TRAF6 Protein Couples Toll-like Receptor 4 Signaling to Src Family Kinase Activation and Opening of Paracellular Pathway in Human Lung Microvascular Endothelia

Anguo Liu,Ping Gong,Sang W Hyun,Kent Z.Q Wang,Elizabeth A Cates,Darren Perkins,Douglas D Bannerman,Adam C Puché,Vladimir Y Toshchakov,Shengyun Fang,Philip E Auron,Stefanie N Vogel,Simeon E Goldblum

doi:10.1074/jbc.m111.310102

Abstract

Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.

Highlights

Bacterial lipopolysaccharide (LPS) disrupts endothelial barrier integrity
We found that four members of the SFK family, c-Src, Fyn, Yes, and Lyn, were expressed in HMVEC-Ls and that selective silencing of c-Src, 3 The abbreviations used are: TLR4, Toll-like receptor 4; EC, endothelial cell; Ad, adenovirus; DN, dominant-negative; HMVEC-L, human lung microvascular EC; Meprin and TRAF Homology (MATH) domain, Meprin and TRAF homology domain; m.o.i., multiplicity of infection; RING, really interesting new gene; SFK, Src family kinase; SH domain, Src homology; SP, scrambled peptide; Toll/IL-1 receptor resistance (TIR) domain, Toll/ IL-1 receptor resistance homology domain; TIRAP, TIR domain-containing adapter protein; aa, amino acid
TRAF6 Required for LPS-induced Barrier Disruption—Having established that TRAF6 is required for LPS-induced SFK activation (Fig. 3), that SFK activation is required for its association to TRAF6 (Fig. 6), and having previously found that SFK activation is a prerequisite to LPS-induced endothelial barrier dysfunction [5], we evaluated whether TRAF6 might be necessary for LPS-induced barrier disruption

Summary

Background

Bacterial lipopolysaccharide (LPS) disrupts endothelial barrier integrity. Results: LPS increases association of a TRAF6 proline-rich SH3-binding motif (aa 461– 469) with c-Src and Fyn, followed by their ubiquitination and activation, which in turn increases endothelial paracellular permeability. Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream It engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVECLs) to open the paracellular pathway through Src family kinase (SFK) activation. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls. Gram-negative sepsis and its attendant endotoxemia can be complicated by life-threatening vascular leak syndromes, including the acute respiratory distress syndrome [1]. TRAF6 physically interacts with SFKs, and during this interaction, TRAF6 catalyzes Lys63-linked ubiquitination of SFKs that in turn reciprocally tyrosine phosphorylate TRAF6 in response to LPS

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION