traf6-flag , traf6-flag ( or traf6 298522 ) , traf2flag ( or traf2 87501 ) ,   myd88-au1 , myd88-au1 ( or myd88 152296 ) , irak ,   nik(kk - aa ) , and ha - p46sapk-pcdna3 expression vectors   have been described ( 8 , 18 ) . expression vectors for nh2-terminal   flag - tagged htoll and htoll ( amino acids 1666 ) were constructed by inserting pcr - generated cdna fragments lacking   the coding sequence for the signal peptide , into the mammalian   expression vector pflag - cmv-1 ( eastman kodak co. , rochester , ny ) . human embryonic 293 or 293 t cells were transiently transfected by the calcium phosphate method with the indicated plasmids . the total amount of dna was kept constant by supplementation with an empty vector ( pcdna3 ; invitrogen corp . , monocytes were separated from   fresh blood of healthy donors as described previously ( 19 ) . 2436 h after transfection ,   cells were lysed in 0.5 ml buffer ( 1% np-40 , 150 mm nacl , 50   mm tris , 1 mm edta , and protease inhibitor cocktail ) . cell lysates were adjusted to 0.7 m nacl , and the indicated antibodies   were added for 14 h at 4c . after extensive washing ( in lysis buffer with the   addition of 0.1% sds ) , the sepharose beads were boiled in sample buffer , and eluted proteins were fractionated by sds - page . cells were transfected with endothelial leukocyte adhesion molecule  luciferase reporter plasmid   ( nf-b luc ; 0.1 g ) , pcmv--galactosidase ( gal ; 0.2 g ) , and   the indicated expression vectors . relative nf-b activity was   calculated by normalizing relative luciferase activity with gal   activity as described previously ( 8) . data shown are from one out   of two to five independent experiments with similar qualitative   results . cells were transfected with   nf-b luc ( 0.5 g ) , ha - p46sapk-pcdna3 ( 2 g ) , and the   indicated expression vectors . 48 h after transfection , cells were   lysed in ripa buffer containing 0.5 mm dithiothreitol , 20 mm   -glycerophosphate , 1 mm sodium orthovanadate , 10 mm sodium fluoride , 1 mm pmsf , leupeptin ( 20 g / ml ) , and aprotinin   ( 20 g / ml ) . lysates were cleared by centrifugation , and protein   concentration was measured using a commercial bradford protein   assay ( promega corp . , madison , wi ) . equal amounts of each lysate ( usually 500 g ) were incubated on ice with 2 g antiserum   to ha ( rabbit polyclonal ; santa cruz biotechnology , inc . , santa   cruz , ca ) for 2 h at 4c . precipitates were boiled in sample buffer and run   onto an sds - polyacrylamide gel . finally , immunoblotting was   performed to detect the active phosphorylated form of sapk   ( pthr-183/ptyr-185 of hjnk1 ) , or sapk as a control , by using   specific antibodies ( anti - pjnk mouse monoclonal , and anti - ha ;   santa cruz biotechnology , inc . ) . an aliquot of the cell lysate was   also analyzed for nf-b activation as above . traf6-flag , traf6-flag ( or traf6 298522 ) , traf2flag ( or traf2 87501 ) ,   myd88-au1 , myd88-au1 ( or myd88 152296 ) , irak ,   nik(kk - aa ) , and ha - p46sapk-pcdna3 expression vectors   have been described ( 8 , 18 ) . expression vectors for nh2-terminal   flag - tagged htoll and htoll ( amino acids 1666 ) were constructed by inserting pcr - generated cdna fragments lacking   the coding sequence for the signal peptide , into the mammalian   expression vector pflag - cmv-1 ( eastman kodak co. , rochester , ny ) . human embryonic 293 or 293 t cells were transiently transfected by the calcium phosphate method with the indicated plasmids . the total amount of dna was kept constant by supplementation with an empty vector ( pcdna3 ; invitrogen corp . , monocytes were separated from   fresh blood of healthy donors as described previously ( 19 ) . 2436 h after transfection ,   cells were lysed in 0.5 ml buffer ( 1% np-40 , 150 mm nacl , 50   mm tris , 1 mm edta , and protease inhibitor cocktail ) . cell lysates were adjusted to 0.7 m nacl , and the indicated antibodies   were added for 14 h at 4c . after extensive washing ( in lysis buffer with the   addition of 0.1% sds ) , the sepharose beads were boiled in sample buffer , and eluted proteins were fractionated by sds - page . cells were transfected with endothelial leukocyte adhesion molecule  luciferase reporter plasmid   ( nf-b luc ; 0.1 g ) , pcmv--galactosidase ( gal ; 0.2 g ) , and   the indicated expression vectors . relative nf-b activity was   calculated by normalizing relative luciferase activity with gal   activity as described previously ( 8) . data shown are from one out   of two to five independent experiments with similar qualitative   results . cells were transfected with   nf-b luc ( 0.5 g ) , ha - p46sapk-pcdna3 ( 2 g ) , and the   indicated expression vectors . 48 h after transfection , cells were   lysed in ripa buffer containing 0.5 mm dithiothreitol , 20 mm   -glycerophosphate , 1 mm sodium orthovanadate , 10 mm sodium fluoride , 1 mm pmsf , leupeptin ( 20 g / ml ) , and aprotinin   ( 20 g / ml ) . lysates were cleared by centrifugation , and protein   concentration was measured using a commercial bradford protein   assay ( promega corp . , madison , wi ) . equal amounts of each lysate ( usually 500 g ) were incubated on ice with 2 g antiserum   to ha ( rabbit polyclonal ; santa cruz biotechnology , inc . , santa   cruz , ca ) for 2 h at 4c . precipitates were boiled in sample buffer and run   onto an sds - polyacrylamide gel . finally , immunoblotting was   performed to detect the active phosphorylated form of sapk   ( pthr-183/ptyr-185 of hjnk1 ) , or sapk as a control , by using   specific antibodies ( anti - pjnk mouse monoclonal , and anti - ha ;   santa cruz biotechnology , inc . ) . an aliquot of the cell lysate was   also analyzed for nf-b activation as above . the expression and eventual regulation of specific transcripts encoding for htoll were analyzed in distinct cell   types that play a critical role in the natural immune response . in particular , human monocytes were separated from   healthy donors and treated with the bacterial product lps   for different periods of time . as shown in fig . 1 a , specific   transcripts for htoll were present in these cells and were   induced significantly after treatment with lps . these observations suggest that modulation of a nonclonal receptor ,   namely htoll , after exposure to infectious agents may play   a regulatory role in the natural immune response . of note ,   pmn and dendritic cells also transcribed htoll mrna at   different levels ( our unpublished observations ) . a chimeric version of htoll in which the extra   cellular portion was substituted with the corresponding region of cd4 ( cd4/htoll ) has been shown previously to induce nf-b activation in jurkat cells ( 2 ) . we engineered   distinct expression constructs encoding for flag epitope tagged htoll ( htoll - flag ) or for a truncated version of htoll   that lacks most of the cytoplasmic portion ( htoll - flag ) . ectopic expression of htoll - flag but not htoll - flag induced nf-b activation in human embryonic 293 t cells at   levels similar to the cd4/toll chimeric protein that served   as a positive control ( fig . 1 , b and c ) . from these observations , it is apparent that either cd4-driven or ectopic expression  forced aggregation of the cytoplasmic portions of   distinct htoll receptors is sufficient to trigger a signaling cascade that leads ultimately to activation of the transcription   factor nf-b . given this , one could speculate that an as yet   unidentified htoll ligand binds to htoll and induces its oligomerization and subsequent signaling cascade , in a manner   similar to il-1 and tnf with their cognate receptors . htoll shares significant sequence similarity with distinct   members of the il-1r family , including il-1ri , il-1racp ,   and myd88 ; of note , phe 513 and trp 514 in il-1ri , which   are conserved in all of these proteins , have been shown to   be essential for il-1ri to signal ( fig . since we   have shown recently a homophilic interaction to occur between the il-1racp and myd88 throughout their homologous domains ( 8) , we asked whether htoll and the   adapter protein myd88 could interact . upon coexpression ,   myd88 and htoll formed an immunoprecipitable complex ; in contrast , a mutant version of htoll , which lacks the   region of homology to myd88 and which was unable to   induce nf-b activation , failed to bind myd88 ( fig . 2 b ) .   a mutant version of myd88 ( myd88 ) , encoding only   for the cooh - terminal il-1r  like domain , abrogates il-1ri / il-1racp  induced nf-b activation ( 8) ; given this ,   we analyzed whether myd88 could act as a dominant   negative inhibitor of htoll - induced nf-b activation . as   predicted , myd88 specifically inhibited htoll - induced   but not tnfr-2induced nf-b activity , lending functional credence to the interaction occurring between htoll   and myd88 ( fig . it is apparent that both il-1r and htoll recruit the adapter protein myd88 to their respective signaling complex . irak and irak-2 are two additional proximal mediators of the il-1r signaling complex ; irak is recruited to   the il-1racp , whereas irak-2 preferentially binds il-1ri ( 8 , 10 ) . given this , we asked whether irak or   irak-2 could interact with htoll . upon ectopic expression , irak and in contrast , irak-2 bound only weakly to htoll   compared with il-1ri ( fig . 3 , a and b ) , thus suggesting   that it may not represent a relevant htoll signal transducer .   nf-b activation induced by a number of cytokine receptors is mediated by members of the traf adapter family . traf2 , for example , plays a critical role in nf-b activation mediated by tnfr-1 and tnfr-2 ( 21 , 22 ) . traf6 has been implicated in the il-1 signaling pathway   and has been shown to complex with irak and irak-2   downstream to the receptor signaling complex ( 8 , 11 ) . therefore , we determined whether dominant negative versions of either could act to inhibit htoll - induced nf-b   activity . traf6 but not traf2 significantly impaired   htoll - induced nf-b activity , suggesting that traf6   may act as an additional downstream mediator of the   htoll - induced nf-b activation cascade ( fig . the protein kinase nik has been shown to act as a general mediator of traf - induced nf-b activation ( 12 ) ;   once activated , nik directly phosphorylates and activates   the ikk/ complex , which is responsible for i-b phosphorylation and subsequent nf-b activation ( 1317 ) .   dominant negative versions of nik , in which the critical   lysine has been mutated to alanine [ nik 429430 ( kk - aa ) ] , act to inhibit nf-b activation induced by fas ,   tnf , and il-1 ( 12 ) . given this , we asked whether   nik(kk - aa ) could inhibit htoll - induced nf-b activation . nik(kk - aa ) abrogated nf-b activity triggered by   htoll ectopic expression ( fig . in addition to inducing activation of nf-b , distinct inflammatory cytokines also induce sapk , also known as   jnk . the active phosphorylated form of sapk binds to   and phosphorylates the transcription factors c - jun , activating transcription factor 2 ( atf2 ) , and ternary complex factor ( tcf)/elk1 ( 2325 ) . in particular , activation of sapk/ jnk by the tnfr-1 occurs through a traf2-dependent   pathway , as a dominant negative version of traf2 acts to   inhibit both nf-b and c - jun activation induced by tnf   ( 18 , 26 , 27 ) . in contrast , a dominant negative version of   nik , which abrogates tnf - induced nf-b activation ,   fails to inhibit c - jun phosphorylation , supporting a model   wherein tnf - mediated nf-b and c - jun pathways bifurcate at traf2 ( 28 , 29 ) . ectopic expression of increasing amounts of htoll - flag but not htoll - flag resulted in   activation of sapk as indicated by specific phosphorylation at thr 183 and tyr 185 ( fig . 4 a ) . overexpression of   traf6 also activated sapk as described previously ( 28 ) .   to identify mediators of htoll - mediated jnk activation , we   cotransfected 293 cells with htoll and dominant negative   versions of either myd88 or traf6 . surprisingly ,   myd88 but not tra6 acted to inhibit htoll - triggered   jnk phosphorylation ( fig . 4 b ) . importantly , under the   same experimental conditions , both myd88 and traf6   abrogated htoll - induced nf-b activation ( 96 and 90% inhibition , respectively ) . additionally , traf6 alone , as well   as myd88 , failed to activate jnk ( data not shown ) . collectively , these observations indicate that although ectopic   expression of traf6 induced sapk , a dominant negative   version of traf6 failed to inhibit htoll - induced jnk   phosphorylation . given this , one could speculate that although traf6 overexpression is sufficient to activate jnk/ sapk , endogenous traf6 does not provide a significant   contribution to jnk / sapk activation by htoll ( fig . , myd88 appears to represent the most upstream mediator of the htoll - mediated signaling cascade ,   which ultimately activates nf-b and c - jun , thus driving   transcriptional activation of several cytokines and costimulatory molecules . therefore , it may represent a potentially   useful therapeutic target for controlling the molecular   switch from the innate to the adaptive immune response . ( a ) human monocytes were treated with lps ( 100 ng / ml )   for different periods of time and analyzed for their htoll   mrna content by northern blotting . ( b ) ectopic expression of htoll - flag and   cd4/toll but not the mutant version htoll - flag activate   nf-b in 293 t cells in a dose - dependent manner , as measured by nf-b reporter gene activity . ( c ) equal amounts   of htoll - flag and htoll - flag are produced upon ectopic   expression in 293 t cells ( 3.2 g of each expression construct were used for this experiment ) . ( a ) sequence alignment of human   myd88 ( amino acids 152296 ) , htoll ( 667 840 ) , hil-1racp ( 391570 ) , and hil-1ri   ( 381569 ) . dots , conserved amino acids that are   essential for il-1ri to signal ( reference 20 ) . ( b )   myd88 associates with htoll but not with a   truncated version of htoll ( htoll ) lacking the   cytoplasmic region sharing sequence similarity   with myd88 . 293 t cells were transfected with   htoll - flag , htoll - flag , or il-1racp  flag as   a positive control together with au1-tagged   myd88 . the presence of myd88 that coprecipitated with the receptors was detected by   immunoblotting with a rabbit polyclonal antiserum to myd88 . ( c ) myd88 inhibits htoll - induced but not the unrelated tnfr-2induced   nf-b activity data are expressed   as the percentage of relative receptor - induced   nf-b activity . ( a ) irak is recruited to htoll but not to the inactive truncated version of htoll ( htoll ) . cells were transfected and analyzed as in fig . 2 b. ( b ) irak-2 binds very   weakly to htoll . cells were transfected and analyzed as in a. ( c ) traf6 but not the unrelated   traf2 attenuates cd4/toll - induced nf-b activity . a dominant   negative mutant version of the downstream molecule nik [ nik(kk - aa ) ] also inhibits cd4/htoll - induced nf-b activity . ( a ) htoll activates sapk / jnk in a dose - dependent manner , as   determined by the presence of the active phosphorylated form of jnk   ( pjnk ) . ( b ) myd88 ( 152296 ) ( myd88 ) but not traf6   ( 289522 ) ( traf6 ) abolishes htoll - induced sapk / jnk phosphorylation . the   diagram shows that the bifurcation of nf-b and jnk / sapk activation   by htoll or tnfr-1 occurs at different levels with respect to the specific   trafs .