Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase during lymphocyte mitogenesis.
Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase during lymphocyte mitogenesis.
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淋巴细胞有丝分裂过程中环磷酸腺苷依赖性蛋白激酶的激活和鸟氨酸脱羧酶的诱导。
DOI：
10.4049/jimmunol.123.2.817
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发表时间：
1979
影响因子：
4.4
通讯作者：
D. Lucas
中科院分区：
文献类型：
--
作者：
G. Klimpel;C. Byus;D. Russell;D. Lucas
在用有丝分裂原、非有丝分裂凝集素或二丁酰环 AMP (DBcAMP) 进行体外培养后，研究了环 AMP 依赖性蛋白激酶的激活和鸟氨酸脱羧酶 (ODC) 的诱导，以及人外周血淋巴细胞中的 RNA 和 DNA 合成。促细胞分裂素植物血凝素 (PHA)、伴刀豆球蛋白 A (Con A) 和美洲商陆促细胞分裂剂 (PWM) 均会导致可溶性环 AMP 依赖性蛋白激酶的激活，诱导 ODC 活性水平增加，并增加 (3H)-胸苷（DNA 合成）和 (3H)-尿苷（RNA 合成）掺入酸不溶性物质的速率。高浓度的 PHA 或 Con A 会导致最大程度的环 AMP 依赖性蛋白激酶激活，但仅具有轻微的促有丝分裂作用。每种有丝分裂原的蛋白激酶最大激活时间各不相同，但在每种情况下，总是先于 ODC 诱导和 RNA 和 DNA 合成增加。在 Con A 刺激的培养物中，环 AMP 依赖性蛋白激酶激活在培养 4 小时时达到最大，并在 24 小时和 48 小时时保持升高。在这些培养物中，首先在培养 6 至 8 小时时观察到 ODC 活性增加，然后在培养 12 小时时可检测到 RNA 合成增加。在所有有丝分裂原刺激的培养物中，首先在第 48 小时观察到 DNA 合成，最大 (3H)-胸苷掺入出现在培养 72 小时。
环 AMP 依赖性蛋白激酶的激活、ODC 的诱导以及 RNA 和 DNA 合成的增加都依赖于淋巴细胞与 Con A 的结合，并且可以通过早期添加 α-甲基甘露糖苷来阻断。培养 12 小时后，添加 α-甲基甘露糖苷对 ODC 的 Con A 诱导或对 24 至 72 小时测量的 RNA 和 DNA 合成几乎没有影响。相比之下，当 α-甲基甘露糖苷晚至 12 或 20 小时添加到 Con A 刺激的培养物中时，在 24 小时测量的环 AMP 依赖性蛋白激酶激活可能会被完全抑制。非有丝分裂凝集素、麦芽凝集素 (WGA) 和双孢蘑菇凝集素 (ABL) 未能增加任何测试参数。 DBcAMP 也不会诱导 ODC 或导致任何 RNA 或 DNA 合成增加。然而，DBcAMP (10-3 M) 确实在培养 15 分钟时就引起了环 AMP 依赖性蛋白激酶的激活。蛋白激酶的 DBcAMP 激活具有浓度依赖性，在 12 小时时达到最大，并在培养 48 小时内保持升高状态。向 Con A 刺激的培养物中添加 DBcAMP (10-3 M) 会导致 ODC 活性以及 RNA 和 DNA 合成抑制 90% 至 95%。这种抑制是浓度依赖性的，并且与每个浓度引起的环AMP依赖性蛋白激酶的激活程度平行。
这些结果表明，环AMP依赖性蛋白激酶的选择性激活和ODC活性增加的诱导是与淋巴细胞有丝分裂相关并调节淋巴细胞有丝分裂的早期整体事件。
Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase (ODC) were studied along with RNA and DNA synthesis in human peripheral blood lymphocytes following in vitro culture with either mitogens, nonmitogenic agglutinins, or dibutyryl cyclic AMP (DBcAMP). The mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) all resulted in activation of soluble cyclic AMP-dependent protein kinase, the induction of increased levels of ODC activity, and increases in the rate of incorporation of (3H)-thymidine (DNA synthesis) and (3H)-uridine (RNA synthesis) into acid-insoluble material. High concentrations of PHA or Con A, which resulted in maximal cyclic AMP-dependent protein kinase activation, were only slightly mitogenic. The time at which maximum activation of protein kinase occurred differed for each mitogen but, in each case, always preceded ODC induction and increased RNA and DNA synthesis. In Con A-stimulated cultures, cyclic AMP-dependent protein kinase activation was maximal at 4 hr of culture and remained elevated at 24 and 48 hr. In these cultures increased ODC activity was first observed at 6 to 8 hr of culture and preceded increased RNA synthesis which was detectable at 12 hr of culture. DNA synthesis in all mitogen-stimulated cultures was first observed at 48 hr with maximum (3H)-thymidine incorporation being at 72 hr of culture.
Cyclic AMP-dependent protein kinase activation, induction of ODC, and increased RNA and DNA synthesis were all shown to be dependent upon lymphocyte-Con A binding and were blocked by early addition of α-methyl mannoside. After 12 hr of culture, α-methyl mannoside addition had little effect on Con A induction of ODC or on RNA and DNA synthesis measured at 24 to 72 hr. In contrast, cyclic AMP-dependent protein kinase activation, measured at 24 hr, could be completely inhibited when α-methyl mannoside was added as late as 12 or 20 hr to Con A-stimulated cultures. The nonmitogenic agglutinins, wheat germ agglutinin (WGA) and Agaricus bisporus lectin (ABL), failed to increase any of the parameters tested. DBcAMP also did not induce ODC or cause any increased RNA or DNA synthesis. However, DBcAMP (10-3 M) did cause activation of cyclic AMP-dependent protein kinase as early as 15 min of culture. DBcAMP activation of protein kinase was concentration-dependent, maximal at 12 hr, and remained elevated through 48 hr of culture. The addition of DBcAMP (10-3 M) to Con A-stimulated cultures caused a 90 to 95% inhibition of ODC activity and RNA and DNA synthesis. This inhibition was concentration-dependent and paralleled the degree of activation of cyclic AMP-dependent protein kinase elicited by each concentration.
These results suggest that the selective activation of cyclic AMP-dependent protein kinase and induction of increased ODC activity are early integral events linked to and regulating lymphocyte mitogenesis.