Laboratory of Biochemistry, School of Medicine, University of Thessaly, Mezourlo 41110, Larissa, Greece.
J Cell Sci. 2010 Sep 1;123(Pt 17):2976-86. doi: 10.1242/jcs.068122. Epub 2010 Aug 10.
Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.
缺氧诱导因子 1（HIF-1）是一种转录激活因子，介导细胞对缺氧的反应，是抗肿瘤治疗的有前途的靶点，对于适应低氧环境、胚胎发生和肿瘤进展至关重要。HIF-1 是 HIF-1α的异二聚体，其表达受氧水平以及各种氧非依赖性机制的控制，以及 HIF-1β（或 ARNT），其表达是组成型的。在这项工作中，我们研究了 HIF-1α的 N 端异二聚化（PAS）结构域的磷酸化，并鉴定出丝氨酸 247 是由酪蛋白激酶 1δ（CK1δ）体外修饰的主要位点。令人惊讶的是，将该位点突变为丙氨酸增强了 HIF-1α的转录活性，这一结果通过在缺氧条件下抑制 CK1δ或使用小干扰 RNA（siRNA）介导的沉默来模拟。相反，CK1δ的过表达或丝氨酸 247 至天冬氨酸的磷酸模拟突变抑制了 HIF-1α的活性，而不影响其稳定性或核积累。免疫沉淀和体外结合实验表明，CK1 依赖性磷酸化 HIF-1α的丝氨酸 247 会损害其与 ARNT 的结合，这一观点也得到了 HIF-1α与 ARNT PAS-B 结构域之间复合物结构建模的支持。我们认为，CK1 对 HIF-1α的修饰代表了一种通过调节其两个亚基之间的相互作用来控制 HIF-1 在缺氧期间活性的新机制。
