Patent Application: US-32042405-A

Abstract:
the present invention relates to ubiquitinated tnf receptor 2 , and the uses thereof . more specifically , the invention relates to the use of tnf receptor 2 ubiquitination to deplete tnf receptor 2 from the cell membrane and cytoplasm , and relocalize it in the insoluble cell fraction . such relocalization can be used to modulate the signaling activity of the tnf receptor 2 and to treat tnf receptor 2 - related diseases . the invention relates further to the use of smurf 2 to ubiquitinate tnf receptor 2 and to the use of traf2 to mediate tnf receptor 2 ubiquitination .

Description:
a further aspect of the invention is the use of tnf - r2 ubiquitination to modulate inflammation and / or autoimmune disease . preferably , inflammation and / or autoimmune disease is selected from the group consisting of crohn &# 39 ; s disease , rheumatoid arthritis , systemic lupus erythematosus , sepsis , chronic hepatitis virus infection , acute pancreatitis , acute respiratory distress syndrome and aids . indeed , tnf - r shedding results in a soluble receptor corresponding to the extracellular region and alters rapidly the number of functionally active tnf - r . these soluble tnf - rs , originally identified as tnf - binding proteins , have been found in urine , serum , ovarian ascites and synovial and cerebral spinal fluids of patients with various diseases ( nophar et al ., 1990 , cope et al ., 1992 ; grosen et al ., 1993 ). several inflammatory disorders are associated with increased production of soluble tnf - rs , in which a correlation between the disease progression and the soluble tnf - r level can be found . for soluble tnf - r2 , these include rheumatoid arthritis ( cope et al ., 1992 ), systemic lupus erythematosus ( gabay et al ., 1997 ), sepsis ( schroder et al ., 1995 ); chronic hepatitis virus infection ( marinos et al ., 1995 ); acute pancreatitis ( de beaux et al ., 1996 ); acute respiratory distress syndrome ( lucas et al ., 1997 ) and aids ( hober et al ., 1996 ). moreover , chronic production of human tnf - r2 in tnf - r2 transgenic mice has detrimental effects leading to multi - organ inflammatory syndrome involving mainly the pancreas , liver , kidney and lung ( douni and kollias , 1998 ). in addition , t - cell - deficient scid mice reconstituted with cd4 + cd62l + t cells from tnf - r2 transgenic mice develop more severe experimental colitis than when wild - type t cells were used for reconstitution . this observation correlates with the up - regulated expression of tnf - r2 on the cd4 + t cells observed in patients with crohn &# 39 ; s disease ( holtmann et al ., 2002 ). the invention is further described with the aid of the following illustrative examples . human embryonic kidney cells ( hek293t ) were a kind gift of dr . m . hall ( university of birmingham , birmingham , uk ) and were grown in dmem supplemented with 10 % fetal bovine serum , 2 mm glutamin , 0 . 4 mm sodium pyruvate , 106 u / l penicillin and 100 mg / l streptomycin . pc60htnf - r55 r75 is a rat t - lymphoma and mouse ctl hybrid cell line stably transfected with human tnf - r1 and 2 , as described previously ( vandenabeele et al ., 1995 ). cells were grown in rpmi 1640 supplemented with 100 μm β - mercaptoethanol , 10 % fetal bovine serum , glutamin , sodium pyruvate and antibiotics . the proteasome inhibitor mg - 132 was purchased from biomol research laboratories , inc . ( butler pike , pa ., usa ). recombinant human tnf was produced in e . coli in our laboratory and purified to 99 % homogeneity . tnf has a specific biological activity of 8 . 