Fung P A, Labrecque R, Pederson T
Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, MA 01545, USA.
Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1064-8. doi: 10.1073/pnas.94.4.1064.
The human C1 heterogeneous nuclear ribonucleoprotein particle protein (hnRNP protein) undergoes a cycle of phosphorylation-dephosphorylation in HeLa cell nuclear extracts that modulates the binding of this protein to pre-mRNA. We now report that hyperphosphorylation of the C1 hnRNP protein is mediated by a kinase activity in nuclear extracts that is RNA-dependent. Although the basal phosphorylation of the C1 hnRNP protein in nuclear extracts reflects a casein kinase II-type activity, its RNA-dependent hyperphosphorylation appears to be mediated by a different kinase. This is indicated by the unresponsiveness of the RNA-stimulated hyperphosphorylation to casein kinase II inhibitors, and the distinct glycerol gradient sedimentation profiles of the basal versus RNA-stimulated C1 hnRNP protein phosphorylation activities from nuclear extracts. RNA-dependent phosphorylation was observed both for a histidine-tagged recombinant human C1 hnRNP protein added to nuclear extracts and also for the endogenous C1 hnRNP protein. Additional results rule out protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, and double-stranded RNA-activated protein kinase as the enzymes responsible for the RNA-dependent hyperphosphorylation of the C1 hnRNP protein. These results reveal the existence in nuclear extracts of an RNA-dependent protein kinase activity that hyperphosphorylates a known pre-mRNA binding protein, and define an additional element to be integrated into the current picture of how nuclear proteins are regulated by phosphorylation.
人C1异质性核核糖核蛋白颗粒蛋白（hnRNP蛋白）在HeLa细胞核提取物中经历磷酸化-去磷酸化循环，该循环调节此蛋白与前体mRNA的结合。我们现在报告，C1 hnRNP蛋白的过度磷酸化由核提取物中一种依赖RNA的激酶活性介导。虽然核提取物中C1 hnRNP蛋白的基础磷酸化反映了一种酪蛋白激酶II型活性，但其依赖RNA的过度磷酸化似乎由一种不同的激酶介导。这表现为RNA刺激的过度磷酸化对酪蛋白激酶II抑制剂无反应，以及核提取物中基础与RNA刺激的C1 hnRNP蛋白磷酸化活性具有不同的甘油梯度沉降图谱。对于添加到核提取物中的组氨酸标签重组人C1 hnRNP蛋白以及内源性C1 hnRNP蛋白，均观察到了依赖RNA的磷酸化。其他结果排除了蛋白激酶A、蛋白激酶C、钙调蛋白依赖性蛋白激酶II和双链RNA激活的蛋白激酶作为负责C1 hnRNP蛋白依赖RNA过度磷酸化的酶。这些结果揭示了核提取物中存在一种依赖RNA的蛋白激酶活性，该活性使一种已知的前体mRNA结合蛋白过度磷酸化，并确定了一个额外的要素，可纳入当前关于核蛋白如何通过磷酸化进行调节的图景中。
