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TLR4 Signaling SASH1 Is a Scaffold Molecule in Endothelial SASH1 Is a Scaffold Molecule in Endothelial TLR4 Signaling Shauna M. Dauphinee, Ashley Clayton, Angela Hussainkhel, Cindy Yang, Yoo-Jin Park, Megan E. Fuller, Josip Blonder, This information is current as Timothy D. Veenstra and Aly Karsan of September 27, 2021. J Immunol 2013; 191:892-901; Prepublished online 17 June 2013; doi: 10.4049/jimmunol.1200583 http://www.jimmunol.org/content/191/2/892 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/06/17/jimmunol.120058 Material 3.DC1 http://www.jimmunol.org/ References This article cites 46 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/191/2/892.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology SASH1 Is a Scaffold Molecule in Endothelial TLR4 Signaling Shauna M. Dauphinee,*,† Ashley Clayton,*,† Angela Hussainkhel,*,‡ Cindy Yang,x Yoo-Jin Park,x Megan E. Fuller,*,{ Josip Blonder,‖ Timothy D. Veenstra,‖ and Aly Karsan*,†,‡,x,{ Recognition of microbial products by TLRs is critical for mediating innate immune responses to invading pathogens. In this study, we identify a novel scaffold protein in TLR4 signaling called SAM and SH3 domain containing protein 1 (SASH1). Sash1 is expressed across all microvascular beds and functions as a scaffold molecule to independently bind TRAF6, TAK1, IkB kinase a, and IkB kinase b. This interaction fosters ubiquitination of TRAF6 and TAK1 and promotes LPS-induced NF-kB, JNK, and p38 activation, culminating in increased production of proinflammatory cytokines and increased LPS-induced endothelial migra- tion. Our findings suggest that SASH1 acts to assemble a signaling complex downstream of TLR4 to activate early endo- thelial responses to receptor activation. The Journal of Immunology, 2013, 191: 892–901. Downloaded from ecognition of bacterial and viral products by the mam- Fas-associated death domain protein (FADD) is a negative reg- malian innate immune system is mediated, in part, by ulator of TLR4 signaling in endothelial cells and FADD-null R a class of receptors called TLRs (1). The intracellular cells show hyperactivation of NF-kB and JNK in response to signaling cascades that are initiated by ligation of TLRs leads to LPS in a TRAF6-dependent manner (6, 7). Because LPS signaling activation of transcription factors such as NF-kB and IFN regu- components have been shown to localize to lipid-rich microdomains http://www.jimmunol.org/ latory factor (IRF), resulting in production of proinflammatory in the plasma membrane (8), we hypothesized that the hyper- cytokines (2, 3). Signaling through TLR4 follows recognition of activation of the LPS signal in FADD-null cells is due, in part, to an LPS and the subsequent recruitment of intracellular adaptor increase in LPS signaling molecules in lipid-rich microdomains. molecules TIRAP, MyD88, and TRIF. Downstream of MyD88, Thus, we used proteomic analysis to identify proteins that are dif- IL-1R–associated kinases (IRAKs) are recruited to activate the ferentially expressed in the lipid-rich microdomains of FADD wild- E3 ubiquitin ligase TRAF6. This results in TRAF6 autoubiquiti- type (WT) versus FADD knockout (KO) cells. The hyperactivation nation of lysine 63 (K63)–linked ubiquitin chains culminating in of the signaling pathway in FADD KO cells would facilitate de- activation of NF-kB and MAPKs (4). Signaling downstream of TRIF tection of proteins present in the cell in limiting amounts. This can also result in recruitment of TRAF6 to the receptor complex and approach resulted in the identification of SAM and SH3 domain by guest on September 27, 2021 subsequent activation of NF-kB and MAPK. Alternatively, recruit- containing protein 1 (SASH1) as a putative candidate for positive ment of TRAF3 leads to induction of IFN-related genes (5). regulation of TLR4 signaling. SASH1 is a large protein with a predicted molecular mass of 137 kDa, and it was originally identified by expression profiling and in silico analysis as a gene that is downregulated in breast and other *Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British solid cancers, such as lung and thyroid (9). Subsequently, SASH1 Columbia V5Z 1L3, Canada; †Experimental Medicine Program, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada; ‡Department of Interdis- mRNA has been shown to be reduced in colon cancer and post- ciplinary Oncology, University of British Columbia, Vancouver, British Columbia operative metastases in the liver (10). However, these studies are x V6T 2B5, Canada; Department of Pathology and Laboratory Medicine, British correlative and no direct evidence for SASH1 as a tumor sup- Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada; {Depart- ment of Pathology and Laboratory Medicine, University of British Columbia, Van- pressor has been described. SASH1 belongs to a family of SAM couver, British Columbia V6T 2B5, Canada; and ‖Laboratory of Proteomics and and SH3 adaptor proteins, consisting of SH3 domain expressed Analytical Technologies, Advanced Technology Program, Science Applications In- in lymphocytes (SLY1) and hematopoietic adaptor containing ternational Corp.–Frederick Inc., National Cancer Institute–Frederick, Frederick, MD 21702 SH3 and SAM domains 1 (HACS1, also called SLY2) (11, 12). Received for publication February 22, 2012. Accepted for publication May 17, 2013. The SLY family has been implicated in regulation of the adaptive This work was supported by Canadian Institutes for Health Research Grant MOP immune response, suggesting that SASH1 may also function in 97744 (to A.K.). S.M.D. was supported by studentships from the Canadian Institutes the immune system. Indeed, SASH1 was recently identified in for Health Research and the Michael Smith Foundation for Health Research. A.H. a global phosphoproteome analysis as a protein that is phos- was supported by a Canadian Institutes for Health Research studentship and a Uni- versity of British Columbia doctoral fellowship. A.K. is a Senior Scholar of the phorylated at multiple sites in response to LPS (13). However, Michael Smith Foundation for Health Research. a molecular function for SASH1 in the immune system has not Address correspondence and reprint requests to Dr. Aly Karsan, Genome Sciences been reported, and thus we aimed to determine whether SASH1 Centre, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, British functions in innate immune signaling. Columbia V5Z 1L3, Canada. E-mail address: [email protected] In this study, we show that Sash1 is preferentially expressed The online version of this article contains supplemental material. in microvascular endothelial cells of various organs, as well as Abbreviations used in this article: FADD, Fas-associated death domain protein; HA, parenchymal cells, but not in lymphocytes. SASH1 positively hemagglutinin; HMEC, human microvascular endothelial cell; IKK, IkB kinase; IP-10, IFN-g–inducible protein 10; IRAK, IL-1R–associated kinase; ISRE, IFN- regulates signaling through TLR4 in human endothelial cells to stimulated responsive element; KO, knockout; MEF, mouse embryonic fibroblast; increase activation of NF-kB and MAPKs, culminating in in- MS, mass spectrometry; SASHI, SAM and SH3 domain containing protein 1; sh, short hairpin; SLY1, SH3 domain expressed in lymphocytes; SLY2, hematopoietic creased production of proinflammatory cytokines. Although SASH1 adaptor containing SH3 and SAM domains 1; WT, wild-type. does not bind to MyD88 or the IRAKs, it interacts with TRAF6 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200583 The Journal of Immunology 893 throughaconservedTRAF6bindingdomaininSASH1and termates by tail digestion with proteinase K followed by PCR using pri- regulates TRAF6 ubiquitination. SASH1 also binds to TAK1 mers specific to the insertion site of the gene-trap construct to distinguish and the IkB kinase (IKK) complex, independent of TRAF6, to homozygous mice. All animal studies have been reviewed and approved by the University of British Columbia. assemble a signaling hub that permits downstream activation of NF-kB and MAPKs. Collectively, these results demonstrate that b-galactosidase staining SASH1 functions as a novel scaffold protein to regulate innate Adult mouse tissues from Sash1+/2 mice and their WT littermate controls immune signaling downstream of TLR4. were dissected and immediately fixed in 2% paraformaldehyde at 4˚C for 4 h. Tissues were cryopreserved and placed into OCT compound at 280˚C until sectioning. Sections (10 mm) were fixed with 0.2% glutaraldehyde Materials and Methods solution and stained with buffer containing 100 mM sodium phosphate (pH Recombinant plasmids, Abs, and reagents 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 1 mg/ml 5-bromo-4-chloro-3-indolyl b-D-galactoside, 5 mM ferrocyanide, Hemagglutinin (HA)-SASH1, Flag-SASH1, and Flag-TAK1 constructs and 5 mM ferricyanide. were generated by PCR and cloned into pcDNA3 by restriction digest. Myc-SASH1DT6BD was obtained by inverse PCR to generate a deletion Isolation of mouse endothelial cells of amino acids 852-860. Flag-TRAF6DN, Flag-TRAF6DC, and Flag- TRAF6DCC were cloned from Flag-TRAF6 into pcDNA3 by restriction Adult lung tissue was harvested from C57BL/6 mice, digested with 2 mg/ml digest. Other plasmids were obtained as follows: Flag-TRAF6 (Tularik, collagenase IV, and incubated with 0.2 mg/ml DNase. Tissue homogenate was San Francisco, CA); Flag-IKKb,Flag-IKKa, and Flag-IKKg (T.D.
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