8 × 10 6 iu / mg purified protein , as determined with the international standard code 87 / 650 ( national institute for biological standards and control , potters bar , uk ). the tnf - r2 - specific monoclonal agonistic utr - 1 and non - agonistic utr - 4 antibodies were obtained from dr . w . lesslauer ( roche basel , basel , switzerland ), polyclonal anti - caspase 14 antibody from s . lippens ( dmbr , ghent university — vib , ghent , belgium ), polyclonal anti - tnf - r2 antibody from dr . w . a . buurman ( university of maastricht , the netherlands ), and monoclonal anti - cmyc antibody from dr . n . mertens ( dmbr , ghent university — vib , ghent , belgium ). monoclonal anti - ha antibody was purchased from crp ( richmond , calif ., usa ), polyclonal anti - traf2 ( sc - 876 ) and monoclonal anti - ubiquitin ( sc - 8017 ) antibodies from santa cruz biotechnology ( santa cruz , calif ., usa ), anti - gfp ( green fluorescent protein ) antibody ( jl - 8 ) from clontech ( palo alto , calif ., usa ), monoclonal anti - flag and monoclonal anti - flag - horseradish peroxidase ( hrp )- linked antibodies from sigma - aldrich ( st . lois , mo ., usa ), monoclonal anti - e tag and monoclonal anti - e - hrp - linked antibodies from amersham pharmacia biotech ( rainham , uk ). anti - mouse and anti - rabbit hrp - linked antibodies were obtained from amersham pharmacia biotech . ha - smurf2 and cmyc - smurf2 ( c716g ) cdna were a kind gift from dr . y . zhang ( laboratory of cellular and molecular biology , national cancer institute , bethesda , md ., usa ), and cloned in pcaggs expression vectors . cmyc - smurf2 wt was obtained by cutting ha - smurf2 wt and cmyc - smurf2 ( c716g ) with scai and ligating the resulting cmyc - containing fragment to the smurf2 wt - containing fragment . pcmv5b - flag - smurf2 wt and several flag - tagged deletion mutants lacking the c2 domain ( smurf2δc2 ), the first , second or third ww domains or combinations ( smurf2δww1 , smurf2δww2 , smurf2δww3 , smurf2δww2 / 3 ), the c2 domain and the first ww domain ( smurf2δc2 / ww1 ) or the hect domain ( smurf2δhect ) were a kind gift from dr . j . wrana ( samuel lunenfeld research institute , mount sinai hospital , toronto , canada ). pcdnai - tnf - r2 wt and mutants were obtained from dr . w . declercq ( dmbr , ghent university — vib , ghent , belgium ) and were described previously ( declercq et al ., 1998 ). pcdna3 - caspase 14δpro was kindly given by s . lippens and pef6 - e by b . depuydt ( dmbr , ghent university — vib , ghent , belgium ). pcdna3 - ha - ubiquitin was kindly provided by dr . k . dimarco - burns ( university of lausanne , lausanne , switzerland ). pef6 - e - murine ( m ) traf2 was obtained after pcr cloning of mtraf2 ( forward primer ( seq id no : 2 ), 5 ′- c gggatccg cggccgctatggctgcagccagtgtgac - 3 ′ and reverse primer ( seq id no : 3 ), 5 ′- gcatagtttagcggccgcttat ctagag agtcctgttaggtccacaa - 3 ′), cutting the pcr product with noti , and cloning into the noti site of pef6 - e . for immunoprecipitation , 1 . 2 × 10 6 hek293t cells were plated on 10 - cm petri dishes and transiently transfected with a total amount of 5 μg dna using the dna calcium phosphate coprecipitation method ( o &# 39 ; mahoney and adams , 1994 ). 1 μg of each expression plasmid was used , except if mentioned otherwise in the figure legends . thirty - six to 48 hours later , cells were lysed in the indicated lysis buffer supplemented with protease -( 10 μg / ml leupeptin , 200 u / ml aprotinin and 1 mm pmsf ) and phosphatase - inhibitors ( 10 mm naf , 1 mm na - vanadate and 5 . 5 mg / ml β - glycerophosphate ). cell lysates were incubated with the indicated antibodies and immobilized to protein a trisacryl beads ( pierce chemicals , rockford , ill ., usa ). beads were washed six times with buffers as indicated in figure legends and binding proteins were eluated with 1 × laemmli buffer . co - precipitating proteins were separated by sds - page and analyzed by western blotting using renaissance - enhanced chemiluminescence system ( nen , boston , mass ., usa ). 2 × 10 5 hek293t cells were transiently transfected with 100 ng tnf - r2 and / or 1 μg ha - smurf2 wt or cmyc - smurf2 ( c716g ). the following day , total rna was isolated via rnazol ( wak chemie , medical gmbh , steinbach , germany ) and quantified . after electrophoresis on a 1 % formaldehyde agarose gel , the rna was transferred by capillary elution to a hybond - n + membrane . hybridization of the northern blot was performed with full length tnf - r2 cdna ( a 1 . 4 kb large kpni / hindiii fragment of pcdnai - htnf - r2 ) used as a probe . the intensity of the tnf - r2 signal , determined using a phosphorimager and an imagequant ® program ( molecular dynamics , sunnyvale , calif . ), is expressed relative to the intensity of the gapdh signal of the same setups . 2 × 10 6 hek293t cells were transiently transfected with 0 . 5 μg ha - ubiquitin , 0 . 5 μg of tnf - r2 and / or e - traf2 with or without 3 μg cmyc - smurf2 wt or mutant ( c716g ). twenty - four to 30 hours later , cells were lysed in tnf - r2 lysis buffer ( 10 mm tris . hcl ph 7 . 5 , 250 mm nacl , 5 mm edta , 1 % np - 40 ) supplemented with protease - and phosphatase - inhibitors . tnf - r2 or e - traf2 immunoprecipitation was performed with utr - 1 or anti - e antibody , respectively . for endogenous tnf - r2 ubiquitination , 1 × 10 7 pc60 htnf - r55 r75 cells were used . cells were pretreated with 30 μm mg - 132 for 30 minutes prior to stimulation with 1 μg / ml htnf for indicated times and lysed in tnf - r2 lysis buffer supplemented with protease - and phosphatase - inhibitors . lysates were incubated with utr - 4 antibody . all immunoprecipitates were immobilized on protein a trisacryl beads . beads were washed twice with lysis buffer , twice with lysis buffer containing 0 . 75 m nacl and twice with lysis buffer . bound proteins were eluated with laemmli buffer and analyzed by immunoblotting with indicated antibodies . 2 × 10 5 hek293t cells were transiently transfected with 100 ng tnf - r2 with or without 1 μg ha - smurf2 wt or cmyc - smurf2 ( c716g ). forty - eight hours later , cells were lysed in 100 μl ripa buffer ( 1 × pbs , 1 % np - 40 , 0 . 1 % sds , 0 . 5 % sodium deoxycholate ) supplemented with protease - and phosphatase - inhibitors for 15 minutes at 4 ° c . the soluble fraction was separated from the insoluble fraction by centrifugation for ten minutes at 4 ° c . at 14 , 000 rpm in an eppendorf centrifuge . 100 μl 2 × laemmli was added to the supernatant fractions , whereas cell pellets were washed with lysis buffer and resolved in 200 μl 1 × laemmli . total cell lysates were obtained by immediate lysis of cells in 200 μl 1 × laemmli . 50 μl of each fraction was used for sds - page followed by immunoblotting with indicated antibodies . for the cell fractionation assay of pc60htnf - r55 r75 cells , 2 × 10 6 cells were pretreated for 30 minutes with 20 μm mg - 132 prior to stimulation with 1 μg / ml htnf for indicated times . cell lysis was performed in tnf - r2 lysis buffer supplemented with protease and phosphatase inhibitors . the assay was continued as described for hek293t . to determine nf - κb activation , 2 × 10 5 hek293t cells were grown in six - well plates and transiently transfected by dna calcium phosphate coprecipitation method . the next day , 1 / 10 th of the cells were seeded out in 24 - well plates in triplicate . forty - eight hours after transfection , cells were lysed in 200 μl lysis buffer ( 25 mm tris - phosphate ph 7 . 8 , 2 mm dithiotreitol , 2 mm 1 , 2 - cyclohexanediaminetetraacetic acid , 10 % glycerol and 1 % triton x - 100 ). luciferase and β - galactosidase activities were analyzed as described previously ( carpentier et al ., 1998 ). luciferase values were normalized for β - galactosidase values to correct for differences in transfection efficiency . [ 2 × 10 5 hek293t cells were grown in six - well plates and transiently transfected by dna calcium phosphate coprecipitation method . twenty - four hours later , cells were left untreated or treated with 1000 iu / ml htnf for ten minutes , rinsed quickly with ice cold phosphate buffered saline ( pbs ) and lysed on ice in 200 μl sds sample buffer ( 62 . 5 mm tris - hcl ( ph 6 . 8 ), 2 % w / v sds , 10 % glycerol , 50 mm dithiotreitol , 0 . 