BACKGROUND
cullin-ring ubiquitin ligase  family of e <dig> enzymes participates in diverse cellular and physiological processes  <cit> .
each crl complex contains a cullin family member that serves as a scaffold to assemble a functional e <dig> complex.
the c-terminal globular domain of cullin interacts with the small ring protein rbx <dig>  forming the catalytic core, while the substrate-recognizing module assembles at the n-terminal cullin repeats domain  <cit> .
in scf  complexes, the substrate-recognizing module consisting of skp <dig> and an f-box protein such as skp <dig> interacts specifically with cul <dig>  <cit> .
similarly, elongin b/c-vhl complex interacts with cul <dig>  <cit> , the btb domain substrate adaptor binds to cul <dig>  while ddb <dig> serves as a cul <dig> adaptor  <cit> .
ddb <dig> belongs to a family of proteins with significant sequence homology  <cit>  that includes sap130/sf3b- <dig>  a component of transcription and rna splicing complex  <cit>  and cpsf <dig>  cleavage and polyadenylation specificity factor  <cit> .
the latter two proteins have not been reported to have functions involving the ubiquitin system.
human cells express seven different cullins, most of which if not all can be modified by an ubiquitin-like protein nedd <dig>  on a conserved lysine residue near the c-terminus of the cullin subunit  <cit> .
nedd <dig> conjugation to cullins  is catalyzed by nedd8-specific e <dig> and e <dig> enzymes  <cit> , while its removal  is mediated by cop <dig> signalosome   <cit> .
the de-neddylated  cullins are specifically bound by cand <dig>  <cit> .
neddylation is shown to facilitate polyubiquitination by crls, while de-neddylation is necessary to maintain the stability of crl components  <cit> .
it was recently shown that skp1-skp <dig> complex triggers dissociation of cand <dig> from cul <dig> and, when charged with substrate p <dig>  induces cul <dig> neddylation by inhibiting csn mediated de-neddylation  <cit> .
this study shows that the substrate-binding module with the charged substrate can drive cullin neddylation.
csn is a functionally pleiotropic complex composed of eight subunits designated csn <dig> to csn <dig>  each having unique roles and specific functions  <cit> .
in addition to the de-neddylase activity that involves csn <dig> and csn5/jab <dig>  <cit> , csn also interacts and recruits de-ubiquitin enzymes and protein kinases  <cit> .
deletions of csn cause drastic alterations in the gene expression profile and early lethality of multi-cellular model organisms such as arabidopsis, drosophila, and mouse  <cit> .
we have shown previously that csn <dig> central and c-terminal regions are necessary for csn complex integrity in both plant and animal cells  <cit> .
the n-terminal domain  of csn <dig> is not involved in complex assembly but carries an activity that inhibits ap- <dig> dependent transcription in mammalian cells  <cit> .
in arabidopsis, deletion of csn1-ntd causes early lethality despite that the mutant  can assemble a csn complex   <cit> .
to understand the functions associated with csn1-ntd, we initiated a search for proteins interacting with this domain.
we report here identification of sap130/sf3b <dig>  a member of ddb <dig> family proteins and an established component of transcription complex and rna processing complex.
our data show that sap <dig> is a general cullin binding protein that normally associates with neddylated form of endogenous cullins in vivo.
the preference of sap <dig> for neddylated cullins is dependent on cand <dig>  a protein that specifically binds un-neddylated cullins.
sap <dig> predominantly binds c-terminal domain of cullin and forms tertiary complex with fully assembled crls.
RESULTS
sap130/sf3b <dig> is a csn1-ntd binding protein
to isolate csn1-ntd interacting proteins, we took a biochemical approach previously described by lyapina et al.
<cit> .
the bait protein ht-fs1-ntd, which contains an n-terminus  <dig> × his-tag linked to the tev protease cleavage site upstream of a flag-tagged csn1-ntd  , was immobilized and incubated with nih3t <dig> whole cell extract.
upon tev protease cleavage, csn1-ntd and its bound proteins were gently eluted and visualized by coomassie blue staining .
individual protein bands were excised and identified by mass-spectrometry and peptide sequencing .
as anticipated, all of these proteins were substoichiometric to the bait, suggesting that the interaction with csn1-ntd is weak or transient in contrast to stable subunit-subunit interactions within csn complex.
besides heat shock  <dig> and  <dig> families of proteins, the following proteins were identified: sap130/sf3b- <dig>  ddx <dig>  cf im <dig> and erp <dig> .
sap <dig> is a component of sf3b complex, which is an integral part of 17s u <dig> snrnp particle  <cit> .
sap <dig>  but not other sf3b subunits, is also a stoichiometric component of gcn5-containing transcription complexes such as staga and tftc complexes   <cit> .
ddx <dig>  and cf im <dig> are involved in rna processing, while erp <dig> plays a key role in thiol-mediated retention in er.
