Advertisement

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

Open AccessPublished:March 23, 2012DOI:https://doi.org/10.1074/jbc.M111.310102
      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 [14C]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 Lys63-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.

      Introduction

      Gram-negative sepsis and its attendant endotoxemia can be complicated by life-threatening vascular leak syndromes, including the acute respiratory distress syndrome (
      • Brigham K.L.
      • Meyrick B.
      Endotoxin and lung injury.
      ). The Gram-negative bacterial component responsible for this loss of endothelial barrier integrity is the outer membrane constituent, endotoxin or lipopolysaccharide (LPS) (
      • Bannerman D.D.
      • Goldblum S.E.
      Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation.
      ,
      • Bannerman D.D.
      • Fitzpatrick M.J.
      • Anderson D.Y.
      • Bhattacharjee A.K.
      • Novitsky T.J.
      • Hasday J.D.
      • Cross A.S.
      • Goldblum S.E.
      Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction. (1998).
      ). We have previously established that LPS directly opens the endothelial paracellular pathway (
      • Bannerman D.D.
      • Goldblum S.E.
      Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation.
      ,
      • Bannerman D.D.
      • Fitzpatrick M.J.
      • Anderson D.Y.
      • Bhattacharjee A.K.
      • Novitsky T.J.
      • Hasday J.D.
      • Cross A.S.
      • Goldblum S.E.
      Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction. (1998).
      ) and requires LPS-binding protein (
      • Goldblum S.E.
      • Brann T.W.
      • Ding X.
      • Pugin J.
      • Tobias P.S.
      Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.
      ), soluble CD14 (
      • Goldblum S.E.
      • Brann T.W.
      • Ding X.
      • Pugin J.
      • Tobias P.S.
      Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.
      ), and Toll-like receptor 4 (TLR4)
      The abbreviations used are: TLR4
      Toll-like receptor 4
      EC
      endothelial cell
      Ad
      adenovirus
      DN
      dominant-negative
      HMVEC-L
      human lung microvascular EC
      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
      TIR domain
      Toll/IL-1 receptor resistance homology domain
      TIRAP
      TIR domain-containing adapter protein
      aa
      amino acid.
      expression (
      • Gong P.
      • Angelini D.J.
      • Yang S.
      • Xia G.
      • Cross A.S.
      • Mann D.
      • Bannerman D.D.
      • Vogel S.N.
      • Goldblum S.E.
      TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. (2008).
      ) for optimal presentation to the endothelial cell (EC) surface. In human lung microvascular EC (HMVEC-L), LPS activates Src family kinase (SFK) and increases tyrosine phosphorylation of components within the EC-EC adherens junction or zonula adherens multiprotein complex, coincident with barrier disruption (
      • Gong P.
      • Angelini D.J.
      • Yang S.
      • Xia G.
      • Cross A.S.
      • Mann D.
      • Bannerman D.D.
      • Vogel S.N.
      • Goldblum S.E.
      TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. (2008).
      ). Prior broad spectrum SFK inhibition protected against both zonula adherens protein tyrosine phosphorylation and barrier disruption (
      • Gong P.
      • Angelini D.J.
      • Yang S.
      • Xia G.
      • Cross A.S.
      • Mann D.
      • Bannerman D.D.
      • Vogel S.N.
      • Goldblum S.E.
      TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. (2008).
      ). In these same studies, 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, Fyn, and Yes, but not Lyn, each diminished both LPS-induced tyrosine phosphorylation of vascular endothelial-cadherin and p120 catenin and barrier disruption (
      • Gong P.
      • Angelini D.J.
      • Yang S.
      • Xia G.
      • Cross A.S.
      • Mann D.
      • Bannerman D.D.
      • Vogel S.N.
      • Goldblum S.E.
      TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. (2008).
      ).
      The structure of SFKs is highly conserved (
      • Engen J.R.
      • Wales T.E.
      • Hochrein J.M.
      • Meyn 3rd, M.A.
      • Banu Ozkan S.
      • Bahar I.
      • Smithgall T.E.
      Structure and dynamic regulation of Src family kinases.
      ,
      • Benati D.
      • Baldari C.T.
      SRC family kinases as potential therapeutic targets for malignancies and immunological disorders.
      ). Each contains an NH2-terminal myristoylated region, a 50–70-amino acid region unique to each family member, an Src homology (SH)3 domain, an SH2 domain, a short linker region, a catalytic domain, and the regulatory COOH terminus. Upon activation, the inactive enzyme, which resides in a perinuclear location, is translocated to the cell periphery where the myristoylated NH2 terminus facilitates attachment to the plasma membrane. The SH3 domain recognizes proline-rich sequences, whereas the SH2 domain recognizes phosphotyrosine-containing proteins. The kinase domain contains a Tyr416 within its activation loop that is autophosphorylated upon activation. The COOH terminus contains a Tyr527 that is phosphorylated in the restrained state. Low affinity intramolecular associations between the SH2 domain and phosphorylated Tyr527 and between SH3 and the short linker region maintain the SFK in an autoinhibited conformation. High affinity binding partners for the SH2 and/or SH3 domains that competitively disrupt these autoinhibitory intramolecular interactions activate SFK activity (
      • Ingley E.
      Src family kinases. Regulation of their activities, levels, and identification of new pathways.
      ).
      TLR4 is the principal signal-transducing receptor for LPS (
      • Poltorak A.
      • He X.
      • Smirnova I.
      • Liu M.Y.
      • Van Huffel C.
      • Du X.
      • Birdwell D.
      • Alejos E.
      • Silva M.
      • Galanos C.
      • Freudenberg M.
      • Ricciardi-Castagnoli P.
      • Layton B.
      • Beutler B.
      Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice. Mutations in Tlr4 gene.
      ,
      • Lien E.
      • Means T.K.
      • Heine H.
      • Yoshimura A.
      • Kusumoto S.
      • Fukase K.
      • Fenton M.J.
      • Oikawa M.
      • Qureshi N.
      • Monks B.
      • Finberg R.W.
      • Ingalls R.R.
      • Golenbock D.T.
      Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.
      ). TLR4 is a membrane-spanning protein composed of an NH2-terminal ectodomain with leucine-rich repeats that binds an extracellular protein, MD2, that is required for ligand recognition, a transmembrane region, and an intracellular region that includes a conserved Toll/IL-1 receptor resistance (TIR) homology domain that participates in protein-protein interactions and downstream signaling (
      • Jin M.S.
      • Lee J.O.
      Structures of the toll-like receptor family and its ligand complexes.
      ,
      • Carpenter S.
      • O'Neill L.A.
      Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signaling proteins.
      ). TIR domain-containing adapter protein (TIRAP), also called MyD88 adapter-like (Mal), constitutively associates with the TIR motif of TLR4 (
      • Jin M.S.
      • Lee J.O.
      Structures of the toll-like receptor family and its ligand complexes.
      ,
      • O'Neill L.A.
      • Bowie A.G.
      The family of five. TIR-domain-containing adaptors in Toll-like receptor signaling.
      ). TIRAP/Mal contains a phosphatidylinositol 4,5-bisphosphate-binding domain that mediates its recruitment to the plasma membrane (
      • Kagan J.C.
      • Medzhitov R.
      Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling.
      ). Upon LPS interaction with MD2, TLR4 dimerizes (
      • Carpenter S.
      • O'Neill L.A.
      Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signaling proteins.
      ). Myeloid differentiation factor 88 (MyD88), another TIR domain-containing adapter molecule that also contains an NH2-terminal death domain, is recruited to and associates with TLR4 and TIRAP/Mal through TIR domain interactions (
      • O'Neill L.A.
      • Bowie A.G.
      The family of five. TIR-domain-containing adaptors in Toll-like receptor signaling.
      ,
      • Sheedy F.J.
      • O'Neill L.A.
      The Troll in Toll. Mal and Tram as bridges for TLR2 and TLR4 signaling.
      ). TIRAP-facilitated recruitment of MyD88 to the TLR4 receptor complex initiates the MyD88-dependent signaling pathway that has been implicated in LPS-induced tyrosine phosphorylation events (
      • Zeisel M.B.
      • Druet V.A.
      • Sibilia J.
      • Klein J.P.
      • Quesniaux V.
      • Wachsmann D.
      Cross-talk between MyD88 and focal adhesion kinase pathways.
      ,
      • Davis C.N.
      • Tabarean I.
      • Gaidarova S.
      • Behrens M.M.
      • Bartfai T.
      IL-1β induces a MyD88-dependent and ceramide-mediated activation of Src in anterior hypothalamic neurons.
      ). MyD88 recruits the serine/threonine kinases, IL-1 receptor-associated kinase (IRAK) 1 and 4 (
      • Akira S.
      • Takeda K.
      Toll-like receptor signaling.
      ). IRAK4 phosphorylates IRAK1 that undergoes a conformational change and autophosphorylation, after which IRAK1 dissociates from the TLR4 complex and forms a complex with tumor necrosis factor (TNF) α-associated factor (TRAF) 6 in the cytoplasm (
      • Akira S.
      • Takeda K.
      Toll-like receptor signaling.
      ). More recently, IRAK2 has been reported to play a more active role than IRAK1 in TLR4-mediated TRAF6 activation (
      • Keating S.E.
      • Maloney G.M.
      • Moran E.M.
      • Bowie A.G.
      IRAK-2 participates in multiple toll-like receptor signaling pathways to NFκB via activation of TRAF6 ubiquitination.
      ,
      • Lin S.C.
      • Lo Y.C.
      • Wu H.
      Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signaling.
      ,
      • Boch J.A.
      • Yoshida Y.
      • Koyama Y.
      • Wara-Aswapati N.
      • Peng H.
      • Unlu S.
      • Auron P.E.
      Characterization of a cascade of protein interactions initiated at the IL-1 receptor.
      ). TRAF6 forms a complex with transforming growth factor-β-activated kinase (TAK1), TAK1-binding protein (TAB) 1 and TAB2, which through activation of multiple transcription factors up-regulates proinflammatory gene expression (
      • Deng L.
      • Wang C.
      • Spencer E.
      • Yang L.
      • Braun A.
      • You J.
      • Slaughter C.
      • Pickart C.
      • Chen Z.J.
      Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.
      ,
      • Lamothe B.
      • Besse A.
      • Campos A.D.
      • Webster W.K.
      • Wu H.
      • Darnay B.G.
      Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation.
      ). Although it is well known that LPS elicits a wide range of host cell responses, studies of downstream TLR4 signaling have largely focused on signaling events that promote such gene expression. Furthermore, much of our understanding of TLR4 signaling has been established in cells of monocyte/macrophage lineage where both TLR4 and CD14 are highly expressed on the cell surface (
      • Dobrovolskaia M.A.
      • Vogel S.N.
      Toll receptors, CD14, and macrophage activation and deactivation by LPS.
      ). In ECs, a major target for circulating intravascular LPS, far less is understood (
      • Henneke P.
      • Golenbock D.T.
      Innate immune recognition of lipopolysaccharide by endothelial cells.
      ,
      • Dauphinee S.M.
      • Karsan A.
      Lipopolysaccharide signaling in endothelial cells.
      ). Hence, the one or more signaling molecules that couple the TLR4-LPS interaction in ECs to SFK activation and barrier disruption are not known.
      A candidate molecule that might extend the TLR4 signaling pathway to SFK activation is the cytoplasmic adapter protein TRAF6. In multiple non-EC systems, including osteoclasts, monocytes, dendritic cells, and glioblastoma and embryonic cell lines, TRAF6 interacts with c-Src after ligation of the type 1 interleukin (IL)-1 receptor (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Nakamura I.
      • Kadono Y.
      • Takayanagi H.
      • Jimi E.
      • Miyazaki T.
      • Oda H.
      • Nakamura K.
      • Tanaka S.
      • Rodan G.A.
      • Duong le T.
      IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.
      ,
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
      ) and members of the TNF receptor superfamily, including CD40 (
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ) and TNF-related activation-induced cytokine-R (
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ). Furthermore, TRAF6−/− and c-Src−/− mice share an unusual phenotype, osteopetrosis (
      • Lomaga M.A.
      • Yeh W.C.
      • Sarosi I.
      • Duncan G.S.
      • Furlonger C.
      • Ho A.
      • Morony S.
      • Capparelli C.
      • Van G.
