Structural and functional studies of the human RIO kinases

描述(申请人提供)：在没有核糖体生物发生的情况下，癌细胞不能增殖。癌细胞中核仁大小的增加和几个癌基因上调核糖体生物合成就证明了这一点。因此，调节核糖体合成的速度可以提供一种阻止癌症进展的方法。核糖体RNA(RRNA)是核糖体的主要组成部分，在核仁中以复杂的、循序渐进的过程成熟。这涉及到几个在分子水平上仍知之甚少的加工因素。我的长期目标是利用X射线结晶学和生物化学来研究rRNA加工因子的分子机制。我将从RIO激酶的研究开始这项工作，这是一组古老的、高度保守的、非典型的丝氨酸激酶，据报道，它是18S rRNA最终成熟步骤所必需的。18S rRNA是小核糖体亚基的RNA成分。基于我们以前对古生代RIO激酶的结构研究，我假设RIO激酶必须以一种不同于经典蛋白激酶的独特方式与多肽底物和ATP相互作用。为了研究这一假设，我建议1.确定人Rio1和Rio3的底物特异性，以及2.确定人Rio1和Rio3的底物结合部位。这将展示RIO酶是如何与多肽底物相互作用的，并确定底物上直接由RIO酶进行磷酸化的残基。我将通过提纯人类Rio1和Rio3，确定每个蛋白质上的自动磷酸化位点，测量定点突变对磷酸化转移反应的影响以评估特定残基的重要性，基于磷酸化位点确定哺乳动物细胞中的假定底物，并解决具有结合底物的人类Rio1和Rio3的结构，从而实现这些目标。 这项工作将提供成功抑制RIO激酶所需的结构和功能信息，这将允许抑制rRNA加工。由于rRNA加工是核糖体生物发生的重要步骤，而核糖体生物发生是肿瘤进展所必需的，这可能为癌症治疗的发展提供机会。因此，这项研究将为评价RIO激酶作为癌症治疗发展的靶点提供信息。

DESCRIPTION (provided by applicant): Cancer cell proliferation cannot occur in the absence of ribosome biogenesis. This is evidenced by an increase in the size of nucleoli in cancer cells and the upregulation of ribosome biogenesis by several oncogenes. Therefore, modulating the rate of ribosome synthesis could provide a means of blocking cancer progression. Ribosomal RNA (rRNA), which comprises most of the ribosome, are matured in a complex, stepwise process in the nucleoli. This involves several processing factors which are still poorly understood at the molecular level. My long term goal is to use X-ray crystallography and biochemistry to study the molecular mechanisms of rRNA processing factors. I will begin this work with the study of the RIO kinases, a group of ancient, highly conserved, atypical serine kinases reported to be necessary for the final maturation step of the 18S rRNA. The 18S rRNA is the RNA component of the small ribosomal subunit. Based on our previous structural studies of archaeal RIO kinases, I hypothesize the RIO kinases must interact with peptide substrates and ATP in a unique manner distinct from that seen for canonical protein kinases. To investigate this hypothesis, I propose to 1. Determine the substrate specificity of human Rio1 and Rio3, and 2. Determine the substrate binding site of human Rio1 and Rio3. This will demonstrate how RIO kinases interact with peptide substrate and identify residues on the substrate which direct phosphorylation by RIO kinases. I will address these aims by purifying human Rio1 and Rio3, determining the autophosphorylation sites on each protein, measuring the effect of site-directed mutations on the phosphoryl transfer reaction to evaluate the importance of specific residues, identifying putative substrates in mammalian cells based on phosphorylation site determination and solving the structure or human Rio1 and Rio3 with bound substrates. This work will provide the structural and functional information required for the successful inhibition of the RIO kinases, which will allow inhibition of rRNA processing. Since rRNA processing is a major step of ribosome biogenesis, and ribosome biogenesis is necessary for cancer progression, this may provide an opportunity for the development of cancer therapeutics. Therefore, this study will provide information for the evaluation of the RIO kinases as targets for the development of cancer treatment.