title: TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia.
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.
authors.affiliation: Mucosal Biology Research Center, University of Maryland, Baltimore, Maryland 21201, USA.
authors.forename: Anguo
authors.initials: A
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Gong
authors.forename: Sang W
authors.initials: SW
authors.lastname: Hyun
authors.forename: Kent Z Q
authors.initials: KZ
authors.lastname: Wang
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Cates
authors.forename: Darren
authors.initials: D
authors.lastname: Perkins
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Bannerman
authors.forename: Adam C
authors.initials: AC
authors.lastname: Puché
authors.forename: Vladimir Y
authors.initials: VY
authors.lastname: Toshchakov
authors.forename: Shengyun
authors.initials: S
authors.lastname: Fang
authors.forename: Philip E
authors.initials: PE
authors.lastname: Auron
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
authors.forename: Simeon E
authors.initials: SE
authors.lastname: Goldblum
mesh_terms: D020816:Amino Acid Motifs; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D048029:TNF Receptor-Associated Factor 6 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D008562:Membrane Glycoproteins; D053594:Myeloid Differentiation Factor 88; D010455:Peptides; D017472:Receptors, Interleukin-1; C434984:TIRAP protein, human; C495345:TLR4 protein, human; D048029:TNF Receptor-Associated Factor 6; D051197:Toll-Like Receptor 4; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases