Preparing your results

Our searching services are busy right now. Your search will reload in five seconds.

X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

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

http://www.ncbi.nlm.nih.gov/pubmed/22447928

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 RIS Download

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

Research resources used in this publication

None found

Research tools detected in this publication

None found

Data used in this publication

None found

Associated grants

  • Agency: NIAID NIH HHS, Id: AI082299
  • Agency: NIAID NIH HHS, Id: AI18797
  • Agency: NHLBI NIH HHS, Id: HL089179
  • Agency: BLRD VA, Id: I01 BX002352
  • Agency: NIAID NIH HHS, Id: R01 AI018797
  • Agency: NIAID NIH HHS, Id: R01 AI082299

Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.