We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial cell (EC) cytoskeletal remodeling. In smooth muscles protein kinase C (PKC) has been proposed to mediate inhibition of MLCP in response to various agonists via phosphorylation/activation of CPI-17, a specific inhibitor of MLCP. We found that similar to smooth muscle, human lung microvascular endothelial cells (HLMVEC) expressed CPI-17. However, the properties and role of CPI-17 in endothelium are unknown. Ectopic expression of CPI-17 in pulmonary artery EC had no effect on the cytoskeleton under non-stimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions and MLC phosphorylation. Similar to PMA, inflammatory agonist histamine caused activation of CPI-17. Depletion of endogenous CPI-17 in HLMVC significantly attenuated histamine-induced increase in endothelial permeability. Together these data indicate a tight link between CPI-17 activation and changes in EC cytoskeleton. Up to now, no protein partners that bind to CPI-17 have been identified. To further evaluate a role of CPI-17 in EC cytoskeleton we employed BacterioMatch Two-Hybrid System (Stratagene) using Escherichia coli as a host for screening of human lung cDNA library and detection putative CPI-17 binding proteins. Sequence analysis of obtained clones revealed a potential interaction of CPI-17 with 30 proteins, including several key cytoskeletal proteins: alpha spectrin, plakoglobin, plectin and gelsolin. Collectively, these data indicate critical involvement of CPI-17 in the regulation of EC cytoskeleton. HL 58064; HL 67307; HL 68062; AHA and ALA of Maryland.