TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia

J Biol Chem. 2012 May 11;287(20):16132-45. doi: 10.1074/jbc.M111.310102. Epub 2012 Mar 23.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Capillary Permeability / drug effects
  • Capillary Permeability / physiology*
  • Cells, Cultured
  • Endothelial Cells
  • Enzyme Activation / drug effects
  • Enzyme Activation / physiology
  • Gene Silencing
  • Humans
  • Interleukin-1 Receptor-Associated Kinases / genetics
  • Interleukin-1 Receptor-Associated Kinases / metabolism
  • Lipopolysaccharides / pharmacology
  • Lung
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Myeloid Differentiation Factor 88 / genetics
  • Myeloid Differentiation Factor 88 / metabolism
  • Peptides / pharmacology
  • Protein Binding / drug effects
  • Protein Binding / genetics
  • Proto-Oncogene Proteins c-fyn / genetics
  • Proto-Oncogene Proteins c-fyn / metabolism
  • Receptors, Interleukin-1 / genetics
  • Receptors, Interleukin-1 / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • TNF Receptor-Associated Factor 6 / agonists
  • TNF Receptor-Associated Factor 6 / genetics
  • TNF Receptor-Associated Factor 6 / metabolism*
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • Ubiquitination / drug effects
  • Ubiquitination / genetics
  • src-Family Kinases / genetics
  • src-Family Kinases / metabolism*

Substances

  • Lipopolysaccharides
  • MYD88 protein, human
  • Membrane Glycoproteins
  • Myeloid Differentiation Factor 88
  • Peptides
  • Receptors, Interleukin-1
  • TIRAP protein, human
  • TLR4 protein, human
  • TNF Receptor-Associated Factor 6
  • Toll-Like Receptor 4
  • FYN protein, human
  • Proto-Oncogene Proteins c-fyn
  • src-Family Kinases
  • IRAK1 protein, human
  • Interleukin-1 Receptor-Associated Kinases