we are particularly interested in sap <dig> because it is highly homologous to ddb <dig>  <cit> , a known component of cul4-based ubiquitin ligase complex that is tightly regulated by the csn complex  <cit> .
to verify interaction of sap <dig> with csn <dig>  sap130-myc was transiently expressed with representative csn subunits in hela cells .
flag-csn <dig>  csn1n  and flag-csn <dig>  but not flag-csn <dig>  readily co-precipitated sap130-myc.
immunoprecipitation of endogenous csn <dig> from nuclear extract weakly but consistently pulled down endogenous sap <dig> .
in addition, recombinant gst-csn <dig>  but not gst itself, could pull down in vitro translated sap <dig> from the lysate .
similarly, another member of ddb <dig> family, cpsf <dig> can also co-immunoprecipitate csn subunits when transiently expressed in cultured cells .
therefore, all three members of the ddb <dig> family in humans can interact with csn.
preferential association of sap <dig> with neddylated cullins is dependent on cand1
given that ddb <dig> functions as a component of cul4a ubiquitin ligase complex, we investigated possible interaction of sap <dig> with cullins.
transiently expressed sap <dig> co-immunoprecipitated endogenous cul <dig>  cul <dig>  cul <dig> and sap <dig>  a known component of sf3b complex .
we noticed that sap <dig> appeared to pull-down only the slower migrating cullin band that normally corresponds to neddylated form of cullins.
to definitively verify this, sap <dig> was in vitro translated in rabbit reticulocyte lysate  supplemented with recombinant gst-nedd <dig>  gst-ubiquitin or gst proteins.
in this system, gst-nedd <dig> can be conjugated to endogenous cul <dig> in the lysate generating cul2gst-nedd <dig> .
both anti-cul <dig> and anti-gst blots showed that sap <dig> specifically co-immunoprecipitated cul2gst-nedd <dig> and cul2nedd <dig>  but not un-neddylated cul <dig> .
this result explicitly demonstrates that sap <dig> preferentially associates with neddylated cul <dig> in the lysate.
taken together the data in figures 2a and 2b, sap <dig> most likely interacts with other neddylated cullins such as cul <dig> and cul <dig>  the observation that cul <dig> was not co-immunoprecipitated by sap <dig> as shown in figure 2a was probably due to the fact that neddylated cul <dig> could not be detected in the total cell extract by the anti-cul <dig> antibody .
it should be mentioned that because neddylated cullins tend to be unstable, the experiments shown above were carried out in the presence of proteasome inhibitor mg <dig> .
we next asked whether sap <dig> binds nedd <dig> itself by examining un-conjugated gst-nedd <dig> monomer in sap <dig> immunoprecipitates.
as shown in figure 2c, gst-nedd <dig>  similar to gst itself, was not bound by sap <dig>  indicating that sap <dig> did not have specific affinity to un-conjugated nedd <dig>
it has been shown that cand <dig>  specifically binds un-neddylated cullins forming stable cand1-cullin complexes in the cells  <cit> .
to determine whether the level of cand <dig> may affect the selective association of sap <dig> with neddylated cullins, we knocked down cand <dig> level in hela cells by sirna.
reduction of cand <dig> amount did not affect the steady state neddylation level of cul <dig>  however sap <dig> no longer exhibited neddylation preference as it co-immunoprecipitated both neddylated and un-neddylated cul <dig> .
concomitant with the loss of neddylation preference was an increase in the amount of cul <dig> that was associated with sap <dig> .
this data suggests that cand <dig> inhibited sap130-cul <dig> interaction and that cand <dig> was necessary for preferential association of sap <dig> with neddylated cul <dig> in the cells.
although the precise mechanism is not understood, it seems possible that cand <dig> sequesters un-neddylated cullins from sap <dig>  regardless, this data showed that neddylation is not necessary for sap <dig> to bind cullins and that sap <dig> has no intrinsic specificity for nedd <dig>
sap <dig> forms tertiary complexes with crls and associates with ubiquitin e <dig> activity
next we asked whether sap <dig> could form tertiary complex with the substrate-binding components of crls.
the cul <dig> substrate receptor ha-vhl and flag-sap <dig> were expressed in hek <dig> cells.
vhl and sap <dig> clearly co-immunoprecipitated each other  supporting the idea that sap <dig> can form a tertiary complex with the cul2-vhl e <dig> complex.
noticeably, while vhl pulled down both neddylated and un-neddylated cul <dig>  sap <dig> pulled down predominantly the neddylated cul <dig>  in addition, sap <dig> also co-precipitated endogenous skp <dig> and skp <dig>  as well as cul <dig> adaptor ddb <dig>  and a transiently expressed ddb <dig> .
these results suggest that in contrast to cand <dig>  sap <dig> is able to form tertiary complexes with different crl e3s including scf, cul2-vhl and cul4-ddb <dig> complexes.
