Novel mechanisms of tyrosine kinase signaling in heart

描述(由申请人提供)：尽管我们对心脏中非受体酪氨酸激酶的了解有了很大的进步，但对于这些家族中个别成员的功能缺乏明确的蓝图。特别是，BMX酪氨酸激酶的存在是最近才在心肌细胞中发现的，这种蛋白在心脏中的功能实际上是未知的。在非心肌细胞中，BMX在细胞存活中起着既定的作用，它的激活被认为涉及依赖于磷脂酰肌醇-3激酶(PI-SK)的转位，随后被Src和其他激酶磷酸化。与这一观点一致，我们的初步研究支持PI-3K和Src都是心脏中的BMX相关蛋白，为心肌细胞中存在这一信号通路提供了生化基础。我们对BMX在一氧化氮(NO)供体诱导的心脏保护中的作用进行了初步研究。我们观察到，在给予NO供体24小时后，BMX蛋白表达增强，膜定位和酪氨酸磷酸化，伴随着NO供体提供的非梗死性表型。这些发现是令人兴奋的，因为它们提高了归因于酪氨酸激酶的细胞内任务可能特定地由这种新的心脏蛋白BMX执行的可能性。重要的是，与来自非心肌细胞的传统观点不同，我们观察到BMX定位于多个细胞内隔室，包括线粒体。此外，我们的初步数据显示，用PP2抑制这一途径完全取消了NO供体治疗在线粒体和整个心脏水平上的心脏保护作用。同样，我们的初步蛋白质组学分析揭示了BMX信号网络中的线粒体蛋白质子集，为BMX调节心脏功能的机制提供了线索。我们的中心假设是，心脏保护涉及BMX的磷酸化，导致其激活和在细胞内重新分布。我们推测，BMX的激活促进了心肌细胞在缺血/再灌注损伤中的存活，部分是通过调节线粒体功能来实现的。我们将研究NO供体对BMX激活的机制，并确定PI-3K和Src在BMX细胞内定位、激酶活性和磷酸化状态中的作用。我们将鉴定完整的BMX多蛋白复合体，并检测心脏中BMX靶标的磷酸化。我们将使用BMX KO小鼠明确地询问BMX在基础缺血/再灌注损伤易感性中的作用，并将检验先天保护方式对该分子存在的依赖。最后，我们将研究BMX在调节线粒体功能和应激反应中的通透性转变中的作用。目前的应用结合了生物化学、蛋白质组学、高分辨率分子成像和动物/细胞生理学，以阐明正常和受保护心脏中BMX信号网络的基本生物学。

DESCRIPTION (provided by applicant): Despite significant advances in our knowledge of non-receptor tyrosine kinases in the heart, a clear blueprint for the functions of individual members of these families is lacking. In particular, the presence of Bmx tyrosine kinase was only recently discovered in cardiac cells, and the function of this protein in the heart is virtually unknown. Bmx has an established role in cell survival in non-cardiac cells and its activation is thought to involve phosphoinositide-3 kinase (PI-SK)-dependent translocation, followed by phosphorylation by Src and other kinases. In agreement with this notion, our preliminary studies support that both PI-3K and Src are Bmx-associated proteins in the heart, providing biochemical foundation for the existence of this signaling pathway in cardiac cells. We conducted pilot studies on the role of Bmx in nitric oxide (NO) donor-induced cardioprotection. We observed enhanced Bmx protein expression, membrane localization, and tyrosine phosphorylation 24 h after NO donor administration, concomitant with the infarct-sparing phenotype afforded by the NO donor. These findings were exciting, because they raised the possibility that intracellular tasks attributed to tyrosine kinases may specifically be carried out by this novel cardiac protein, Bmx. Importantly, in contrast to conventional wisdom from non-cardiac cells, we observe localization of Bmx to multiple intracellular compartments, including mitochondria. Furthermore, our preliminary data show that inhibition of this pathway with PP2 completely abolishes the cardioprotective effects of NO donor-treatment, both at the level of the mitochondria and the whole heart. Likewise, our preliminary proteomic analyses revealed a sub-set of mitochondrial proteins within the Bmx signaling network, providing clues regarding the mechanisms by which Bmx may regulate cardiac function. Our central hypothesis is that cardioprotection involves phosphorylation of Bmx, leading to its activation and intracellular redistribution. We hypothesize that Bmx activation promotes cardiac cell survival during ischemia/reperfusion injury, in part through regulation of mitochondrial function. We will examine mechanisms of Bmx activation in response to NO donors and define the roles of PI-3K and Src in Bmx intracellular localization, kinase activity and phosphorylation state. We will characterize intact Bmx multiprotein complexes and examine phosphorylation of Bmx targets in the heart. We will definitively interrogate the role of Bmx in basal susceptibility to ischemia/reperfusion injury using Bmx KO mice and will examine the reliance of innate protective modalities on the presence of this molecule. Lastly, we will investigate the role of Bmx to regulate mitochondrial function and permeability transition in response to stress. The present application combines biochemistry, proteomics, high-resolution molecular imaging and animal/cell physiology to elucidate the fundamental biology of the Bmx signaling network in the normal and protected heart.