MK2-mediated mechanisms of apoptosis during endothelial barrier disruption

Johns Hopkins University, Baltimore MD

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Acute Respiratory Distress Syndrome (ARDS) remains a frequent occurrence in the intensive care unit and is associated with high mortality. A crucial determinant of ARDS is endothelial cell (EC) apoptosis leading to endothelial barrier dysfunction, increased vascular permeability, and resulting hypoxia. Understanding the molecular mechanisms that regulate EC apoptosis following injurious stimuli is crucial to improving outcomes in ARDS. Mitogen activated protein kinase activated protein kinase 2 (MK2) is part of a critical stress activated protein kinase pathway that has been implicated in initiating the apoptotic cascade, though how MK2 interacts with the apoptotic cascade is unclear. Activation of the apoptotic cascade culminates in activation of the executioner caspase-3 (casp3). Published data from our lab has demonstrated that MK2 phosphorylates casp3, and I have generated preliminary data showing the kinase activity of MK2 potentiates casp3 activity in a model cell line. Interestingly, our lab has also identified that MK2 directly binds with casp3 to facilitate nuclear transport and promote execution of apoptosis independent of casp3 phosphorylation. In total, these data suggest MK2 interacts with the apoptotic cascade and specifically casp3 in two distinct ways: it phosphorylates casp3 via its kinase function and binds directly to casp3 for nuclear transport via a chaperone function. This application serves to provide a training vehicle as I elucidate further details about these two distinct mechanisms. In Aim 1, I will determine the role of MK2-dependent phosphorylation of caspase-3 in potentiating casp3 activation and apoptosis during endothelial barrier disruption. In Aim 2, I will evaluate binding between the docking region of MK2 and the putative binding domain of casp3 and the impact of this binding on nuclear translocation of casp3 and subsequent apoptosis and endothelial barrier disruption. Investigating these dual functions MK2 would provide novel mechanisms into the regulation of casp3 activity and execution of apoptosis following injury, and possible identification of a therapeutic target to improve vascular barrier integrity in ARDS. Through this proposal, I will learn not only highly specialized laboratory skills including techniques to evaluate protein-protein interactions and endothelial cell isolation, but also biology of kinase signaling and apoptosis. In addition, I will hone leadership and communication skills necessary for academic career development. The data I generate will create a strong foundation for a future K08 application, with the goal of becoming an independently funded physician-scientist studying molecular mechanisms of lung endothelial cell dysfunction in ARDS.