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Role of Cellular Ca ++ in Phosphorylation of 21 K and 19 K Polypeptides in Cultured Thyroid Cells: Effects of Phorbol Ester, Trifluoperazine, and 8-Diethylamino-Octy1-3,4,5- Trimethoxybenzoate Hydrochloride*

1987; Oxford University Press; Volume: 121; Issue: 1 Linguagem: Inglês

10.1210/endo-121-1-175

1945-7170

Masahiro Ikeda, William J. Deery, M S Ferdows, Thor B. Nielsen, James B. Field,

Mass Spectrometry Techniques and Applications

Cultured dog thyroid cells contain 21 and 19 kilodalton (K) phosphoproteins which by several criteria have been identified as light chains of myosin (MLC). TSH causes a reduction in the phosphorylation state of the 21 K-19 K proteins, at least in part through activating adenylate cyclase and increasing cAMP levels. We now report that 12-O-tetradecanoylphorbol- 13-acetate (TPA) also decreases the 21 K-19 K protein phosphorylation state, but in contrast to that due to TSH, the TPA-induced decrease is not associated with elevated cAMP levels. The effect of TPA was not additive to that of TSH. Because Ca++ is a major factor regulating MLC kinase and TPAstimulated protein kinase C in other systems, the role of Ca++ in the phosphorylation of the 21 and 19 K polypeptides in dog thyroid was examined. In intact cells, both (8-diethylamino)- octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (1 × 10-4 M) and trifluoperazine (TFP) (4 × 10-5 M) increase basal 21 K-19 K protein phosphorylation and inhibit the decrease in phosphorylation caused by TSH and TPA without affecting cAMP levels. Ionophore A23187 (5 × 10-6 M) counteracts TMB- 8-and TFP-stimulated phosphorylation as well as TMB-8 and TFP inhibition of TSH-and TPA-reduced 21 K-19 K phosphorylation. Incubation of 32PO4-labeled dog thyroid cells in the absence of extracellular Ca++ or with verapamil does not significantly affect basally phosphorylated 21 K-19 K proteins or the decreased 21 K-19 K phosphorylation state caused by TSH. These results strongly suggest that the phosphorylation state of the 21 and 19 K proteins is affected more significantly by intracellular Ca++ pools than by extracellular Ca++, and implicate a kinase(s) other than Ca++-calmodulin-dependent MLC kinase in the phosphorylation of MLC in the dog thyroid. (Endocrinology121: 175–181, 1987)