we reasoned that by binding to active crls, sap <dig> may associate with e <dig> ubiquitin ligase activities.
flag-sap <dig> and flag-cpsf <dig> immunocomplexes were isolated from cultured cells and in vitro e <dig> activity assay was performed .
in this assay, the anti-ubiquitin blot was used to detect the polyubiquitin products generated by the immunocomplexes.
as anticipated, both sap <dig> and cpsf <dig> immunocomplexes exhibited robust ubiquitin e1/e <dig> dependent polyubiquitinating activities in vitro .
sap <dig> binding domains in cullins
in crl complexes, the substrate-binding module assembles at the n-terminal cullin repeats, while cand <dig> binds to both n- and c-terminal domains of cullins  <cit> .
to define the sap <dig> binding domains on cullins, we constructed the following truncations in ha-cul <dig> : cul1n <dig> , cul1c <dig>  and cul1c <dig> .
as expected, skp <dig> was co-precipitated by full-length cul <dig> and cul1n <dig> but not cul1c <dig> or cul1c <dig> .
cand <dig> only interacted with full-length cul <dig> but none of the truncations, while csn <dig> could associate with both the n-terminal and c-terminal cul <dig> deletions .
sap <dig> strongly interacted with cul <dig> c-terminal fragment c <dig> and was also associated with c <dig> and n <dig> .
this result shows that sap <dig> can bind to both n-terminal as well as the c-terminal domains of cul <dig>
we noticed that while sap <dig> preferred neddylated form of endogenous cul <dig>  it could bind both neddylated and un-neddylated forms of ectopically expressed ha-cul <dig> .
to understand how endogenous cul <dig> may differ from ectopically expressed ha-cul <dig> in neddylation dynamics, we compared the changes in the neddylation level in response to csn inactivation .
we previously reported that the de-neddylation activity of csn could be blocked by anti-csn <dig> antibody  <cit> .
specifically, incubation of the cell extract with anti-csn <dig> antibody blocks the de-neddylation reaction but not the cellular neddylation activity, causing neddylated cullins to accumulate.
hela cell extracts containing over-expressed ha-cul <dig> or vector were incubated with anti-csn <dig> antibody or a pre-immune serum.
while endogenous cul <dig> became completely neddylated by anti-csn <dig> treatment , the neddylation level of ha-cul <dig> did not change .
this result suggests that the neddylation reaction on ha-cul <dig> was slower or deficient compared to endogenous cul <dig>  under the identical condition when de-neddylation was blocked, ha-cul <dig> failed to be fully neddylated like endogenous cul <dig> , suggesting that overexpressed ha-cul <dig> is a poor neddylation substrates in vivo.
we reasoned that ha-cul <dig> overexpression system is unsuitable to study the neddylation and de-neddylation dynamics.
however, this system is still valid for defining the cul <dig> domains involved in sap <dig> binding.
in fact, sap <dig> interaction with cullins does not require neddylation as shown earlier.
to extend the observations on sap130-cul <dig> interaction, we carried out a similar experiment on cul <dig> truncations .
ha-cul2c <dig>  containing the wh-a and wh-b domains and the nedd <dig> attachment site  can be effectively neddylated, as indicated by the nedd <dig> blot after ha-precipitation .
as predicted, both the full-length cul <dig> and the n <dig> fragment could pull-down elongin b, while c <dig> could not .
this result is consistent with our understanding that the n-terminal region of cul <dig> is necessary and sufficient for binding to elongin b/c-vhl substrate-binding module.
as in cul <dig> binding experiment, cand <dig> was associated only with full-length cul <dig> but not with any of the terminal deletions .
we found that sap <dig> specifically pulled down the c-terminal fragments c <dig> and Δn <dig> , but not n <dig> , indicating that sap <dig> interacted with cul <dig> c-terminal wh-a/b domain.
in a similar experiment, csn <dig> was found to bind ha-cul <dig> and all of the truncation mutants tested  suggesting that csn <dig> or the csn complex has more than one contact site in cul <dig> as in cul <dig>  these results show that sap <dig> predominantly binds the c-terminal domain  of cullins, but it can also weakly interact with the n-terminal domain; while csn <dig> binds to both n- and c-terminal regions of cullins.
csn regulates sap130-cullin interaction
to investigate the role of csn <dig> in sap130-cul <dig> interaction, recombinant gst-csn <dig> or gst protein was incubated with in vitro translated ha-sap <dig> in rrl.
we found that addition of the recombinant proteins did not affect cul <dig> stability or its neddylation level .
this is consistent with the genetic analysis in arabidopsis that csn1-ntd is not required for csn mediated de-neddylation of cul <dig>  <cit> .
notably, the amounts of cul2gst-nedd <dig> that co-precipitated with sap <dig> clearly declined when the lysate was supplemented with increasing amounts of gst-csn <dig>  but not gst .