      • Kaufman S.
      • van der Heiden A.
      • Itie A.
      • Wakeham A.
      • Khoo W.
      • Sasaki T.
      • Cao Z.
      • Penninger J.M.
      • Paige C.J.
      • Lacey D.L.
      • Dunstan C.R.
      • Boyle W.J.
      • Goeddel D.V.
      • Mak T.W.
      TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.
      ,
      • Soriano P.
      • Montgomery C.
      • Geske R.
      • Bradley A.
      (1991) Targeted disruption of the c-Src proto-oncogene leads to osteopetrosis in mice.
      ). All six members of the TRAF family contain a TRAF domain composed of a coiled-coil region in tandem with a more highly conserved COOH-terminal immunoglobulin-like Meprin and TRAF Homology (MATH) domain (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ,
      • Arch R.H.
      • Gedrich R.W.
      • Thompson C.B.
      Tumor necrosis factor receptor-associated factors (TRAFs). A family of adapter proteins that regulates life and death.
      ). The MATH domain participates in recruitment of TRAF proteins to receptors, multimerization with other TRAF molecules, and binding to IRAKs (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ,
      • Arch R.H.
      • Gedrich R.W.
      • Thompson C.B.
      Tumor necrosis factor receptor-associated factors (TRAFs). A family of adapter proteins that regulates life and death.
      ,
      • Ye H.
      • Arron J.R.
      • Lamothe B.
      • Cirilli M.
      • Kobayashi T.
      • Shevde N.K.
      • Segal D.
      • Dzivenu O.K.
      • Vologodskaia M.
      • Yim M.
      • Du K.
      • Singh S.
      • Pike J.W.
      • Darnay B.G.
      • Choi Y.
      • Wu H.
      Distinct molecular mechanism for initiating TRAF6 signaling.
      ). TRAF2–6 each contain an NH2-terminal RING finger domain with multiple zinc finger-like motifs (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ,
      • Arch R.H.
      • Gedrich R.W.
      • Thompson C.B.
      Tumor necrosis factor receptor-associated factors (TRAFs). A family of adapter proteins that regulates life and death.
      ). The RING domain, in concert with the first zinc finger region, permits TRAF6 to function as an autocatalytic Lys63 ubiquitin ligase required for downstream MAPK and NF-κB activation (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Arch R.H.
      • Gedrich R.W.
      • Thompson C.B.
      Tumor necrosis factor receptor-associated factors (TRAFs). A family of adapter proteins that regulates life and death.
      ,
      • Chen H.
      • Wu Y.
      • Zhang Y.
      • Jin L.
      • Luo L.
      • Xue B.
      • Lu C.
      • Zhang X.
      • Yin Z.
      Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination.
      ,
      • Lamothe B.
      • Campos A.D.
      • Webster W.K.
      • Gopinathan A.
      • Hur L.
      • Darnay B.G.
      The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL.
      ). Of relevance to this study, TRAF6 contains within its MATH domain a proline-rich, putative SH3-binding motif, RPTIPRNPK (aa 461–469), which is required for its physical association with c-Src (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ). However, this same TRAF6 sequence is insufficient for SFK activation (
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ), suggesting that additional sequences in TRAF6 regulate SFK catalytic activity. In this study, we define the molecular interaction through which TRAF6 couples LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls. More specifically, 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.

      DISCUSSION

      In this study, we have established the absolute requirement for TRAF6 in HMVEC-Ls for LPS-induced SFK activation (Fig. 3) and barrier disruption (Fig. 9). In HMVEC-Ls, LPS increases TRAF6 polyubiquitination and its association with IRAK1 (Fig. 2). Furthermore, LPS increased TRAF6 association with each of two SFKs, c-Src (Fig. 4) and Fyn (Fig. 5), and this TRAF6-SFK association required both an intact proline-rich putative SH3-binding motif in TRAF6 (Figs. 4B and 5C) and a catalytically active state in the SFKs (Fig. 6). TRAF6-catalyzed Lys63-linked ubiquitination of Fyn and possibly c-Src (Fig. 7) and SFK(s) increased tyrosine phosphorylation of TRAF6 (Fig. 8). Together, these data establish TRAF6 as the pivotal signaling molecule that couples LPS engagement of TLR4 to SFK activation and loss of endothelial barrier integrity.
      TLR4 activation can transduce signals downstream through both MyD88-dependent and MyD88-independent signaling pathways (
      • Carpenter S.
      • O'Neill L.A.
      Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signaling proteins.
      ,
      • Sheedy F.J.
      • O'Neill L.A.
      The Troll in Toll. Mal and Tram as bridges for TLR2 and TLR4 signaling.
      ,
      • Akira S.
      • Takeda K.
      Toll-like receptor signaling.
      ). In the MyD88-dependent pathway, recruitment of MyD88 to the TLR4 signaling complex is facilitated by the bridging adapter molecule, TIRAP/Mal (
      • Sheedy F.J.
      • O'Neill L.A.
      The Troll in Toll. Mal and Tram as bridges for TLR2 and TLR4 signaling.
      ). TIRAP/Mal also contains a putative TRAF6-binding motif that enables it to interact directly with and recruit TRAF6 transiently to the plasma membrane (
      • Mansell A.
      • Brint E.
      • Gould J.A.
      • O'Neill L.A.
      • Hertzog P.J.
      Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-κB activation by toll-like receptor (TLR)-2 and TLR4.
      ,
      • Verstak B.
      • Nagpal K.
      • Bottomley S.P.
      • Golenbock D.T.
      • Hertzog P.J.
      • Mansell A.
      MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4-mediated NF-kappaB proinflammatory responses.
      ). TIRAP/Mal is central to the MyD88-dependent pathway in the cases of TLR2 and TLR4. That either prior knockdown of TIRAP/Mal or overexpression of a TIRAP/Mal DN in HMVEC-Ls protected against LPS-induced SFK activation and barrier disruption (Fig. 1) indicates that these LPS-induced EC responses require an intact MyD88-dependent pathway. The MyD88-independent TLR4 signaling pathway utilizes another bridging adapter, TRAM, to recruit TRIF (also referred to as TICAM-1) to TLR4 (
      • Carpenter S.
      • O'Neill L.A.
      Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signaling proteins.
