Patel R C, Stanton P, McMillan N M, Williams B R, Sen G C
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195, USA.
Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8283-7. doi: 10.1073/pnas.92.18.8283.
The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.
干扰素诱导的双链（ds）RNA激活蛋白激酶（PKR）具有抗病毒、抗细胞和抗肿瘤活性。其通过自身磷酸化进行酶促激活的机制以及酶无活性突变体所观察到的显性负抑制表型引发了PKR二聚化的观点。在此，我们提供直接证据支持PKR-PKR相互作用。我们表明，在不存在dsRNA的情况下，放射性标记的PKR能与基质结合的未标记PKR特异性相互作用。自我缔合活性部分存在于170个残基的N端区域，该区域也构成双链RNA结合结构域（DRBD）。DRBD能与基质结合的PKR或基质结合的DRBD结合。通过化学交联直接证明了DRBD的二聚化。亲和层析和电泳迁移超迁移分析表明，无法结合dsRNA的突变体仍可表现出蛋白质-蛋白质相互作用。在哺乳动物细胞的双杂交转录激活分析中也能观察到PKR-PKR相互作用，因此这很可能是PKR在体内活性的一个重要特征。
