Protein kinase A activation by retinoic acid in the nuclei of HL60 cells.
Protein kinase A activation by retinoic acid in the nuclei of HL60 cells.
复制标题
HL60 细胞核中视黄酸激活蛋白激酶 A。
DOI：
10.1016/j.bbagen.2016.11.039
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
N.
中科院分区：
文献类型：
--
作者：
Sakai;A.;Imai;M.;Takahashi;K.;Hasegawa;S.;Yamasaki;M.;Ohba;T.;and Takahashi;N.
背景蛋白激酶 A (PKA) 的激活发生在视黄酸 (RA) 诱导的人早幼粒细胞白血病 HL60 细胞的粒细胞分化过程中。众所周知，PKA 的 RIIα 调节亚基在分化早期被 RA（视黄酰化）修饰。我们研究了细胞分化过程中RA对PKA的影响，以了解该过程在RIIα亚基视黄酰化中的潜在意义。方法采用免疫印迹、免疫沉淀、共聚焦显微镜、PCR和PKA活性测定来表征RA对PKA的影响。结果我们发现RA诱导HL60细胞中RIIα的细胞内移动和PKA的激活。在 RA 处理的 HL60 细胞中观察到 RIIα 水平增加。免疫印迹和免疫染色分析检测到，RA 治疗改变了 PKA 亚基、RIIα 和 Cα 的细胞内定位，并增加了它们在细胞核中的蛋白质水平。与核 Cα 的增加一致，与对照细胞相比，RA 处理的 HL60 细胞显示 PKA 的蛋白质磷酸化活性和核级分中磷酸化蛋白质的水平均有所增加。此外，与对照细胞相比，RA 处理的 HL60 细胞中 RIIα 蛋白稳定。结论这些结果表明 RA 稳定 RIIα 蛋白并激活细胞核中的 PKA，从而增加核蛋白的磷酸化。一般意义我们的证据表明 PKA 的视黄酰化可能有助于其稳定和激活，这可能参与 RA 诱导 HL60 细胞粒细胞分化的能力。
BackgroundActivation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits.MethodsImmunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA.ResultsWe found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells.ConclusionsThese results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins.General significanceOur evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.