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重大进展，但缺乏这些家族单个成员功能的清晰蓝图。特别是，Bmx酪氨酸激酶的存在是最近才发现的心脏细胞中，这种蛋白质在心脏中的功能几乎是未知的。Bmx在非心脏细胞中的细胞存活中具有确定的作用，并且其活化被认为涉及磷酸肌醇-3激酶（PI-SK）依赖性易位，随后通过Src和其他激酶磷酸化。与此一致，我们的初步研究支持PI-3 K和Src都是心脏中的Bmx相关蛋白，为心脏细胞中存在这种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。我们观察到增强Bmx蛋白表达，膜定位，酪氨酸磷酸化24小时后，NO供体管理，伴随着由NO供体提供的梗死保留表型。这些发现令人兴奋，因为它们提出了一种可能性，即酪氨酸激酶的细胞内任务可能是由这种新型心脏蛋白Bmx专门执行的。重要的是，与非心脏细胞的传统观点相反，我们观察到Bmx定位于多个细胞内隔室，包括线粒体。此外，我们的初步数据表明，用PP 2抑制该途径完全消除了NO供体治疗的心脏保护作用，无论是在线粒体水平还是在整个心脏水平。同样，我们的初步蛋白质组学分析揭示了Bmx信号网络中线粒体蛋白的子集，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，导致其激活和细胞内再分布。我们推测，Bmx激活促进心肌细胞存活在缺血/再灌注损伤，部分通过调节线粒体功能。我们将研究Bmx激活的机制，在响应NO供体和定义PI-3 K和Src在Bmx细胞内定位，激酶活性和磷酸化状态的作用。我们将表征完整的Bmx多蛋白复合物，并检查心脏中Bmx靶点的磷酸化。我们将明确询问的作用Bmx在基础易感性缺血/再灌注损伤使用Bmx基因敲除小鼠，并将检查依赖的先天性保护模式的存在下，这种分子。最后，我们将研究Bmx在调节线粒体功能和渗透性转换中的作用。本申请结合生物化学、蛋白质组学、高分辨率分子成像和动物/细胞生理学来阐明正常和受保护心脏中Bmx信号传导网络的基础生物学。

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.