this result indicated that excess csn <dig> protein could interfere with the interaction between sap <dig> and cul <dig>
we next determined the effect of csn <dig> on sap <dig> associated e <dig> activity.
whole cell extract from a stable flag-sap <dig> line was pre-incubated with recombinant gst-csn <dig>  gst, or biochemically purified csn complex.
flag-sap <dig> immunocomplex was subsequently isolated and used for the in vitro e <dig> activity assay .
sap <dig> associated polyubiquitination activity was detectably reduced upon addition of excess gst-csn <dig>  and was dramatically reduced upon addition of csn complex .
the observation that sap <dig> associated e <dig> activity was sensitivity to csn complex further reinforced the notion that the polyubiquitin chains produced by the sap <dig> immunocomplex were mediated by crls rather than non-specifically bound e3s.
we also found that cul <dig> neddylation was reduced upon addition of csn deneddylase as expected , but remained unchanged when supplemented with gst-csn <dig> .
since csn <dig> can inhibit sap130-cul <dig> interaction , we suggest that csn <dig> affects sap <dig> associated e <dig> activity by preventing sap130-crl interaction.
taken together, our data indicate that csn complex negatively regulates sap <dig> associated polyubiquitination activity by both cullin de-neddylation and blocking the recruitment of crl e <dig> complexes.
discussion
crl ubiquitin e <dig> ligases are essential ubiquitination machineries in eukaryotes that are involved in a wide array of fundamental processes from genome stability to cell cycle control  <cit> .
nedd <dig> attachment and removal from the cullin subunit represents an important mechanism by which crl functions are regulated  <cit> .
while the enzymes mediating cullin neddylation and de-neddylation have been identified, the regulatory mechanism and the functional consequences of this modification remain obscure.
to this end, we have identified sap <dig> as a new type of cullin binding protein that interacts with multiple cullins predominantly via cullin c-terminal domain.
under normal conditions when cand <dig> was present at physiological levels, sap <dig> preferred neddylated cullins and associated with fully assembled crl e <dig> ligases.
sap <dig> immunocomplex exhibited a polyubiquitination activity that could be inhibited by excess amount of csn complex or csn <dig> protein.
the role of sap <dig> in cullin neddylation pathway remains elusive.
ectopic expression or moderate knock-down of sap <dig> in cultured cells did not cause obvious changes in cullin neddylation level .
we found that neddylation is not an essential signal for sap130-cullin association, as sap <dig> can bind unneddylated cullins when cul <dig> or cul <dig> were overexpressed in cultured cells or when cand <dig> level was knocked down.
this result suggests that cand <dig> is the key factor that limits sap130-cullin interaction and renders preferential association of sap <dig> with neddylated cullins in vivo.
along this line, our data showed that csn de-neddylase negatively regulates sap <dig> associated ubiquitination activity .
sap <dig> does not appear to discriminate between different cullins.
this cullin binding capability distinguishes it from conventional crl adaptors such as skp <dig>  elongin b/c and btb proteins, which exhibit specificity to different cullins by binding to the n-terminal cullin repeats.
nonetheless, since sap <dig> weakly associates with the n-terminal region of cul <dig> , it may act as a substrate adaptor like ddb <dig> under certain circumstances.
it is also possible that sap <dig> acts as a general crl adaptor to assist substrate ubiquitination by binding to cullin c-terminal domain.
along these lines, it is interesting to note that commd <dig>  a nf-κb inhibitor, was found to promote ubiquitination of nf-κb by binding to the c-terminal domain of cul <dig> in the elongin b/c-cul2-socs <dig> e <dig> complex  <cit> .
commd <dig> regulates the stability of rela-chromatin complex and the recruitment of rel-a to the e <dig> complex in response to stimuli  <cit> .
bornstein et al.
<cit>  showed that increased availability of the cul <dig> substrate binding components skp1-skp <dig> along with its substrate could trigger cul <dig> neddylation.
this process is accomplished by a sequential step that involves the dissociation of cand <dig> from cul <dig> and inactivation of csn mediated de-neddylation.
this finding together with our studies raises the question of whether sap <dig> may play an active role in facilitating substrate dependent cullins neddylation, particularly during the gene expression processes.
sap <dig> is a known component of rna processing and transcription complexes.
the sf3b complex, which contains sap <dig> or sf3b- <dig>  is involved in binding to the intron's branch point of the pre-mrna during the early step of pre-spliceosome assembly.
however, sap <dig> is the only subunit of sf3b that does not cross-link to rna  <cit> .
similarly, though a component of staga/tftc complexes which bind damaged dna template and contain histone acetyltransferase activity   <cit> , sap <dig> is neither required for the dna binding nor the hat activity  <cit> .
to date, the specific role of sap <dig> in these complexes is yet unknown.
increasing evidence show that transcription and co-transcriptional rna processing are dependent on the ubiquitin-proteasome system and the neddylation pathway.