      ,
      • Sheedy F.J.
      • O'Neill L.A.
      The Troll in Toll. Mal and Tram as bridges for TLR2 and TLR4 signaling.
      ,
      • Akira S.
      • Takeda K.
      Toll-like receptor signaling.
      ). Recently, in a yeast two-hybrid system, the NH2-terminal portion of TRIF was shown to interact with the COOH terminus of TRAF6, and abrogation of this interaction strongly inhibited TRIF-mediated interferon-β induction (
      • Sasai M.
      • Tatematsu M.
      • Oshiumi H.
      • Funami K.
      • Matsumoto M.
      • Hatakeyama S.
      • Seya T.
      Direct binding of TRAF2 and TRAF6 to TICAM-1/TRIF adaptor participates in activation of the Toll-like receptor 3/4 pathway.
      ). Although our data establish both LPS-induced SFK activation and barrier disruption as TIRAP/Mal-dependent, thereby implicating involvement of the MyD88-dependent pathway, in these studies, participation of the TRIF-dependent pathway has not been formally excluded.
      The NH2-terminal RING domain of TRAF6 is an E3 ubiquitin ligase that, in concert with the dimeric E2 enzyme Ubc/Uev1A, catalyzes its own autoubiquitination via site-specific Lys63-linked polyubiquitin chains (
      • Deng L.
      • Wang C.
      • Spencer E.
      • Yang L.
      • Braun A.
      • You J.
      • Slaughter C.
      • Pickart C.
      • Chen Z.J.
      Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.
      ,
      • Lamothe B.
      • Besse A.
      • Campos A.D.
      • Webster W.K.
      • Wu H.
      • Darnay B.G.
      Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation.
      ). TRAF6 autoubiquitination is a prerequisite to selected downstream signaling events, including IκB kinase and MAPK activation (
      • Deng L.
      • Wang C.
      • Spencer E.
      • Yang L.
      • Braun A.
      • You J.
      • Slaughter C.
      • Pickart C.
      • Chen Z.J.
      Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.
      ,
      • Lamothe B.
      • Besse A.
      • Campos A.D.
      • Webster W.K.
      • Wu H.
      • Darnay B.G.
      Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation.
      ). Whether TRAF6 autoubiquitination is required for its association with SFKs was unknown. Because LPS increased SFK association with unmodified E. coli-derived GST-TRAF6 (FIGURE 4, FIGURE 5), TRAF6 ubiquitination is clearly not required for this interaction. When our prokaryote-generated GST-TRAF6 was processed for ubiquitin immunoblotting, as anticipated, ubiquitin could not be detected.
      A. Liu, personal communication.
      TRAF6 also has been reported to catalyze Lys63-linked ubiquitination of other signaling molecules such as AKT (
      • Yang W.L.
      • Wang J.
      • Chan C.H.
      • Lee S.W.
      • Campos A.D.
      • Lamothe B.
      • Hur L.
      • Grabiner B.C.
      • Lin X.
      • Darnay B.G.
      • Lin H.K.
      The E3 ligase TRAF6 regulates Akt ubiquitination and activation.
      ) and other ubiquitin ligases, including cIAP1 and -2 (
      • Tseng P.H.
      • Matsuzawa A.
      • Zhang W.
      • Mino T.
      • Vignali D.A.
      • Karin M.
      Different modes of ubiquitination of the adaptor TRAF3 selectively activate the expression of type I interferons and proinflammatory cytokines.
      ). In several reports, activated SFKs were shown to be ubiquitinated and targeted for degradation by the proteasome (
      • Oda H.
      • Kumar S.
      • Howley P.M.
      Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination. (1999).
      ,
      • Harris K.F.
      • Shoji I.
      • Cooper E.M.
      • Kumar S.
      • Oda H.
      • Howley P.M.
      Ubiquitin-mediated degradation of active Src tyrosine kinase.
      ,
      • Rao N.
      • Miyake S.
      • Reddi A.L.
      • Douillard P.
      • Ghosh A.K.
      • Dodge I.L.
      • Zhou P.
      • Fernandes N.D.
      • Band H.
      Negative regulation of Lck by Cbl ubiquitin ligase.
      ), presumably via Lys48-linked ubiquitination. We asked whether LPS activation of TRAF6 might lead to Lys63-linked ubiquitination of one or more SFKs. In HMVEC-Ls, increased Lys63-linked ubiquitination of Fyn, as detected by re-immunoprecipitation (Fig. 7G, lane 4) and possibly c-Src (Fig. 7A, lane 4), was detected in response to LPS. It is conceivable that SDS treatment during re-immunoprecipitation denatured and rendered the anti-c-Src antibody nonimmunogenic. Furthermore, TRAF6 was required for this modification (Fig. 7, B–D). Although TRAF6 autoubiquitination may be required under some conditions for multiple signaling events (
      • Deng L.
      • Wang C.
      • Spencer E.
      • Yang L.
      • Braun A.
      • You J.
      • Slaughter C.
      • Pickart C.
      • Chen Z.J.
      Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.
      ,
      • Lamothe B.
      • Besse A.
      • Campos A.D.
      • Webster W.K.
      • Wu H.
      • Darnay B.G.
      Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation.
      ,
      • Rao N.
      • Miyake S.
      • Reddi A.L.
      • Douillard P.
      • Ghosh A.K.
      • Dodge I.L.
      • Zhou P.
      • Fernandes N.D.
      • Band H.
      Negative regulation of Lck by Cbl ubiquitin ligase.
      ,
      • Walsh M.C.
      • Kim G.K.
      • Maurizio P.L.
      • Molnar E.E.
      • Choi Y.
      TRAF6 autoubiquitination-independent activation of the NFκB and MAPK pathways in response to IL-1 and RANKL.
      ,
      • Wang K.Z.
      • Galson D.L.
      • Auron P.E.
      TRAF6 is autoinhibited by an intramolecular interaction which is counteracted by trans-ubiquitination.
      ), this modification does not appear to regulate the TRAF6-SFK interaction. Whether Lys63-linked ubiquitination of SFKs influences either their catalytic state or their association with TRAF6 remains to be determined.
      TRAF6 and c-Src have been reported to interact physically in signaling pathways in non-EC systems (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Nakamura I.
