Submitted on April 2, 2008
Revised on July 7, 2008
Accepted on July 29, 2008
Crosstalk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids
Anna A Birukova1, Elena Alekseeva1, Ivan Cokic1, Christopher E Turner2, and Konstantin G Birukov3*
1 The University of Chicago
2 SUNY Upstate Medical University
3 University of Chicago
* To whom correspondence should be addressed. E-mail: kbirukov{at}medicine.bsd.uchicago.edu.
We have previously reported the barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on the pulmonary endothelial cells (EC), delineated the role of Rac- and Cdc42-dependent mechanisms, and described the involvement of focal adhesion protein paxillin in the enhancement of EC barrier upon OxPAPC challenge. This study examined a potential role of paxillin in the feedback mechanism of Rac regulation by focal adhesions in the OxPAPC-stimulated EC. Our results demonstrate that OxPAPC induced Rac-dependent, Rho-independent peripheral accumulation of paxillin-containing focal adhesions accompanied by time-dependent paxillin phosphorylation. Molecular inhibition of Rac decreased association of paxillin with the Rac-specific guanine nucleotide exchange factor 
PIX. Molecular inhibition of paxillin also attenuated OxPAPC-induced enhancement of adherens junctions critical for EC barrier protective response, accumulation of VE-cadherin in the membrane fractions, and decreased activation of Rac and its effector PAK1. Expression of paxillin with a mutated PAK1-dependent phosphorylation site (S273A) attenuated OxPAPC-induced PAK1 activation and EC barrier-protective response. These results suggest that PAK1-specific paxillin phosphorylation at Ser-273 is critically involved in the positive-feedback regulation of Rac/PAK1 pathway and may contribute to the sustained enhancement of EC barrier caused by oxidized phospholipids.