in our recent study on csn <dig> knockout mice, we found that deletion of csn <dig> abolished csn-mediated deneddylation of cullins such as cul <dig>  cul <dig>  and cul4a  <cit> .
however the upset in cul <dig> neddylation level did not compromise degradation of scf substrates, but caused dramatic transcription defects in a subset of genes  <cit> .
many crl components such as f-box proteins, elongin b/c and vhl have also been implicated in transcription control  <cit> .
our finding that sap <dig> associates with cullins raises a tantalizing hypothesis that sap <dig> links crl ubiquitination complexes and the gene expression machinery in a process regulated by csn.
it remains to be elucidated in the future whether sap <dig> plays a role in crl mediated substrate ubiquitination, particularly at the vicinity of rna processing and transcription complexes.
CONCLUSIONS
cullin-ring ubiquitin e <dig> ligases are critical ubiquitination machineries in eukaryotes.
in this study, we identified sap <dig> as a new cullin binding protein that is likely involved in the nedd <dig> pathway.
sap <dig> interacts with neddylated cullins in vivo in a manner dependent on cand <dig> level.
sap <dig> forms tertiary complexes with active cullin-ring e <dig> ligases and is associated with polyubiquitination activity that could be modulated by csn.

 cullin-ring ubiquitin ligase family of e enzymes participates in diverse cellular and physiological processes. each crl complex contains a cullin family member that serves as a scaffold to assemble a functional e complex. the c-terminal globular domain of cullin interacts with the small ring protein rbx forming the catalytic core, while the substrate-recognizing module assembles at the n-terminal cullin repeats domain. in scf complexes, the substrate-recognizing module consisting of skp and an f-box protein such as skp interacts specifically with cul. similarly, elongin b/c-vhl complex interacts with cul, the btb domain substrate adaptor binds to cul while ddb serves as a cul adaptor. ddb belongs to a family of proteins with significant sequence homology that includes sap130/sf3b- a component of transcription and rna splicing complex and cpsf cleavage and polyadenylation specificity factor. the latter two proteins have not been reported to have functions involving the ubiquitin system. human cells express seven different cullins, most of which if not all can be modified by an ubiquitin-like protein nedd on a conserved lysine residue near the c-terminus of the cullin subunit. nedd conjugation to cullins is catalyzed by nedd8-specific e and e enzymes, while its removal is mediated by cop signalosome. the de-neddylated cullins are specifically bound by cand. neddylation is shown to facilitate polyubiquitination by crls, while de-neddylation is necessary to maintain the stability of crl components. it was recently shown that skp1-skp complex triggers dissociation of cand from cul and, when charged with substrate p induces cul neddylation by inhibiting csn mediated de-neddylation. this study shows that the substrate-binding module with the charged substrate can drive cullin neddylation. csn is a functionally pleiotropic complex composed of eight subunits designated csn to csn each having unique roles and specific functions. in addition to the de-neddylase activity that involves csn and csn5/jab, csn also interacts and recruits de-ubiquitin enzymes and protein kinases. deletions of csn cause drastic alterations in the gene expression profile and early lethality of multi-cellular model organisms such as arabidopsis, drosophila, and mouse. we have shown previously that csn central and c-terminal regions are necessary for csn complex integrity in both plant and animal cells. the n-terminal domain of csn is not involved in complex assembly but carries an activity that inhibits ap- dependent transcription in mammalian cells. in arabidopsis, deletion of csn1-ntd causes early lethality despite that the mutant can assemble a csn complex. to understand the functions associated with csn1-ntd, we initiated a search for proteins interacting with this domain. we report here identification of sap130/sf3b a member of ddb family proteins and an established component of transcription complex and rna processing complex. our data show that sap is a general cullin binding protein that normally associates with neddylated form of endogenous cullins in vivo. the preference of sap for neddylated cullins is dependent on cand a protein that specifically binds un-neddylated cullins. sap predominantly binds c-terminal domain of cullin and forms tertiary complex with fully assembled crls.  sap130/sf3b is a csn1-ntd binding protein to isolate csn1-ntd interacting proteins, we took a biochemical approach previously described by lyapina et al.. the bait protein ht-fs1-ntd, which contains an n-terminus × his-tag linked to the tev protease cleavage site upstream of a flag-tagged csn1-ntd, was immobilized and incubated with nih3t whole cell extract. upon tev protease cleavage, csn1-ntd and its bound proteins were gently eluted and visualized by coomassie blue staining. individual protein bands were excised and identified by mass-spectrometry and peptide sequencing. as anticipated, all of these proteins were substoichiometric to the bait, suggesting that the interaction with csn1-ntd is weak or transient in contrast to stable subunit-subunit interactions within csn complex. besides heat shock and families of proteins, the following proteins were identified: sap130/sf3b- ddx cf im and erp. sap is a component of sf3b complex, which is an integral part of 17s u snrnp particle. sap but not other sf3b subunits, is also a stoichiometric component of gcn5-containing transcription complexes such as staga and tftc complexes. ddx and cf im are involved in rna processing, while erp plays a key role in thiol-mediated retention in er. we are particularly interested in sap because it is highly homologous to ddb, a known component of cul4-based ubiquitin ligase complex that is tightly regulated by the csn complex. to verify interaction of sap with csn sap130-myc was transiently expressed with representative csn subunits in hela cells. flag-csn csn1n and flag-csn but not flag-csn readily co-precipitated sap130-myc. immunoprecipitation of endogenous csn from nuclear extract weakly but consistently pulled down endogenous sap. in addition, recombinant gst-csn but not gst itself, could pull down in vitro translated sap from the lysate. similarly, another member of ddb family, cpsf can also co-immunoprecipitate csn subunits when transiently expressed in cultured cells. therefore, all three members of the ddb family in humans can interact with csn. preferential association of sap with neddylated cullins is dependent on cand1 given that ddb functions as a component of cul4a ubiquitin ligase complex, we investigated possible interaction of sap with cullins. transiently expressed sap co-immunoprecipitated endogenous cul cul cul and sap a known component of sf3b complex. we noticed that sap appeared to pull-down only the slower migrating cullin band that normally corresponds to neddylated form of cullins. to definitively verify this, sap was in vitro translated in rabbit reticulocyte lysate supplemented with recombinant gst-nedd gst-ubiquitin or gst proteins. in this system, gst-nedd can be conjugated to endogenous cul in the lysate generating cul2gst-nedd. both anti-cul and anti-gst blots showed that sap specifically co-immunoprecipitated cul2gst-nedd and cul2nedd but not un-neddylated cul. this result explicitly demonstrates that sap preferentially associates with neddylated cul in the lysate. taken together the data in figures 2a and 2b, sap most likely interacts with other neddylated cullins such as cul and cul the observation that cul was not co-immunoprecipitated by sap as shown in figure 2a was probably due to the fact that neddylated cul could not be detected in the total cell extract by the anti-cul antibody. it should be mentioned that because neddylated cullins tend to be unstable, the experiments shown above were carried out in the presence of proteasome inhibitor mg. we next asked whether sap binds nedd itself by examining un-conjugated gst-nedd monomer in sap immunoprecipitates. as shown in figure 2c, gst-nedd similar to gst itself, was not bound by sap indicating that sap did not have specific affinity to un-conjugated nedd it has been shown that cand specifically binds un-neddylated cullins forming stable cand1-cullin complexes in the cells. to determine whether the level of cand may affect the selective association of sap with neddylated cullins, we knocked down cand level in hela cells by sirna. reduction of cand amount did not affect the steady state neddylation level of cul however sap no longer exhibited neddylation preference as it co-immunoprecipitated both neddylated and un-neddylated cul. concomitant with the loss of neddylation preference was an increase in the amount of cul that was associated with sap. this data suggests that cand inhibited sap130-cul interaction and that cand was necessary for preferential association of sap with neddylated cul in the cells. although the precise mechanism is not understood, it seems possible that cand sequesters un-neddylated cullins from sap regardless, this data showed that neddylation is not necessary for sap to bind cullins and that sap has no intrinsic specificity for nedd sap forms tertiary complexes with crls and associates with ubiquitin e activity next we asked whether sap could form tertiary complex with the substrate-binding components of crls. the cul substrate receptor ha-vhl and flag-sap were expressed in hek cells. vhl and sap clearly co-immunoprecipitated each other supporting the idea that sap can form a tertiary complex with the cul2-vhl e complex. noticeably, while vhl pulled down both neddylated and un-neddylated cul sap pulled down predominantly the neddylated cul in addition, sap also co-precipitated endogenous skp and skp as well as cul adaptor ddb and a transiently expressed ddb. these results suggest that in contrast to cand sap is able to form tertiary complexes with different crl e3s including scf, cul2-vhl and cul4-ddb complexes. we reasoned that by binding to active crls, sap may associate with e ubiquitin ligase activities. flag-sap and flag-cpsf immunocomplexes were isolated from cultured cells and in vitro e activity assay was performed. in this assay, the anti-ubiquitin blot was used to detect the polyubiquitin products generated by the immunocomplexes. as anticipated, both sap and cpsf immunocomplexes exhibited robust ubiquitin e1/e dependent polyubiquitinating activities in vitro. sap binding domains in cullins in crl complexes, the substrate-binding module assembles at the n-terminal cullin repeats, while cand binds to both n- and c-terminal domains of cullins. to define the sap binding domains on cullins, we constructed the following truncations in ha-cul: cul1n, cul1c and cul1c. as expected, skp was co-precipitated by full-length cul and cul1n but not cul1c or cul1c. cand only interacted with full-length cul but none of the truncations, while csn could associate with both the n-terminal and c-terminal cul deletions. sap strongly interacted with cul c-terminal fragment c and was also associated with c and n. this result shows that sap can bind to both n-terminal as well as the c-terminal domains of cul we noticed that