      • Kadono Y.
      • Takayanagi H.
      • Jimi E.
      • Miyazaki T.
      • Oda H.
      • Nakamura K.
      • Tanaka S.
      • Rodan G.A.
      • Duong le T.
      IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.
      ,
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ,
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ). In murine osteoclasts and dendritic cells, TNF-related activation-induced cytokine, also known as RANKL, increased co-immunoprecipitation of c-Src with TRAF6 (
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ). In these same co-expression studies, an NH2-terminal deletion mutant, TRAF6(289–530), was sufficient to co-immunoprecipitate c-Src, whereas a COOH-terminal deletion mutant, TRAF6(1–289), could not. The authors found that GST-c-Src-SH3 interacted directly with in vitro translated TRAF6, and they mapped the interacting motif in TRAF6 to a proline-rich sequence, aa 461–469 (RPTIPRNPK). When one or more of these prolines was substituted with alanine(s), the TRAF6-c-Src association was disrupted. In murine osteoclast-like cells, the human T98G glioblastoma cell line, and human embryonic kidney HEK293T cells, human recombinant IL-1α also increased TRAF6-c-Src association in coimmunoprecipitation assays (
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
      ). Finally, IL-1β increased the association of a fluoroprobe-labeled TRAF6 with labeled c-Src in the cytoplasm of living HEK293 T cells (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ). Here, we now have established that the TRAF6-c-Src interaction can be extended to LPS/TLR4 signaling (Fig. 4), to another SFK, Fyn (Fig. 5), and to HMVEC-Ls (FIGURE 4, FIGURE 5).
      Although it is clear that TRAF6 can physically engage SFKs (FIGURE 4, FIGURE 5) (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Nakamura I.
      • Kadono Y.
      • Takayanagi H.
      • Jimi E.
      • Miyazaki T.
      • Oda H.
      • Nakamura K.
      • Tanaka S.
      • Rodan G.A.
      • Duong le T.
      IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.
      ,
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ,
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ), the relative hierarchical position between these two signaling elements within any one signaling network is unclear. In this study, we found that prior siRNA-induced silencing of TRAF6 completely blocked SFK activation (Fig. 3B), suggesting that TRAF6 is upstream of SFKs. However, we found that prior pharmacological blockade of SFK activation prevented SFK binding to TRAF6 (Fig. 6, B and C), suggesting that prior SFK activation is required for its interaction with TRAF6. Activation of SFKs involves autophosphorylation of Tyr416 within the activation loop and release from the autoinhibited restrained state (
      • Engen J.R.
      • Wales T.E.
      • Hochrein J.M.
      • Meyn 3rd, M.A.
      • Banu Ozkan S.
      • Bahar I.
      • Smithgall T.E.
      Structure and dynamic regulation of Src family kinases.
      ,
      • Benati D.
      • Baldari C.T.
      SRC family kinases as potential therapeutic targets for malignancies and immunological disorders.
      ). Perhaps only in this activated state with its open conformation is the SH3 domain within SFK accessible for binding to the proline-rich putative TRAF6 SH3-binding domain. A number of other studies have addressed this same issue (
      • Nakamura I.
      • Kadono Y.
      • Takayanagi H.
      • Jimi E.
      • Miyazaki T.
      • Oda H.
      • Nakamura K.
      • Tanaka S.
      • Rodan G.A.
      • Duong le T.
      IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.
      ,
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ,
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ), yet the hierarchical relationship between TRAF6 and SFKs remains unresolved. In selected experimental systems, overexpression of TRAF6 stimulated SFK activation (
      • Wong B.R.
      • Besser D.
      • Kim N.
      • Arron J.R.
      • Vologodskaia M.
      • Hanafusa H.
      • Choi Y.
      TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
      ) and SFK-mediated downstream signaling events (
      • Wang K.Z.
      • Wara-Aswapati N.
      • Boch J.A.
      • Yoshida Y.
      • Hu C.D.
      • Galson D.L.
      • Auron P.E.
      TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
      ,
      • Nakamura I.
      • Kadono Y.
      • Takayanagi H.
      • Jimi E.
      • Miyazaki T.
      • Oda H.
      • Nakamura K.
      • Tanaka S.
      • Rodan G.A.
      • Duong le T.
      IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.
      ,
      • Funakoshi-Tago M.
      • Tago K.
      • Sonoda Y.
      • Tominaga S.
      • Kasahara T.
      TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
      ,
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ). However, in cells of monocyte/macrophage lineage, stimulation of CD40, a member of the TNF receptor superfamily, recruits TRAF6 to the receptor and leads to c-Src activation (
      • Mukundan L.
      • Bishop G.A.
      • Head K.Z.
      • Zhang L.
      • Wahl L.M.
      • Suttles J.
      TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
      ). Preincubation of cells with a cell-permeable peptide that competitively inhibits TRAF6 recruitment and binding to CD40 did not prevent c-Src activation, suggesting that c-Src might be upstream of TRAF6. These combined data generated in various host systems do not conclusively indicate which of the two signaling molecules, TRAF6 or SFK(s), is upstream to the other. In this study, we found that TRAF6 catalyzes Lys63-linked ubiquitination of one or more SFKs, while at the same time, SFKs increase tyrosine phosphorylation of TRAF6. The interaction(s) between TRAF6 and SFKs is likely a complex two-way multistep process in which more than one TRAF6 domain participates and SFKs activate other SFKs.
      We now have established TRAF6 as a critical adapter molecule in HMVEC-Ls for LPS/TLR4-induced SFK activation (Fig. 3) and barrier disruption (Fig. 9). Other reports have similarly indicated that TRAF6 is a key mediator of vascular endothelial pathophysiology (
      • Pollet I.
      • Opina C.J.
      • Zimmerman C.
      • Leong K.G.
      • Wong F.
      • Karsan A.
      Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-κB and c-Jun N-terminal kinase.
      ,
      • Min J.K.
      • Cho Y.L.
      • Choi J.H.
      • Kim Y.
      • Kim J.H.
      • Yu Y.S.
      • Rho J.
      • Mochizuki N.
      • Kim Y.M.
      • Oh G.T.
      • Kwon Y.G.
      Receptor activator of nuclear factor (NF)-κB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice.
      ,
      • Choi Y.S.