01 % bromophenol blue ). cell lysates were separated on 10 % sds - page and immunoblotted for detection of phosphorylated p38 mapk or total p38 mapk expression with phospho - p38 mapk ( thr180 / tyr182 ) antibody or p38 mapk antibody , respectively , according to manufacturer &# 39 ; s conditions ( cell signaling technology , beverly , mass ., usa ). traf2 was used as bait in a sos recruitment system ( srs ) yeast two - hybrid screening of a human spleen cdna library to search for novel traf2 interaction partners . from 3 . 7 × 10 6 screened clones , 23 positive clones expressed known interaction partners such as traf2 itself ( 6 ×), traf1 ( 7 ×), tnf - r - associated death domain protein ( tradd ) ( 5 ×), cellular flice inhibitory protein ( c - flip ) ( 1 ×) and the tnf - r superfamily receptors cd40 ( 3 ×) and herpes virus entry mediator ( hvem ) ( 1 ×). six clones represented novel traf2 interacting proteins . one corresponded to a fragment of the e3 ubiquitin ligase smurf2 , which has previously been shown to play a role in tgfβ signaling ( kavsak et al ., 2000 ; lin et al ., 2000 ). this clone ( amino acids 385 - 748 ) contained nearly the whole catalytic hect domain ( amino acids 368 - 748 ). the latter domain contains two potential traf2 binding sites ([ psat ]- x -[ qe ]- e ), a sree motif ( amino acids 398 - 401 ) and an aiee motif ( amino acids 738 - 741 ) ( ye et al ., 1999 ). full length smurf2 also interacted with traf2 in mammalian cells , since traf2 was able to co - immunoprecipitate with smurf2 in hek293t cells that were transiently transfected with an expression plasmid for ha - tagged smurf2 and e - tagged traf2 ( fig1 , panel b ). deletion mutants of smurf2 lacking the c2 domain ( δc2 ), the first , second or third ww domains or combinations ( δww1 , δww2 , δww3 , δww2 / 3 ), the c2 domain and the first ww domain ( δc2 / ww1 ) or the hect domain ( ahect ), were used to define the interaction domain ( s ) of smurf2 with traf2 ( see also , fig1 , panel a ). smurf2 with a deletion of one or more ww domains ( δww1 , δww2 , δww3 , δww2 / 3 ) still retained its ability to interact with traf2 , although deletion of ww2 , ww3 , or their combination , significantly reduced the binding to traf2 . in contrast , interaction was even improved when deleting the first ww - domain . deletion of the c2 or hect domain abolished completely the interaction with traf2 , indicating a requirement for the c2 domain and the hect domain . the role of the hect domain is consistent with the yeast two - hybrid data , in which the hect domain was isolated as a traf2 - interacting partner ( fig1 , panel c ). the interaction between traf2 and smurf2 could not be improved by the use of a ligase inactive smurf2 ( c716g ) mutant , in which the cysteine that is believed to conjugate ubiquitin was replaced by a glycine ( huibregtse et al ., 1995 ). smurf2 is an e3 ubiquitin ligase for smad 1 , − 2 , − 7 and tgf - β receptor - 1 ( tβr - i ), leading to their proteolysis ( lin et al ., 2000 ; kavsak et al ., 2000 ; zhang et al ., 2001 ). likewise , the ubiquitin e3 ligases cellular inhibitor of apoptosis ( ciap ) 1 and siah1a have been implicated in the degradation of traf2 ( li et al ., 2002 ; habelhah et al ., 2002 ). therefore , it was assessed whether smurf2 could also target traf2 for degradation . for this purpose , hek293t cells were transiently transfected with increasing amounts of a smurf2 expression plasmid and endogenous traf2 expression levels were detected by immunoblotting . cell lysis was performed in ripa buffer , as this leaves relatively little insoluble material . no obvious effect of smurf2 on traf2 protein levels could be seen ( fig2 ). similarly , no effect of smurf2 could be seen on the expression of traf2 , when the corresponding traf2 expression vector was cotransfected . upon receptor stimulation of cd30 , cd40 , tnf - r2 or latent membrane protein - 1 ( lmp - 1 ), traf2 interacts transiently with these receptors at the plasma membrane , followed by a cytoplasmic depletion of traf2 and a redistribution of traf2 to a detergent - insoluble fraction reminiscent of membrane rafts . cd30 , cd40 and tnf - r2 , but not lmp - 1 triggering subsequently led to traf2 degradation ( duckett and thompson , 1997 ; hostager et al ., 2000 ; chan and lenardo , 2000 ; brown et al ., 2001 ; fotin - mleczek et al ., 2002 ). in the case of cd40 , traf2 degradation has been proposed to be mediated by the ring finger - mediated ubiquitin ligase activity of traf2 itself ( brown et al ., 2002 ), whereas ciap1 or siah1a have been implicated in traf2 degradation in response to tnf - r2 or stress conditions , respectively ( li et al ., 2002 ; habelhah et al ., 2002 ). in this context , it was examined whether the hect ubiquitin ligase smurf2 could induce the down - regulation of traf2 in the presence of tnf - r2 . hek293t cells were cotransfected with equal amounts of tnf - r2 and increasing amounts of smurf2 . as a control for transfection efficiency , cytoplasmic gfp was also cotransfected . despite the presence of tnf - r2 , traf2 degradation could not be seen when smurf2 was cotransfected ( fig3 ). however , smurf2 induced a dose - dependent depletion of the tnf - r2 protein . a similar smurf2 - induced decrease of the steady - state level of tpr - 1 has previously been observed in the presence of smad7 ( kavsak et al ., 2000 ). however , in this case , no traf2 cotransfection was required for the smurf2 - mediated down - modulation of tnf - r2 , probably because hek293t cells already express enough endogenous traf2 . to analyze whether the ligase activity of smurf2 is needed to induce the cytoplasmic down - regulation of tnf - r2 , the effect of co - expression of tnf - r2 with smurf2 wild - type ( wt ) or a mutant smurf2 ( c716g ), which was designed to abolish the e3 ligase activity of smurf2 ( zhang et al ., 2001 ), on tnf - r2 expression was verified . in contrast to wild - type smurf2 , the smurf2 ( c716g ) mutant did not affect the steady - state levels of tnf - r2 ( fig4 ), indicating that the tnf - r2 down - regulation is dependent on the catalytic activity of smurf2 . as a negative control , the effect of smurf2 on the expression of another gene ( caspase - 14 without prodomain ( casp 14δpro )) that had been cloned after the same promotor was also verified , and found to be insensitive to smurf2 co - expression ( fig4 ). to exclude that smurf2 might influence the steady - state level of tnf - r2 by interfering with the transcription or stability of the tnf - r2 mrna , tnf - r2 mrna expression was verified by northern blotting . therefore , rna was isolated from hek293t cells transiently transfected with tnf - r2 , smurf2 wt or smurf2 ( c716g ), followed by northern blot analysis with a tnf - r2 - specific 32 p labeled probe . no significant difference could be noted in the mrna levels of tnf - r2 between cells that were transfected with smurf2 wt or smurf2 ( c716g ), further indicating that smurf2 exerts its effect on the tnf - r2 protein level ( fig5 ). it has previously been demonstrated that smad7 functions as an adaptor to target smurf1 and smurf2 to tβr - i , leading to an enhanced turnover of this receptor ( kavsak et al ., 2000 ; ebisawa et al ., 2001 ). the ability of traf2 to bind tnf - r2 ( rothe et al ., 1994 ) as well as smurf2 , together with the effect of smurf2 on tnf - r2 steady - state levels , raised the interesting possibility that traf2 also functions to recruit smurf2 to tnf - r2 . to address this question , tnf - r2 and smurf2 were co - expressed in hek293t cells in the presence or absence of traf2 , and smurf2 co - immunoprecipitation with tnf - r2 was analyzed . to avoid smurf2 - induced tnf - r2 down - regulation , the catalytic smurf2 ( c716g ) mutant was used in these experiments . when smurf2 ( c716g ) was co - expressed with tnf - r2 , smurf2 ( c716g ) could not be co - immunoprecipitated with tnf - r2 . however , in the presence of increasing amounts of traf2 , smurf2 co - immunoprecipitated with tnf - r2 in a dose - dependent manner , demonstrating that traf2 can indeed act as an adaptor between smurf2 and tnf - r2 ( fig6 ). previous data have shown that smurf1 and smurf2 function as e3 ligases in the ubiquitination and subsequent lysosomal and proteasomal degradation of smad7 and tβri ( ebisawa et al ., 2001 ; kavsak et al ., 2000 ). to investigate whether traf2 or tnf - r2 could also undergo smurf2 - mediated ubiquitination , hek293t cells were transiently transfected with e - traf2 , tnf - r2 and cmyc - smurf2 wt or mutant ( c716g ), together with ha - tagged ubiquitin . tnf - r2 ( left panel of fig7 ) or e - traf2 ( right panel of fig7 ) were immunoprecipitated , followed by western blotting with an anti - ha antibody to detect ubiquitin - conjugates of tnf - r2 or traf2 . in the absence of smurf2 , ubiquitination of tnf - r2 could already be observed , probably due to the presence of endogenous smurf2 or ( an ) other ubiquitin ligase ( s ). however , tnf - r2 ubiquitination increased significantly in the presence of smurf2 wt , which was even slightly increased upon co - expression of traf2 . in contrast , smurf2 ( c716g ) was unable to induce tnf - r2 ubiquitination and even decreased the constitutive tnf - r2 ubiquitination . the latter observation suggested that smurf2 ( c716g ) can exert a dominant negative effect , possibly by competing with endogenous smurf2 . it should also be noted that a tnf - r2 mutant lacking the last 37 amino acids , disrupting the binding of traf2 , was no longer ubiquitinated ( data not shown ). in contrast to tnf - r2 , traf2 was not ubiquitinated upon smurf2 expression ( fig7 , right panel ). moreover , the latter experiments also showed that ubiquitinated tnf - r2 can be co - immunoprecipitated with traf2 . in conclusion , these results further strengthen the role of smurf2 and traf2 in the ubiquitination of tnfr2 . several yeast , as well as mammalian receptors , undergo stimulus - dependent ubiquitination , which functions as a marker for internalization by endocytosis ( hicke , 2001a and b ). indeed , several mammalian receptors are able to bind directly or indirectly (“( in ) directly ”) to adaptin complexes that link them to clathrin - coated pits ( hicke , 1999 ; strous and govers , 1999 ; sorkin and von zastrow , 2002 ). on the other hand , it has been suggested that ubiquitin might affect the localization of the modified protein within the plasma membrane , e . g ., relocalizing the ubiquitinated protein to membrane rafts to facilitate endocytosis . membrane rafts are cholesterol -, sphingomyelin - and glycolipid - enriched mini - domains within the plasma membrane ( anderson and jacobson , 2002 ). in this context , two interesting observations on traf2 and tnf - r2 have been made in the past . first , in human umbilical vein endothelial cells ( huvec ), traf2 has been shown to bind with the inner surface coat protein caveolin - 1 of membrane invaginations . moreover , upon overexpression of tnf - r2 , traf2 binding to this receptor results in the recruitment of caveolin - 1 , possibly to localize tnf - r2 complexes to caveolae ( feng et al ., 2001 ). in addition , it has been clearly demonstrated in several cell lines that tnf - r2 engagement leads to the cytoplasmic depletion of traf2 , associated with its translocation to lipid rafts ( duckett and thompson , 1997 ; chan and lenardo , 2000 ; fotin - mleczek et al ., 2002 ). although a main feature of lipid rafts and caveolae is their insolubility in non - ionic detergents at 4 ° c . ( anderson and jacobson , 2002 ), tnf - r2 - mediated redistribution of traf2 to ripa buffer ( containing sodium deoxycholate assures solubilization of rafts ; scott and ibanez , 2001 )- insoluble complexes has been observed previously , suggesting also an interaction of traf2 with macromolecular cell structures such as the cytoskeleton ( arch et al ., 2000 ). binding of traf2 with the actin - binding protein filamin has indeed been described ( leonardi et al ., 2000 ). apparently , filamin and traf2 are first targeted to lipid rafts by the ring finger of traf2 , before they are sequestered into the cytoskeleton ( arron et al ., 2002 ). since our previous experiments were all done with the soluble cell fraction , the above findings encouraged us to