Identification of Novel Protein Kinases Dependent on Phosphocreatine Rather than ATP

依赖于磷酸肌酸而不是 ATP 的新型蛋白激酶的鉴定

• 批准号: 9979867
• 负责人: BRUCE M. SPIEGELMAN
• 金额: $ 82.06万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-31 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979867
• 关键词: Adipocytes; Animals; Area; Award; Binding Sites; Biochemical; Biochemical Reaction; Biochemistry; Biological; Brown Fat; Cell Extracts; Cells; Code; Creatine; Electron Transport; Enzymes; Eukaryota; Event; Futile Cycling; Generations; Genome; Glucagon; Goals; Insulin; Link; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Mus; Mutate; Natural regeneration; Nature; Obesity; Pathway interactions; Peptides; Phosphocreatine; Phosphorylation; Physiological; Physiology; Post-Translational Protein Processing; Protein Kinase; Proteins; Proto-Oncogene Proteins c-akt; Reaction; Regulation; Resolution; Running; Serine; Site; Specificity; System; Thermogenesis; Threonine; Tyrosine; Work; biophysical analysis; catalyst; glycogenolysis; high reward; high risk; in vivo; inhibitor/antagonist; inorganic phosphate; lipid biosynthesis; member; mouse model; novel

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.

抽象的 蛋白质的翻译后修饰是调节蛋白活性的主要机制。 其中，最常见的是一组称为蛋白激酶的酶磷酸化。 蛋白激酶（PK）几乎参与了真核生物中生物学意义的所有途径。 代谢调节需要在每个系统中蛋白激酶的功能，包括胰岛素和胰高血糖素 作用，糖原分解，脂肪形成和适应性热发生。过多的Akt蛋白激酶活性 胰岛素作用的下游也被怀疑是肥胖与癌症之间的关键联系。这 哺乳动物的活性组包括约560个已知酶，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使用ATP作为高能磷酸盐供体，转移 ATP的γ-磷酸在（主要）丝氨酸，苏氨酸或酪氨酸保留。大自然已经发展 另一个含有分子的高能磷酸盐通常比ATP更丰富： 磷酸盐（CRP）。由于ATP是ATP合酶的抑制剂，因此细胞无法存储ATP。相反， ATP的γ-磷酸可以转移到创建（CR），再生ADP并允许电子 运输链继续沿“向前”方向运行。我们最近的工作证明了 热脂肪细胞中CR和CRP的非典型功能，它运行徒劳的创造周期 磷酸化和去磷酸化。这个徒劳的循环探索能量而无需工作，因此 导致热量产生。这项工作证明了创造的更广泛功能，而不是“公正” 储能使我们提出了一个不寻常的问题：是否有优先使用的蛋白激酶 CRP？实际上，我们在这里使用高分辨率蛋白质质谱法证明了棕色 脂肪细胞提取物可以利用CRP磷酸化某些肽位点（在S/T和Y残差下） 当将ATP用作底物时，未修改。我们前进的主要目标是（1）证明 这些磷酸化事件是从CRP到靶蛋白的直接磷酸盐转移反应（2） 为了证明这些磷酸化取决于体内CRP，使用Cr的鼠模型 和CRP缺陷动物（3）纯化并表征了CRP依赖性PK。这些可能是新的 当使用CRP用作肽的肽靶特异性的Kinome或已知PK的成员 底物（4）进行生化和生物物理研究，以表征酶促反应和 识别PKS上的CRP结合位点。 （5）研究CRP PK的身体重要性， 在蛋白质靶标中突变特定的靶位点，并消除依赖CRP的PK本身。这 项目将在生物化学和生理学上开放一个潜在的重要新领域，并代表 “催化剂奖的高风险，高回报的项目。