      • Choi H.J.
      • Min J.K.
      • Pyun B.J.
      • Maeng Y.S.
      • Park H.
      • Kim J.
      • Kim Y.M.
      • Kwon Y.G.
      Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6-mediated endothelial nitric oxide production.
      ,
      • Zirlik A.
      • Bavendiek U.
      • Libby P.
      • MacFarlane L.
      • Gerdes N.
      • Jagielska J.
      • Ernst S.
      • Aikawa M.
      • Nakano H.
      • Tsitsikov E.
      • Schönbeck U.
      TRAF-1, -2, -3, -5, and -6 are induced in atherosclerotic plaques and differentially mediate proinflammatory functions of CD40L in endothelial cells.
      ). In human dermal microvascular ECs, TRAF6 is required for LPS-induced angiogenesis (
      • Pollet I.
      • Opina C.J.
      • Zimmerman C.
      • Leong K.G.
      • Wong F.
      • Karsan A.
      Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-κB and c-Jun N-terminal kinase.
      ), a multistep process that involves hyperpermeable vessels (
      • Carmeliet P.
      Mechanisms of angiogenesis and arteriogenesis.
      ). In fact, TRAF6 has been shown to mediate increased permeability across human umbilical vein EC monolayers in response to both receptor activator of NF-κB ligand (
      • Min J.K.
      • Cho Y.L.
      • Choi J.H.
      • Kim Y.
      • Kim J.H.
      • Yu Y.S.
      • Rho J.
      • Mochizuki N.
      • Kim Y.M.
      • Oh G.T.
      • Kwon Y.G.
      Receptor activator of nuclear factor (NF)-κB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice.
      ) and IL-33 (
      • Choi Y.S.
      • Choi H.J.
      • Min J.K.
      • Pyun B.J.
      • Maeng Y.S.
      • Park H.
      • Kim J.
      • Kim Y.M.
      • Kwon Y.G.
      Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6-mediated endothelial nitric oxide production.
      ). In human carotid arteries, TRAF6 expression is increased in atherosclerotic lesions, and silencing of TRAF6 blocks proatherogenic gene expression in human saphenous vein ECs (
      • Zirlik A.
      • Bavendiek U.
      • Libby P.
      • MacFarlane L.
      • Gerdes N.
      • Jagielska J.
      • Ernst S.
      • Aikawa M.
      • Nakano H.
      • Tsitsikov E.
      • Schönbeck U.
      TRAF-1, -2, -3, -5, and -6 are induced in atherosclerotic plaques and differentially mediate proinflammatory functions of CD40L in endothelial cells.
      ). It is conceivable that TRAF6 is a pivotal signaling element in a final common pathway for the host response to a subset of injurious stimuli, including bacterial LPS, for life-threatening vascular leak syndromes.

      Acknowledgments

      We thank Shirley A. Taylor for excellent secretarial support.

      References

        • Brigham K.L.
        • Meyrick B.
        Endotoxin and lung injury.
        Am. Rev. Respir. Dis. 1986; 133: 913-927
        • Bannerman D.D.
        • Goldblum S.E.
        Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation.
        Am. J. Physiol. 1997; 273: L217-L226
        • Bannerman D.D.
        • Fitzpatrick M.J.
        • Anderson D.Y.
        • Bhattacharjee A.K.
        • Novitsky T.J.
        • Hasday J.D.
        • Cross A.S.
        • Goldblum S.E.
        Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction. (1998).
        Infect. Immun. 1998; 66: 1400-1497
        • Goldblum S.E.
        • Brann T.W.
        • Ding X.
        • Pugin J.
        • Tobias P.S.
        Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.
        J. Clin. Invest. 1994; 93: 692-702
        • Gong P.
        • Angelini D.J.
        • Yang S.
        • Xia G.
        • Cross A.S.
        • Mann D.
        • Bannerman D.D.
        • Vogel S.N.
        • Goldblum S.E.
        TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. (2008).
        J. Biol. Chem. 2008; 283: 13437-13449
        • Engen J.R.
        • Wales T.E.
        • Hochrein J.M.
        • Meyn 3rd, M.A.
        • Banu Ozkan S.
        • Bahar I.
        • Smithgall T.E.
        Structure and dynamic regulation of Src family kinases.
        Cell. Mol. Life Sci. 2008; 65: 3058-3073
        • Benati D.
        • Baldari C.T.
        SRC family kinases as potential therapeutic targets for malignancies and immunological disorders.
        Curr. Med. Chem. 2008; 15: 1154-1165
        • Ingley E.
        Src family kinases. Regulation of their activities, levels, and identification of new pathways.
        Biochim. Biophys. Acta. 2008; 1784: 56-65
        • Poltorak A.
        • He X.
        • Smirnova I.
        • Liu M.Y.
        • Van Huffel C.
        • Du X.
        • Birdwell D.
        • Alejos E.
        • Silva M.
        • Galanos C.
        • Freudenberg M.
        • Ricciardi-Castagnoli P.
        • Layton B.
        • Beutler B.
        Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice. Mutations in Tlr4 gene.
        Science. 1998; 282: 2085-2088
        • Lien E.
        • Means T.K.
        • Heine H.
        • Yoshimura A.
        • Kusumoto S.
        • Fukase K.
        • Fenton M.J.
        • Oikawa M.
        • Qureshi N.
        • Monks B.
        • Finberg R.W.
        • Ingalls R.R.
        • Golenbock D.T.
        Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.
        J. Clin. Invest. 2000; 105: 497-504
        • Jin M.S.
        • Lee J.O.
        Structures of the toll-like receptor family and its ligand complexes.
        Immunity. 2008; 29: 182-191
        • Carpenter S.
        • O'Neill L.A.
        Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signaling proteins.
        Biochem. J. 2009; 422: 1-10
        • O'Neill L.A.
        • Bowie A.G.
        The family of five. TIR-domain-containing adaptors in Toll-like receptor signaling.
        Nat. Rev. Immunol. 2007; 7: 353-364
        • Kagan J.C.
        • Medzhitov R.
        Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling.
        Cell. 2006; 125: 943-955
        • Sheedy F.J.
        • O'Neill L.A.
        The Troll in Toll. Mal and Tram as bridges for TLR2 and TLR4 signaling.
