Identification of Novel Protein Kinases Dependent on Phosphocreatine Rather than ATP

摘要 蛋白质的翻译后修饰是调节蛋白质活性的主要机制。 其中，最常见的是一组被称为蛋白激酶的酶对蛋白质的磷酸化。 蛋白激酶(PKs)参与了真核生物中几乎所有具有生物学意义的途径。 代谢调节需要蛋白激酶在每个系统中发挥作用，包括胰岛素和胰高血糖素 作用、糖原分解、脂肪生成和适应性产热。AKT蛋白激酶活性过高 胰岛素的下游作用也被怀疑是肥胖和癌症之间的关键环节。这个 哺乳动物基因组包括约560个已知的酶，约占整个编码基因组的0.2%。到目前为止 众所周知，这些酶中的每一种都利用三磷酸腺苷作为高能磷酸供体，转移 γ-磷酸作用于(主要)丝氨酸、苏氨酸或酪氨酸残基。大自然已经发展起来了 另一种通常比三磷酸腺苷更丰富的高能磷酸盐分子： 磷酸肌酸(CRP)。因为三磷酸腺苷是三磷酸腺苷合成酶的抑制剂，所以细胞不能储存三磷酸腺苷。相反， 三磷酸腺苷的γ-磷酸可以转移到肌酸(Cr)，再生腺苷二磷酸并允许电子 运输链将继续朝着“前进”的方向运行。我们最近的工作证明了一个 肌酸循环无效的生热脂肪细胞中肌酸和C反应蛋白的非典范作用 磷酸化和去磷酸化。这个无效的循环在不做功的情况下消耗能量，因此 会导致热量的产生。这项工作展示了肌酸比“公正”更广泛的功能 能量储存让我们提出了一个不同寻常的问题：有没有优先利用 C反应蛋白？事实上，我们已经在这里使用高分辨率蛋白质质谱学证明了，棕色 脂肪细胞提取物可以利用C反应蛋白来磷酸化某些肽部位(S/T和Y残基)， 当使用三磷酸腺苷作为底物时，不会被修饰。我们前进的主要目标是(1)演示 这些磷酸化事件是从CRP到目标蛋白的直接磷酸转移反应(2) 为了证明这些磷酸化作用依赖于体内的C反应蛋白，使用小鼠的肌红蛋白模型 和C反应蛋白缺陷型动物(3)纯化和鉴定依赖C反应蛋白的蛋白激酶。这些可能是新的 当使用C反应蛋白作为靶标时，改变多肽靶标特异性的动态体或已知的PKs的成员 底物(4)进行生化和生物物理研究以表征酶反应和 确定PK上的CRP结合位点。(5)研究C反应蛋白蛋白的生理意义，通过 突变蛋白质靶标中的特定靶点，并消融依赖于CRP的PKs本身。这 该项目将在生物化学和生理学领域开辟一个潜在的重要新领域，并代表着 高风险、高回报类型的项目，这是催化剂奖的目标。

Abstract Post-translational modifications of proteins are a major mechanism for the modulation of protein activity. Among these, the most common is protein phosphorylation by a set of enzymes called protein kinases. Protein kinases (PKs) are involved in almost every pathway of biological significance in eukaryotes. Metabolic regulation requires protein kinase function in every system, including insulin and glucagon action, glycogenolysis, adipogenesis and adaptive thermogenesis. Excessive AKT protein kinase activity downstream of insulin action is also suspected of being a key link between obesity and cancer. The mammalian kinome includes some 560 known enzymes, about 0.2% of the entire coding genome. As far as it is known, every one of these enzymes utilizes ATP as a high-energy phosphate donor, transferring the γ-phosphate of ATP onto (mainly) serine, threonine or tyrosine residues. Nature has developed another high-energy phosphate containing molecule that is often more abundant than ATP: phosphocreatine (CrP). Because ATP is an inhibitor of ATP synthase, cells can't store ATP. Instead, the γ-phosphate of ATP can be transferred to creatine (Cr), regenerating ADP and allowing the electron transport chain to continue functioning in the “forward” direction. Our recent work has demonstrated a non-canonical function of Cr and CrP in thermogenic adipose cells, which run a futile cycle of creatine phosphorylation and de-phosphorylation. This futile cycle expends energy without doing work and hence, results in the generation of heat. This work demonstrating a broader function of creatine than “just” energy storage caused us to ask an unusual question: are there protein kinases that preferentially use CrP? In fact, we have demonstrated here, using high-resolution protein Mass Spectrometry, that brown fat cell extracts can utilize CrP to phosphorylate certain peptide sites (at both S/T and Y residues) that are not modified when ATP is used as a substrate. Our key goals moving forward are to (1) demonstrate that these phosphorylation events are direct phosphate transfer reaction from CrP to target proteins (2) to demonstrate that these phosphorylations are dependent on CrP in vivo, using murine models of Cr and CrP-deficient animals (3) purify and characterize the CrP-dependent PKs. These may be new members of the kinome or known PKs that alter peptide target specificity when they use CrP as a substrate (4) perform biochemical and biophysical studies to characterize the enzymatic reactions and identify the CrP binding sites on the PKs. (5) investigate the physiological importance of the CrP PKs, by mutating specific target sites in protein targets and ablating the CrP-dependent PKs themselves. This project will open up a potentially important new area in biochemistry and physiology, and represents a “high-risk, high-reward type of project for which the Catalyst Award is intended.