of cyclic AMP-dependent protein kinases markedly phosphorylated the 4 H M r proteins. The 32P-labeling of H M r proteins using either endogenous cyclic AMP-dependent protein kinase or the purified catalytic subunit was always lower in cells cultured in the presence of TSH (reassociated in follicle-like structures) than in freshly dispersed cells or cells cultured in basal conditions (cells in monolayer). These results suggest that the 4 high molecular mass thyroid phosphoproteins represent structural components, the phosphorylation of which could vary with the cellular organization.