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

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.

Pubmed ID: 22447928

Authors

  • Liu A
  • Gong P
  • Hyun SW
  • Wang KZ
  • Cates EA
  • Perkins D
  • Bannerman DD
  • Puché AC
  • Toshchakov VY
  • Fang S
  • Auron PE
  • Vogel SN
  • Goldblum SE

Journal

The Journal of biological chemistry

Publication Data

May 11, 2012

Associated Grants

  • Agency: NIAID NIH HHS, Id: AI082299
  • Agency: NIAID NIH HHS, Id: AI18797
  • Agency: NHLBI NIH HHS, Id: HL089179
  • Agency: NIAID NIH HHS, Id: R01 AI018797
  • Agency: NIAID NIH HHS, Id: R01 AI082299

Mesh Terms

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