Novel mechanisms of tyrosine kinase signaling in heart

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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.