Cyclic AMP-dependent phosphorylation of high molecular mass proteins in pig thyroid cells.
Four high molecular mass (H Mr) 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 HMr1 to HMr4 determined by polyacrylamide gel electrophoresis in the in the presence of sodium dodecylsulfate was about 310 000 for HMr1, 250 000 for HMr2, 240 000 for Hmr3 and 220 000 for HMr4. HMr1 comigrated with brain MAP1, whereas HMr3 and HMr4 had the same mobility as alpha-and beta-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 HMr proteins. The 32P-labeling of HMr 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.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009218:Myosins / Q000032:analysis; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013049:Spectrin / Q000032:analysis; D013552:Swine; D013961:Thyroid Gland / Q000032:analysis* / Q000166:cytology / Q000201:enzymology
D008565:Membrane Proteins; D010750:Phosphoproteins; D013049:Spectrin; D011494:Protein Kinases; D009218:Myosins