Molecular characterization of Hunk, a novel regulator of pancreatic β-cell function

本研究项目涉及Hunk基因的分子特征，该基因最近在Attie实验室通过数量性状位点分析（QTL）鉴定，并显示出对小鼠分离胰岛中胰岛素分泌的负调节作用。Hunk是一种丝氨酸/苏氨酸蛋白激酶，其直接底物未知。然而，这些底物的鉴定对于理解蛋白激酶调节胰岛素分泌的潜在机制是必要的。为了获得对可能的Hunk底物的初步了解，已经对来自Hunk野生型和Hunk敲除小鼠的胰岛进行了磷酸化蛋白质组分析。结果表明，两种基因型间有243个蛋白质发生了差异磷酸化，其中Pard 3b基因的磷酸化程度最高。有趣的是，人类PARD 3B基因位点已经与墨西哥人群中的2型糖尿病（T2 D）风险相关。然而，磷酸化蛋白质组分析不能区分Hunk的直接底物和作为Hunk敲除的间接结果可能差异磷酸化的蛋白质。因此，本项目的主要研究目标是确定Hunk的直接底物。为了使他们的检测，化学遗传学的方法，计划已经成功地应用于其他几种蛋白激酶的生理底物的识别。该方法首先需要产生所谓的类似物敏感（AS）激酶，其能够结合ATP类似物。因此，使用分子克隆方法，第一个任务将是创建AS-Hunk激酶。随后，该构建体将用于体外激酶测定，其中将应用仅可被AS-Hunk激酶利用的ATP类似物，从而允许确定Hunk特异性磷酸化。因此，来自Hunk基因敲除小鼠以及人类胰岛中的磷酸化底物应该被纯化，蛋白质将通过质谱法鉴定，这将与威斯康星州大学的Lloyd Smith教授合作执行。最后，通过在所鉴定的底物的磷酸化位点中表达模拟磷酸化或非磷酸化状态的特定突变，将在功能性体外测定中进一步研究磷酸化蛋白的潜在影响。

The present research project deals with the molecular characterization of Hunk, a gene which has recently been identified by quantitative trait locus analysis (QTL) in the Attie lab and was shown to negatively regulate insulin secretion in murine isolated pancreatic islets. Hunk is a Serine / Threonine protein kinase whose direct substrates are not known. However, the identification of these substrates is necessary to understand the underlying mechanism by which the protein kinase regulates insulin secretion. To gain preliminary insight into possible Hunk substrates, a phosphoproteome analysis was already conducted on islets from Hunk wild-type and Hunk knockout mice. This analysis revealed 243 proteins differentially phoyphorylated between the two genotypes, the strongest phosphorylation change occurred on the gene Pard3b. Interestingly, the human PARD3B gene locus has already been associated with type 2 diabetes (T2D) risk in Mexican populations. However, the phosphoproteome analysis was not able to distinguish between direct substrates for Hunk, and proteins that may be differentially phosphorylated as an indirect consequence of the Hunk knockout. As a consequence, the primary research objective of this project consists in the identification of the direct substrates for Hunk. To allow their detection, a chemical-genetic approach is planned that has already been successfully applied for the identification of the physiological substrates of several other protein kinases. This method will firstly require the generation of a so called analogue-sensitive (AS) kinase, which is able to bind ATP analogs. Thus, using molecular cloning methods, the first task will be the creation of the AS-Hunk kinase. Subsequently, this construct will be used in an in vitro kinase assay in which ATP analogs will be applied that can only be utilized by the AS-Hunk kinase and thus allows the determination of phosphorylation specifically by Hunk. Consequently, phosphorylated substrates in pancreatic islets from Hunk knockout mice as well as in human islets are supposed to be purified and proteins will be identified by mass spectrometry, which will be executed in cooperation with Professor Lloyd Smith at the University of Wisconsin. Finally, by expressing specific mutations in the phosphorylation sites of the identified substrates that mimic either the phosphorylated- or the unphosphorylated state, potential impacts of the phosphorylated proteins will be further investigated in functional in vitro assays.