while sap preferred neddylated form of endogenous cul it could bind both neddylated and un-neddylated forms of ectopically expressed ha-cul. to understand how endogenous cul may differ from ectopically expressed ha-cul in neddylation dynamics, we compared the changes in the neddylation level in response to csn inactivation. we previously reported that the de-neddylation activity of csn could be blocked by anti-csn antibody. specifically, incubation of the cell extract with anti-csn antibody blocks the de-neddylation reaction but not the cellular neddylation activity, causing neddylated cullins to accumulate. hela cell extracts containing over-expressed ha-cul or vector were incubated with anti-csn antibody or a pre-immune serum. while endogenous cul became completely neddylated by anti-csn treatment, the neddylation level of ha-cul did not change. this result suggests that the neddylation reaction on ha-cul was slower or deficient compared to endogenous cul under the identical condition when de-neddylation was blocked, ha-cul failed to be fully neddylated like endogenous cul, suggesting that overexpressed ha-cul is a poor neddylation substrates in vivo. we reasoned that ha-cul overexpression system is unsuitable to study the neddylation and de-neddylation dynamics. however, this system is still valid for defining the cul domains involved in sap binding. in fact, sap interaction with cullins does not require neddylation as shown earlier. to extend the observations on sap130-cul interaction, we carried out a similar experiment on cul truncations. ha-cul2c containing the wh-a and wh-b domains and the nedd attachment site can be effectively neddylated, as indicated by the nedd blot after ha-precipitation. as predicted, both the full-length cul and the n fragment could pull-down elongin b, while c could not. this result is consistent with our understanding that the n-terminal region of cul is necessary and sufficient for binding to elongin b/c-vhl substrate-binding module. as in cul binding experiment, cand was associated only with full-length cul but not with any of the terminal deletions. we found that sap specifically pulled down the c-terminal fragments c and Δn, but not n, indicating that sap interacted with cul c-terminal wh-a/b domain. in a similar experiment, csn was found to bind ha-cul and all of the truncation mutants tested suggesting that csn or the csn complex has more than one contact site in cul as in cul these results show that sap predominantly binds the c-terminal domain of cullins, but it can also weakly interact with the n-terminal domain; while csn binds to both n- and c-terminal regions of cullins. csn regulates sap130-cullin interaction to investigate the role of csn in sap130-cul interaction, recombinant gst-csn or gst protein was incubated with in vitro translated ha-sap in rrl. we found that addition of the recombinant proteins did not affect cul stability or its neddylation level. this is consistent with the genetic analysis in arabidopsis that csn1-ntd is not required for csn mediated de-neddylation of cul. notably, the amounts of cul2gst-nedd that co-precipitated with sap clearly declined when the lysate was supplemented with increasing amounts of gst-csn but not gst. this result indicated that excess csn protein could interfere with the interaction between sap and cul we next determined the effect of csn on sap associated e activity. whole cell extract from a stable flag-sap line was pre-incubated with recombinant gst-csn gst, or biochemically purified csn complex. flag-sap immunocomplex was subsequently isolated and used for the in vitro e activity assay. sap associated polyubiquitination activity was detectably reduced upon addition of excess gst-csn and was dramatically reduced upon addition of csn complex. the observation that sap associated e activity was sensitivity to csn complex further reinforced the notion that the polyubiquitin chains produced by the sap immunocomplex were mediated by crls rather than non-specifically bound e3s. we also found that cul neddylation was reduced upon addition of csn deneddylase as expected, but remained unchanged when supplemented with gst-csn. since csn can inhibit sap130-cul interaction, we suggest that csn affects sap associated e activity by preventing sap130-crl interaction. taken together, our data indicate that csn complex negatively regulates sap associated polyubiquitination activity by both cullin de-neddylation and blocking the recruitment of crl e complexes. discussion crl ubiquitin e ligases are essential ubiquitination machineries in eukaryotes that are involved in a wide array of fundamental processes from genome stability to cell cycle control. nedd attachment and removal from the cullin subunit represents an important mechanism by which crl functions are regulated. while the enzymes mediating cullin neddylation and de-neddylation have been identified, the regulatory mechanism and the functional consequences of this modification remain obscure. to this end, we have identified sap as a new type of cullin binding protein that interacts with multiple cullins predominantly via cullin c-terminal domain. under normal conditions when cand was present at physiological levels, sap preferred neddylated cullins and associated with fully assembled crl e ligases. sap immunocomplex exhibited a polyubiquitination activity that could be inhibited by excess amount of csn complex or csn protein. the role of sap in cullin neddylation pathway remains elusive. ectopic expression or moderate knock-down of sap in cultured cells did not cause obvious changes in cullin neddylation level. we found that neddylation is not an essential signal for sap130-cullin association, as sap