Piga A, Wickremasinghe R G, Taheri M R, Yaxley J C, Hoffbrand A V
Exp Cell Res. 1985 Jul;159(1):103-12. doi: 10.1016/s0014-4827(85)80041-0.
We have measured changes in tyrosine protein kinase activity in the particulate and soluble fractions of phytohemagglutinin (PHA)-stimulated human lymphocytes using a synthetic tyrosine-containing peptide as substrate. Following PHA stimulation, the activity of this enzyme remained stable for 24 h and then fell sharply in both fractions. Phosphorylation of endogenous substrates was also studied by gel electrophoresis of the same subcellular preparations which had been incubated with [gamma-32P]ATP. Gels were treated with alkali to hydrolyse phosphoserine, dried and autoradiographed. In the particulate fraction from resting lymphocytes, two prominent bands were labelled, one of 55-60 kD and another of 38 kD. Direct analysis showed that these proteins were labelled largely on tyrosine residues. The 38 kD band became less intensely labelled 24 h following PHA addition, and had disappeared by 72 h. Two new bands appeared at this time: a 42 kD band which was phosphorylated mostly on threonine and a 32 kD band phosphorylated on tyrosine. The 55-60 kD band remained unchanged up to 96 h following PHA addition. The final band pattern in stimulated lymphocytes resembled that of the malignant T lymphoblastic cell line JM1. Our data suggest that tyrosine protein kinases in resting lymphocytes are part of a mechanism which transduces external growth signals to the cell interior, and that this mechanism is inactivated once the signal has been transmitted. The pattern of endogenous substrates which are phosphorylated on tyrosine and threonine residues in the malignant T lymphoblastic cell lines JM1 is likely to be characteristic of proliferating T lymphoid cells rather than of the malignant state, since similar bands appear in normal lymphocytes upon PHA stimulation.
我们使用一种含酪氨酸的合成肽作为底物，测定了植物血凝素（PHA）刺激的人淋巴细胞颗粒组分和可溶性组分中酪氨酸蛋白激酶活性的变化。PHA刺激后，该酶的活性在24小时内保持稳定，然后在两个组分中均急剧下降。还通过对与[γ-32P]ATP孵育的相同亚细胞制剂进行凝胶电泳，研究了内源性底物的磷酸化情况。凝胶用碱处理以水解磷酸丝氨酸，干燥并进行放射自显影。在静息淋巴细胞的颗粒组分中，有两条明显的条带被标记，一条为55 - 60 kD，另一条为38 kD。直接分析表明，这些蛋白质主要在酪氨酸残基上被标记。添加PHA后24小时，38 kD条带的标记强度减弱，到72小时时消失。此时出现了两条新条带：一条42 kD的条带主要在苏氨酸上磷酸化，一条32 kD的条带在酪氨酸上磷酸化。添加PHA后96小时内，55 - 60 kD条带保持不变。刺激淋巴细胞中的最终条带模式类似于恶性T淋巴母细胞系JM1的条带模式。我们的数据表明，静息淋巴细胞中的酪氨酸蛋白激酶是将外部生长信号转导至细胞内部的机制的一部分，并且一旦信号传递，该机制就会失活。恶性T淋巴母细胞系JM1中在酪氨酸和苏氨酸残基上磷酸化的内源性底物模式可能是增殖性T淋巴细胞的特征，而不是恶性状态的特征，因为在PHA刺激的正常淋巴细胞中也出现了类似的条带。