assess whether the smurf2 - induced depletion of tnf - r2 did not result from its degradation , but was rather due to a tnf - r2 translocation to the insoluble fraction . to address this question , tnf - r2 expression was studied in the soluble and insoluble cell fraction prepared from hek293t cells , which were transiently transfected with tnf - r2 in the absence or presence of smurf2 wt or smurf2 ( c716g ). co - expression of smurf2 wt indeed resulted in relocalization of tnf - r2 to the insoluble fraction , whereas this could not be seen upon co - expression of the ligase inactive mutant smurf2 ( c716g ) ( fig8 ). similarly , traf2 was also sequestered to the insoluble fraction in the presence of smurf2 wt . because no obvious difference in the total expression levels of tnf - r2 could be detected between the different setups , these results suggested that the down - regulation of tnf - r2 observed in the previous experiments ( in which only the soluble fraction was analyzed ) is due to its translocation to the insoluble fraction . to examine whether tnf - r2 also undergoes tnf - dependent ubiquitination , ubiquitination of endogenous tnf - r2 was assessed in pc60htnf - r55 r75 cells , a rat / t cell hybridoma cell line that contains stably transfected tnf - r1 and tnf - r2 ( vandenabeele et al ., 1995 ). cells were pretreated with 30 μm of the proteasome inhibitor mg - 132 for 30 minutes and subsequently stimulated with 1 μg / ml htnf for 15 or 60 minutes . tnf - r2 immunoprecipitates were analyzed for the presence of ubiquitin - tnf - r2 conjugates via immunoblotting with an anti - ubiquitin antibody . ubiquitinated tnf - r2 could be clearly detected one hour after tnf stimulation ( fig9 ). only one band instead of a smear was detected , thus resembling mono - ubiquitination of tnf - r2 instead of poly - ubiquitination , as has been shown for other membrane receptors ( hicke et al ., 2001a ). similarly , a relocalization of tnf - r2 to the insoluble fraction could be demonstrated after three hours tnf stimulation ( fig1 ), following the time after which we were able to detect tnf - r2 ubiquitination . smurf2 overexpression does not affect traf2 - or tnf - r2 - induced nf - κb and p38 mapk activation taking into account that ectopic expression of smurf2 can induce ubiquitination and subsequent re - localization of tnf - r2 to the insoluble fraction , we wondered whether this might have any effect on tnf - r2 - mediated signal transduction leading to nf - kb and p38 mapk activation . to investigate this , hek293t cells were transiently transfected with tnf - r2 , an nf - κb - dependent luciferase reporter gene and variable amounts of smurf2 wt or smurf2 ( c716g ) mutant . overexpression of tnf - r2 on its own already activated nf - κb - dependent gene expression is most likely caused by the spontaneous oligomerization of receptor molecules ( rothe et al ., 1995 ). although smurf2 wt induces a 40 % reduction in nf - κb activity compared to the control , this inhibition seems not to be related to its ligase activity since the catalytic inactive mutant smurf2 ( c716g ) gives a similar reduction ( fig1 ). it might be that smurf2 competes with another essential signaling protein for binding to traf2 , thus preventing nf - κb activation . similarly , we analyzed whether ectopic expression of smurf2 has any effect on p38 mapk activity in hek293t cells . overexpression of e - traf2 or tnf - stimulation for ten minutes were used as positive controls . as shown in fig1 , smurf2 had no effect on basal or tnf - induced p38 mapk phosphorylation . it should be mentioned , however , that p38 mapk activation in the latter experiment might mainly result from tnf - r1 , thus masking a potential effect of smurf2 on tnf - r2 signaling . all of which are incorporated by this reference in their entirety anderson r . g . and k . jacobson . a role for lipid shells in targeting proteins to caveolae , rafts , and other lipid domains . science , 2002 jun . 7 ; 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