The overall aim of the proposed research is to define the events that initiate and regulate endothelial cell contraction in an effort to elucidate the basis of increased vascular permeability. The primary hypothesis underlying the proposed work dictates that exposure to inflammatory mediators activates the endothelial cell actomyosin based contractile system. This hypothesis states that phosphorylation of myosin II in concert with assembly of the filamentous cytoskeleton (actin, myosin IIA and IIB) is essential for activation of endothelial cell contractile activity. Calcium (Ca2+) stimulated phosphorylation of the myosin II regulatory light chain (RLC) by myosin light chain kinase (MLCK) and Ca2+ independent phosphorylation of myosin II RLC by p-21 activated kinase (PAK2) have been shown to activate endothelial cell contraction. In nonmuscle cells, MLCK-catalyzed phosphorylation results in mono- and diphosphorylation of RLC at site Ser19 or sites Ser19 and Thr18, respectively (36). Within the last 10 years additional pathways of myosin II activation have been discovered and are currently being defined (5,13,19,20,35,48,). In endothelial cells, we have shown that the small GTPase dependent enzyme, PAK2 catalyzes Ca2+ independent activation of nonmuscle myosin II by phosphorylation of the RLC that is restricted to site Ser19 (19). This PAK2-mediated monophosphorylation results in a less forceful contractile response than when the RLC is diphosphorylated by MLCK. More recent studies have shown that PAK2 also phosphorylates unactivated MLCK which results in inhibition of MLCK activation by Ca2+ (35). Collectively, these findings have clearly established a role for PAK2 in mediating and regulating myosin II activity, and thus contractile activity, in endothelial cells. The goal of the proposed studies is to biochemically and morphologically characterize myosin II activation by Ca2+ - dependent and Ca2+ -independent signaling pathways in endothelial cells. The working hypothesis is that specific pools of myosin II, determined by the heavy chain isoform specificity (IIA vs IIB), are activated by enzyme specific (MLCK210, PAK2, Rho- kinase) modifications (monophosphorylation or diphosphorylation). Functionally, regulated differential phosphorylation of myosin II would allow the endothelial cell to react to a variety of physiological signals with graded contractile responses.
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