        J. Leukocyte Biol. 2007; 82: 196-203
        • Zeisel M.B.
        • Druet V.A.
        • Sibilia J.
        • Klein J.P.
        • Quesniaux V.
        • Wachsmann D.
        Cross-talk between MyD88 and focal adhesion kinase pathways.
        J. Immunol. 2005; 174: 7393-7397
        • Davis C.N.
        • Tabarean I.
        • Gaidarova S.
        • Behrens M.M.
        • Bartfai T.
        IL-1β induces a MyD88-dependent and ceramide-mediated activation of Src in anterior hypothalamic neurons.
        J. Neurochem. 2006; 98: 1379-1389
        • Akira S.
        • Takeda K.
        Toll-like receptor signaling.
        Nat. Rev. Immunol. 2004; 4: 499-511
        • Keating S.E.
        • Maloney G.M.
        • Moran E.M.
        • Bowie A.G.
        IRAK-2 participates in multiple toll-like receptor signaling pathways to NFκB via activation of TRAF6 ubiquitination.
        J. Biol. Chem. 2007; 282: 33435-33443
        • Lin S.C.
        • Lo Y.C.
        • Wu H.
        Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signaling.
        Nature. 2010; 465: 885-890
        • Boch J.A.
        • Yoshida Y.
        • Koyama Y.
        • Wara-Aswapati N.
        • Peng H.
        • Unlu S.
        • Auron P.E.
        Characterization of a cascade of protein interactions initiated at the IL-1 receptor.
        Biochem. Biophys. Res. Commun. 2003; 303: 525-531
        • Deng L.
        • Wang C.
        • Spencer E.
        • Yang L.
        • Braun A.
        • You J.
        • Slaughter C.
        • Pickart C.
        • Chen Z.J.
        Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.
        Cell. 2000; 103: 351-361
        • Lamothe B.
        • Besse A.
        • Campos A.D.
        • Webster W.K.
        • Wu H.
        • Darnay B.G.
        Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation.
        J. Biol. Chem. 2007; 282: 4102-4112
        • Dobrovolskaia M.A.
        • Vogel S.N.
        Toll receptors, CD14, and macrophage activation and deactivation by LPS.
        Microbes Infect. 2002; 4: 903-914
        • Henneke P.
        • Golenbock D.T.
        Innate immune recognition of lipopolysaccharide by endothelial cells.
        Crit. Care Med. 2002; 30: S207-S213
        • Dauphinee S.M.
        • Karsan A.
        Lipopolysaccharide signaling in endothelial cells.
        Lab. Invest. 2006; 86: 9-22
        • Wang K.Z.
        • Wara-Aswapati N.
        • Boch J.A.
        • Yoshida Y.
        • Hu C.D.
        • Galson D.L.
        • Auron P.E.
        TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.
        J. Cell Sci. 2006; 119: 1579-1591
        • Nakamura I.
        • Kadono Y.
        • Takayanagi H.
        • Jimi E.
        • Miyazaki T.
        • Oda H.
        • Nakamura K.
        • Tanaka S.
        • Rodan G.A.
        • Duong le T.
        IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.
        J. Immunol. 2002; 168: 5103-5109
        • Funakoshi-Tago M.
        • Tago K.
        • Sonoda Y.
        • Tominaga S.
        • Kasahara T.
        TRAF6 and c-SRC induce synergistic AP-1 activation via PI3-kinase-AKT-JNK pathway.
        Eur. J. Biochem. 2003; 270: 1257-1268
        • Mukundan L.
        • Bishop G.A.
        • Head K.Z.
        • Zhang L.
        • Wahl L.M.
        • Suttles J.
        TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages.
        J. Immunol. 2005; 174: 1081-1090
        • Wong B.R.
        • Besser D.
        • Kim N.
        • Arron J.R.
        • Vologodskaia M.
        • Hanafusa H.
        • Choi Y.
        TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src.
        Mol. Cell. 1999; 4: 1041-1049
        • Lomaga M.A.
        • Yeh W.C.
        • Sarosi I.
        • Duncan G.S.
        • Furlonger C.
        • Ho A.
        • Morony S.
        • Capparelli C.
        • Van G.
        • Kaufman S.
        • van der Heiden A.
        • Itie A.
        • Wakeham A.
        • Khoo W.
        • Sasaki T.
        • Cao Z.
        • Penninger J.M.
        • Paige C.J.
        • Lacey D.L.
        • Dunstan C.R.
        • Boyle W.J.
        • Goeddel D.V.
        • Mak T.W.
        TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.
        Genes Dev. 1999; 13: 1015-1024
        • Soriano P.
        • Montgomery C.
        • Geske R.
        • Bradley A.
        (1991) Targeted disruption of the c-Src proto-oncogene leads to osteopetrosis in mice.
        Cell. 1991; 64: 693-702
        • Arch R.H.
        • Gedrich R.W.
        • Thompson C.B.
        Tumor necrosis factor receptor-associated factors (TRAFs). A family of adapter proteins that regulates life and death.
        Genes Dev. 1998; 12: 2821-2830
        • Ye H.
        • Arron J.R.
        • Lamothe B.
        • Cirilli M.
        • Kobayashi T.
        • Shevde N.K.
        • Segal D.
        • Dzivenu O.K.
        • Vologodskaia M.
        • Yim M.
        • Du K.
        • Singh S.
        • Pike J.W.
        • Darnay B.G.
        • Choi Y.
        • Wu H.
        Distinct molecular mechanism for initiating TRAF6 signaling.
        Nature. 2002; 418: 443-447
        • Chen H.
        • Wu Y.
        • Zhang Y.
        • Jin L.
        • Luo L.
        • Xue B.
        • Lu C.
        • Zhang X.
        • Yin Z.
        Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination.
        FEBS Lett. 2006; 580: 3145-3152
        • Lamothe B.
        • Campos A.D.
        • Webster W.K.
        • Gopinathan A.
        • Hur L.
        • Darnay B.G.
        The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL.
        J. Biol. Chem. 2008; 283: 24871-24880
        • Cates E.A.
        • Connor E.E.
        • Mosser D.M.
        • Bannerman D.D.
        Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-κB activation and apoptosis.
