Ikeda M, Deery W J, Ferdows M S, Nielsen T B, Field J B
Endocrinology. 1987 Jul;121(1):175-81. doi: 10.1210/endo-121-1-175.
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-tetradecanoyl-phorbol-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 TPA-stimulated 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 X 10(-4) M) and trifluoperazine (TFP) (4 X 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 X 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.
培养的犬甲状腺细胞含有21千道尔顿（k）和19千道尔顿的磷蛋白，根据多项标准已被鉴定为肌球蛋白轻链（MLC）。促甲状腺激素（TSH）至少部分通过激活腺苷酸环化酶和提高环磷酸腺苷（cAMP）水平，导致21k-19k蛋白的磷酸化状态降低。我们现在报告，12-O-十四烷酰佛波醇-13-乙酸酯（TPA）也会降低21k-19k蛋白的磷酸化状态，但与TSH导致的情况不同，TPA诱导的降低与cAMP水平升高无关。TPA的作用与TSH的作用并非相加性。由于钙离子（Ca++）是调节其他系统中MLC激酶和TPA刺激的蛋白激酶C的主要因素，因此研究了Ca++在犬甲状腺中21k和19k多肽磷酸化中的作用。在完整细胞中，盐酸（8-二乙氨基）-辛基-3,4,5-三甲氧基苯甲酸酯（TMB-8）（1×10^(-4)M）和三氟拉嗪（TFP）（4×10^(-5)M）均可增加基础21k-19k蛋白的磷酸化，并抑制TSH和TPA引起的磷酸化降低，而不影响cAMP水平。离子载体A23187（5×10^(-6)M）可抵消TMB-8和TFP刺激的磷酸化以及TMB-8和TFP对TSH和TPA降低的21k-19k磷酸化的抑制作用。在无细胞外Ca++或维拉帕米存在的情况下，对32PO4标记的犬甲状腺细胞进行孵育，对基础磷酸化的21k-19k蛋白或TSH引起的21k-19k磷酸化状态降低没有显著影响。这些结果强烈表明，21k和19k蛋白的磷酸化状态受细胞内Ca++池的影响比受细胞外Ca++的影响更显著，并表明在犬甲状腺中MLC的磷酸化涉及一种不同于Ca++-钙调蛋白依赖性MLC激酶的激酶。
