Characterization of a new family of protein kinases

描述(申请人提供)：由线粒体丙酮酸脱氢酶复合体(PDC)催化的反应连接糖酵解和柠檬酸循环。然而，这种反应的生理意义超出了它在能量生产中的作用，因为它也是合成甾醇和脂肪酸的碳源，并提供了控制丙酮酸组织水平的一般手段。因此，这种反应在一般新陈代谢、适应饥饿和缺氧、糖尿病、缺血和癌症中起着重要作用。哺乳动物的PDC是通过丙酮酸脱氢酶激酶(PDHK1、PDHK2、PDHK3和PDHK4)和丙酮酸脱氢酶磷酸酶(PDP1和PDP2)的多种同工酶催化的可逆磷酸化(失活)/去磷酸化(再激活)循环来调节的。可逆的磷酸化是PDC的短期和长期调节的原因。在过去的十年里，PDHK和PDP同工酶的结构和生化特性研究取得了重大进展。另一方面，它们在饥饿、糖尿病或癌症中对PDC的调节作用仍然知之甚少。一般认为，至少在饥饿和糖尿病中，PDC的长期调节在很大程度上反映了同工酶PDHK4的诱导。然而，在PDHK4/小鼠模型中缺乏明确的表型强烈地表明了替代机制的存在(S)。我们的初步数据表明，PDHK2可能在饥饿状态下对PDC的调控起关键作用，而PDHK3可能在癌细胞的Warburg效应中起作用。在本申请中，我们建议使用PDHK2-/-、PDHK3-/-和PDHK4-/-基因敲除小鼠模型来探索这些假说。这将通过以下具体目标来实现：1)确定PDHK2比活性稳定变化的分子基础；2)阐明PDHK2在适应饥饿中的作用；以及3)确定PDHK3在PDC调节中的生理作用。这些目标的实现将为丙酮酸脱氢酶反应在一般碳水化合物和脂肪代谢中的作用、在适应饥饿和癌症中的作用提供新的线索。从长远来看，它可能会导致高度特异的药物的开发，这些药物将减轻与糖尿病、缺血和癌症相关的并发症。与公共健康相关：对PDHK活性的长期调节是适应食物匮乏、低氧、高脂饮食等的核心。然而，当类似的机制在糖尿病、缺血或癌症中被激活时，结果是有害的。因此，揭示调控PDHK的分子机制对于开发新的治疗药物至关重要。

DESCRIPTION (provided by applicant): The reaction catalyzed by mitochondrial pyruvate dehydrogenase complex (PDC) links glycolysis and Citric Acid Cycle. However, physiological significance of this reaction goes beyond its role in energy production because it also serves as a source of carbon for biosynthesis of sterols and fatty acid and provides the general means to control the tissue levels of pyruvate. Consequently, this reaction plays an important role in general metabolism, in adaptation to starvation and hypoxia, in diabetes, ischemia, and cancer. Mammalian PDC is regulated through the reversible phosphorylation (inactivation)/dephosphorylation (re-activation) cycle catalyzed by multiple isozymes of pyruvate dehydrogenase kinase (PDHK1, PDHK2, PDHK3, and PDHK4) and pyruvate dehydrogenase phosphatase (PDP1 and PDP2). The reversible phosphorylation accounts for the short- and long-term regulation of PDC. Within the past decade, significant progress has been made in structural and biochemical characterization of PDHK and PDP isozymes. On the other hand, their roles in regulation of PDC in starvation, diabetes, or cancer remain poorly understood. It is generally believed that, at least in starvation and diabetes, the long-term regulation of PDC largely reflects the induction of isozyme PDHK4. However, the lack of a clear phenotype in PDHK4-/- mouse model strongly suggests the existence of alternative mechanism(s). Our preliminary data indicate that PDHK2 might be crucial for regulation of PDC in starvation, while PDHK3 might contribute to the Warburg effect in cancer cells. In this application, we propose to explore these hypotheses using PDHK2-/-, PDHK3-/-, and PDHK4-/- knockout mouse models. This will be achieved through the following Specific Aims: 1) to identify the molecular basis of stable changes in the specific activity of PDHK2; 2) to elucidate the role of PDHK2 in adaptation to starvation; and 3) to establish the physiological role of PDHK3 in regulation of PDC. Accomplishment of these objectives will shed new light on the role of pyruvate dehydrogenase reaction in general carbohydrate and lipid metabolism, in adaptation to starvation, and in cancer. In the long run, it may lead to the development of highly specific drugs that will alleviate complications associated with diabetes, ischemia, and cancer. PUBLIC HEALTH RELEVANCE: The long-term regulation of PDHK activity is central to adaptation to food deprivation, hypoxia, high-fat diet, etc. However, when similar mechanisms are activated in diabetes, ischemia, or cancer, the outcomes are detrimental. Thus, uncovering the molecular mechanisms responsible for the regulation of PDHK is crucial for the development of new therapeuticals.