        Comp. Immunol. Microbiol. Infect. Dis. 2009; 32: 477-490
        • Yang M.
        • Omura S.
        • Bonifacino J.S.
        • Weissman A.M.
        Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells. Importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes.
        J. Exp. Med. 1998; 187: 835-846
        • Liu A.
        • Garg P.
        • Yang S.
        • Gong P.
        • Pallero M.A.
        • Annis D.S.
        • Liu Y.
        • Passaniti A.
        • Mann D.
        • Mosher D.F.
        • Murphy-Ullrich J.E.
        • Goldblum S.E.
        Epidermal growth factor-like repeats of thrombospondins activate phospholipase Cγ and increase epithelial cell migration through indirect epidermal growth factor receptor activation.
        J. Biol. Chem. 2009; 284: 6389-6402
        • Sui X.F.
        • Kiser T.D.
        • Hyun S.W.
        • Angelini D.J.
        • Del Vecchio R.L.
        • Young B.A.
        • Hasday J.D.
        • Romer L.H.
        • Passaniti A.
        • Tonks N.K.
        • Goldblum S.E.
        Receptor protein-tyrosine phosphatase micro-regulates the paracellular pathway in human lung microvascular endothelia. (2005).
        Am. J. Pathol. 2005; 166: 1247-1258
        • Toshchakov V.U.
        • Basu S.
        • Fenton M.J.
        • Vogel S.N.
        Differential involvement of BB loops of toll-IL-1 resistance (TIR) domain-containing adapter proteins in TLR4- versus TLR2-mediated signal transduction.
        J. Immunol. 2005; 175: 494-500
        • Toshchakov V.Y.
        • Fenton M.J.
        • Vogel S.N.
        Cutting edge. Differential inhibition of TLR signaling pathways by cell-permeable peptides representing BB loops of TLRs.
        J. Immunol. 2007; 178: 2655-2660
        • Yang W.L.
        • Wang J.
        • Chan C.H.
        • Lee S.W.
        • Campos A.D.
        • Lamothe B.
        • Hur L.
        • Grabiner B.C.
        • Lin X.
        • Darnay B.G.
        • Lin H.K.
        The E3 ligase TRAF6 regulates Akt ubiquitination and activation.
        Science. 2009; 325: 1134-1138
        • Tseng P.H.
        • Matsuzawa A.
        • Zhang W.
        • Mino T.
        • Vignali D.A.
        • Karin M.
        Different modes of ubiquitination of the adaptor TRAF3 selectively activate the expression of type I interferons and proinflammatory cytokines.
        Nat. Immunol. 2010; 11: 70-75
        • Mansell A.
        • Brint E.
        • Gould J.A.
        • O'Neill L.A.
        • Hertzog P.J.
        Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-κB activation by toll-like receptor (TLR)-2 and TLR4.
        J. Biol. Chem. 2004; 279: 37227-37230
        • Verstak B.
        • Nagpal K.
        • Bottomley S.P.
        • Golenbock D.T.
        • Hertzog P.J.
        • Mansell A.
        MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4-mediated NF-kappaB proinflammatory responses.
        J. Biol. Chem. 2009; 284: 24192-24203
        • Sasai M.
        • Tatematsu M.
        • Oshiumi H.
        • Funami K.
        • Matsumoto M.
        • Hatakeyama S.
        • Seya T.
        Direct binding of TRAF2 and TRAF6 to TICAM-1/TRIF adaptor participates in activation of the Toll-like receptor 3/4 pathway.
        Mol. Immunol. 2010; 47: 1283-1291
        • Oda H.
        • Kumar S.
        • Howley P.M.
        Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination. (1999).
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9557-9562
        • Harris K.F.
        • Shoji I.
        • Cooper E.M.
        • Kumar S.
        • Oda H.
        • Howley P.M.
        Ubiquitin-mediated degradation of active Src tyrosine kinase.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 13738-13743
        • Rao N.
        • Miyake S.
        • Reddi A.L.
        • Douillard P.
        • Ghosh A.K.
        • Dodge I.L.
        • Zhou P.
        • Fernandes N.D.
        • Band H.
        Negative regulation of Lck by Cbl ubiquitin ligase.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 3794-3799
        • Walsh M.C.
        • Kim G.K.
        • Maurizio P.L.
        • Molnar E.E.
        • Choi Y.
        TRAF6 autoubiquitination-independent activation of the NFκB and MAPK pathways in response to IL-1 and RANKL.
        PLoS One. 2008; 3: e4064
        • Wang K.Z.
        • Galson D.L.
        • Auron P.E.
        TRAF6 is autoinhibited by an intramolecular interaction which is counteracted by trans-ubiquitination.
        J. Cell. Biochem. 2010; 110: 763-771
        • Pollet I.
        • Opina C.J.
        • Zimmerman C.
        • Leong K.G.
        • Wong F.
        • Karsan A.
        Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-κB and c-Jun N-terminal kinase.
        Blood. 2003; 102: 1740-1742
        • Min J.K.
        • Cho Y.L.
        • Choi J.H.
        • Kim Y.
        • Kim J.H.
        • Yu Y.S.
        • Rho J.
        • Mochizuki N.
        • Kim Y.M.
        • Oh G.T.
        • Kwon Y.G.
        Receptor activator of nuclear factor (NF)-κB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice.
        Blood. 2007; 109: 1495-1502
        • Choi Y.S.
        • Choi H.J.
        • Min J.K.
        • Pyun B.J.
        • Maeng Y.S.
        • Park H.
        • Kim J.
        • Kim Y.M.
        • Kwon Y.G.
        Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6-mediated endothelial nitric oxide production.
        Blood. 2009; 114: 3117-3126
        • Zirlik A.
        • Bavendiek U.
        • Libby P.
        • MacFarlane L.
        • Gerdes N.
        • Jagielska J.
        • Ernst S.
        • Aikawa M.
        • Nakano H.
        • Tsitsikov E.
        • Schönbeck U.
        TRAF-1, -2, -3, -5, and -6 are induced in atherosclerotic plaques and differentially mediate proinflammatory functions of CD40L in endothelial cells.
        Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1101-1107
        • Carmeliet P.
        Mechanisms of angiogenesis and arteriogenesis.
        Nat. Med. 2000; 6: 389-395