can bind unneddylated cullins when cul or cul were overexpressed in cultured cells or when cand level was knocked down. this result suggests that cand is the key factor that limits sap130-cullin interaction and renders preferential association of sap with neddylated cullins in vivo. along this line, our data showed that csn de-neddylase negatively regulates sap associated ubiquitination activity. sap does not appear to discriminate between different cullins. this cullin binding capability distinguishes it from conventional crl adaptors such as skp elongin b/c and btb proteins, which exhibit specificity to different cullins by binding to the n-terminal cullin repeats. nonetheless, since sap weakly associates with the n-terminal region of cul, it may act as a substrate adaptor like ddb under certain circumstances. it is also possible that sap acts as a general crl adaptor to assist substrate ubiquitination by binding to cullin c-terminal domain. along these lines, it is interesting to note that commd a nf-κb inhibitor, was found to promote ubiquitination of nf-κb by binding to the c-terminal domain of cul in the elongin b/c-cul2-socs e complex. commd regulates the stability of rela-chromatin complex and the recruitment of rel-a to the e complex in response to stimuli. bornstein et al. showed that increased availability of the cul substrate binding components skp1-skp along with its substrate could trigger cul neddylation. this process is accomplished by a sequential step that involves the dissociation of cand from cul and inactivation of csn mediated de-neddylation. this finding together with our studies raises the question of whether sap may play an active role in facilitating substrate dependent cullins neddylation, particularly during the gene expression processes. sap is a known component of rna processing and transcription complexes. the sf3b complex, which contains sap or sf3b- is involved in binding to the intron's branch point of the pre-mrna during the early step of pre-spliceosome assembly. however, sap is the only subunit of sf3b that does not cross-link to rna. similarly, though a component of staga/tftc complexes which bind damaged dna template and contain histone acetyltransferase activity, sap is neither required for the dna binding nor the hat activity. to date, the specific role of sap in these complexes is yet unknown. increasing evidence show that transcription and co-transcriptional rna processing are dependent on the ubiquitin-proteasome system and the neddylation pathway. in our recent study on csn knockout mice, we found that deletion of csn abolished csn-mediated deneddylation of cullins such as cul cul and cul4a. however the upset in cul neddylation level did not compromise degradation of scf substrates, but caused dramatic transcription defects in a subset of genes. many crl components such as f-box proteins, elongin b/c and vhl have also been implicated in transcription control. our finding that sap associates with cullins raises a tantalizing hypothesis that sap links crl ubiquitination complexes and the gene expression machinery in a process regulated by csn. it remains to be elucidated in the future whether sap plays a role in crl mediated substrate ubiquitination, particularly at the vicinity of rna processing and transcription complexes.  cullin-ring ubiquitin e ligases are critical ubiquitination machineries in eukaryotes. in this study, we identified sap as a new cullin binding protein that is likely involved in the nedd pathway. sap interacts with neddylated cullins in vivo in a manner dependent on cand level. sap forms tertiary complexes with active cullin-ring e ligases and is associated with polyubiquitination activity that could be modulated by csn. 
neddylation is shown to facilitate e complex assembly; while un-neddylated cullins are bound by cand that prevents recruitment of the substrates. it forms tertiary complexes with fully assembled crl e complexes such as scfskp elongin b/c -cul2- vhl and cul4-ddb complex by binding to both n-terminal and c-terminal domain of cullins. cullin-ring ubiquitin e ligases are regulated by modification of an ubiquitin-like protein, nedd on the cullin subunit.
 cullin-ring ubiquitin e ligases are regulated by modification of an ubiquitin-like protein, nedd on the cullin subunit. neddylation is shown to facilitate e complex assembly; while un-neddylated cullins are bound by cand that prevents recruitment of the substrates. the level of nedd modification is critically dependent on the cop signalosome, an eight-subunit protein complex containing nedd isopeptidase activity.  we report isolation of sap as a csn interacting protein. sap is homologous to ddb and is a component of sf3b rna splicing complex and staga/tftc transcription complexes, but its specific function within these complexes is unknown. we show that sap can interact with a variety of cullin proteins. it forms tertiary complexes with fully assembled crl e complexes such as scfskp elongin b/c -cul2- vhl and cul4-ddb complex by binding to both n-terminal and c-terminal domain of cullins. sap preferentially associates with neddylated cullins in vivo. however knock-down of cand abolished this preference and increased association of sap with cul furthermore, we provide evidence that csn regulates sap130-cul interaction and sap130-associated polyubiquitinating activity.  sap is a cullin binding protein that is likely involved in the nedd pathway. the association of sap with various cullin member proteins such as cul cul and cul4a is modulated by cand and csn. as an established component of transcription and rna processing complexes, we hypothesis that sap may link crl mediated ubiquitination to gene expression. 