Identification of the Kinase for Flow- and Stretch-elicited Phosphorylation of Platelet Endothelial Cell Adhesion Molecule-1 in Endothelial Cells

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.
Mechanical forces regulate physiological processes ranging from cellular functions and development of tissues to the health of an organism. One of the best known mammalian systems that are dynamically regulated by mechanical forces is the endothelium. Vascular endothelial cells (ECs) are under the influence of shear stress and stretch due to blood flow, however, the mechanism by which they sense and convert forces into biochemical activities is largely unknown. Platelet endothelial cell adhesion molecule (PECAM)-1 is expressed abundantly at interendothelial adhesion sites. PECAM-1 tyrosine phosphorylation is increased and causes intracellular signaling in ECs that are exposed to flow or when magnetic beads directly pull PECAM-1. We propose that PECAM-1 is a mechanosensor and that PECAM-1 phosphorylation is an initial step of ECs mechanosignaling. Since PECAM-1 has no intrinsic kinase activity, identification of the kinase is essential to elucidating PECAM-1-dependent mechanotransduction. To further establish PECAM-1 mechanosensing function, PECAM-1 activity was investigated in mechanically stretched ECs and detergent extracted EC models. The results demonstrate that stretch triggers PECAM-1 phosphorylation in both intact cells and extracted cell model, indicating that PECAM-1 response is a mechanically trigger event that does not require living cells. They further suggest that the fundamental mechanism, including the kinase, for stretch-induced PECAM-1 phosphorylation is preserved in the extracted cell model. To identify the kinase, a combined approach of pharmacological and molecular biological techniques was developed. Firstly, based on a database on inhibitor sensitivities of 244 kinases, several kinase inhibitors were used to treat the cell model and systematically screen potential kinases for PECAM-1 phosphorylation. Next, ECs were treated with small interfering RNA against each of the 3 kinase candidates obtained from the screening, and PECAM-1 phosphorylation