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知识取得了重大进展，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在心脏细胞中发现的，这种蛋白质在心脏中的功能几乎是未知的。Bmx在非心脏细胞的细胞存活中有明确的作用，其激活被认为涉及磷酸肌醇-3激酶（PI-SK）依赖性易位，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bmx相关蛋白，为该信号通路在心脏细胞中存在提供了生化基础。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，NO供体给药24小时后，Bmx蛋白表达、膜定位和酪氨酸磷酸化增强，并伴有NO供体提供的梗死保护表型。这些发现令人兴奋，因为它们提出了一种可能性，即由酪氨酸激酶引起的细胞内任务可能是由这种新的心脏蛋白Bmx特异性地执行的。重要的是，与非心脏细胞的传统观点相反，我们观察到Bmx定位于多个细胞内室，包括线粒体。此外，我们的初步数据表明，PP2抑制这一途径完全消除了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信号网络中的一组线粒体蛋白，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配。我们假设Bmx激活促进心肌细胞在缺血/再灌注损伤中的存活，部分通过调节线粒体功能。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。我们将描述完整的Bmx多蛋白复合物，并检查心脏中Bmx靶点的磷酸化。我们将使用Bmx KO小鼠明确询问Bmx在对缺血/再灌注损伤的基础易感性中的作用，并将检查先天保护模式对该分子存在的依赖。最后，我们将研究Bmx在应激反应中调节线粒体功能和通透性转变的作用。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/细胞生理学来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.