BACKGROUND
the regulated expression and degradation of proteins are critical to all aspects of cell development and proliferation.
the two main routes for eukaryotic intracellular protein clearance are the ubiquitin-proteasome system  and the autophagy-lysosome pathway.
a key component involved in regulating degradation of proteins by the ups is the cop <dig> signalosome .
the csn is an eight-subunit  protein complex, highly conserved amongst eukaryotes  <cit>  originally identified in arabidopsis as a negative regulator of photomorphogenesis  <cit> .
through its function in the regulation of the ups, the csn has been implicated in the regulation of biological processes as diverse as dna replication and repair, cell-cycle progression and cell development  <cit> .
degradation of cellular proteins by the 26s proteasome  <cit>  is preceded by ubiquitination of target proteins  <cit> , a process mediated by three enzyme complexes; a ubiquitin activating enzyme , a ubiquitin conjugating enzyme  and a ubiquitin ligase   <cit> .
the e <dig> ligase interacts with the protein substrate and thus confers the specificity of the ups  <cit> .
the largest known class of e <dig> ubiquitin ligases comprises the cullin-ring ligases  of which the best studied is the scf  complex  <cit> .
the cullin subunit  of the scf forms a scaffold to recruit and bring into close proximity the e <dig> and its substrate, thereby facilitating ubiquitin transfer from the e <dig> to target proteins .
the ring protein  is the fourth subunit of the scf and is responsible for e <dig> recruitment, whilst the variable f-box protein subunit, recruited to the scf complex via the adaptor protein skp <dig>  binds substrates selectively  <cit> .
in yeast, over  <dig> f-box proteins are known, over  <dig> in a. thaliana, and ~ <dig> in humans  <cit> .
since each cullin  forms complexes with a variable substrate recognition subunit   specificity in crl target protein recruitment is achieved by the large number of variable srs containing crls.
it is thought that, altogether, the human genome may have the capacity to code for as many as  <dig> different crls.
given the potential number and diversity of target proteins requiring crl mediated ubiquitination for degradation, dynamic regulation of the crl complex repertoire in a cell at any given time is essential.
all cullins studied  have been shown to be modified by neddylation  <cit> , which facilitates their ubiquitin ligase activity  <cit>  possibly via increased e <dig> affinity  <cit> .
the deneddylation of cullins is mediated by the csn complex  <cit> .
although initial studies indicated a negative role for deneddylation, further studies have implicated deneddylation in the positive regulation of crl activity  <cit> .
it has since been proposed by several groups that optimal crl activity requires the cyclic neddylation and deneddylation of the cullin subunit  <cit> .
although the exact mechanisms are not fully understood, it is thought that f-box proteins themselves are targeted for degradation in part by autoubiquitination within the scf complex  <cit> .
the deneddylation of cullins by the csn is believed to regulate the autoubiquitination of srss  <cit> , thereby modulating crl composition and activity.
furthermore, the csn has been shown to be associated with a deubiquitinase activity which may further stabilize autoubiquitinated srss  <cit> .
the csn complex is therefore an integral regulator of crl activity and subsequent protein degradation.
in this present study we have investigated the effects of knocking down csn <dig> and csn <dig> in the model k <dig> cell line, a model of human erythrocyte and megakaryocyte progenitors  <cit> .
whilst knockdown of either csn <dig> or csn <dig> resulted in common changes including the sequential loss of f-box proteins skp <dig>  cdc <dig> and β-trcp other important differences occurred.
for example csn <dig> knockdown resulted in apoptotic cell death associated with aberrant mitosis, whereas csn <dig> knockdown cells underwent non-apoptotic cell death that was associated with both inhibition of autophagy and the formation of a novel csn <dig> containing csn subcomplex.
RESULTS
csn <dig> and csn <dig> knockdown and resulting aberrant scf activity
plasmids encoding short hairpin rna  to either csn <dig> or csn <dig> were electroporated into k <dig> cells together with h-2kk plasmid and transfected cells sorted by anti- h-2kk magnetic beads.
three days post transfection, neither csn <dig> nor csn <dig> protein could be detected by western blot analysis in their respective knockdown protein extracts .
furthermore, csn <dig> knockdown of over 99% and csn <dig> knockdown of over 95% was achieved at the mrna level relative to mock transfectants as measured by quantitative real-time pcr  .
this level of knockdown was maintained throughout the time course of the experiments .
transfection with either the h-2kk plasmid alone or co-transfection with h-2kk plasmid and empty vector  had no effect on the level of csn <dig> or csn <dig> mrna or protein relative to mock transfected cells .
co-transfections with h-2kk and a vector encoding a control scrambled shrna sequence had no effect on the level of csn <dig> or csn <dig> mrna or protein relative to cells co-transfected with empty vector , demonstrating that the results were specific and not due to off-target effects.
the instability of particular csn subunits in the absence of another subunit has been reported previously.
for example, csn <dig> knockdown has been shown to result in the loss of csn <dig>  csn <dig> and csn <dig> protein  <cit> .
we therefore determined csn <dig> and csn <dig> protein levels in csn <dig> and csn <dig> knockdown cells, respectively, by western blotting.
a significant reduction in csn <dig> protein was observed in cells lacking csn <dig>  whilst depletion of csn <dig> had no effect on csn <dig> protein or mrna levels relative to vector control cells .
interestingly, csn <dig> knockdown not only resulted in loss of csn <dig> protein, but also in the significant reduction of csn <dig> mrna as determined by qrt-pcr .
consistent with previous studies of csn deregulation  <cit> , both csn <dig> and csn <dig> knockdowns were associated with accumulation of neddylated cul <dig>  indicating functional dysregulation of the csn following knockdown of either subunit.
studies demonstrating cul <dig> hyperneddylation in csn deficient cells have also demonstrated degradation of the f-box protein skp <dig>  <cit> .
in agreement with these observations, complete loss of skp <dig> protein was observed in both the csn <dig> and csn <dig> knockdown cells .
skp <dig> binds to and mediates the ubiquitination of multiple proteins, including the cyclin dependent kinase inhibitor p27kip <dig>  <cit> .
consistent with loss of skp <dig>  knockdown of csn <dig> and csn <dig> resulted in the accumulation of p <dig> .
loss of csn <dig> and csn <dig> results in the sequential loss of f-box proteins
skp <dig> is one of many f-box proteins that can bind to the cul1/roc1/skp <dig> complex, thereby altering the substrate specificity of the scf .
analysis of three such f-box proteins, skp <dig>  cdc <dig> and β-trcp revealed a sequential loss of protein in csn <dig> and csn <dig> knockdown cells .
in csn <dig> knockdown cells, skp <dig> protein was reduced by ~60% by day  <dig> and over 90% by day  <dig>  whereas cdc <dig> was lost at a slower rate with 30–40% lost by day  <dig> increasing to ~70% by day  <dig> .
loss of β-trcp was still more retarded with ~70% protein remaining at day  <dig> .
by day  <dig>  all three f-box proteins were undetectable in csn <dig> knockdown cells .
similar sequential loss of f-box proteins was observed in csn <dig> knockdown cells.
at day  <dig> post transfection skp <dig> protein could not be detected, cdc <dig> protein was greatly reduced but remained detectable, and β-trcp was largely unaffected .
in order to determine the mechanism of f-box protein loss in both csn <dig> and csn <dig> knockdown cells, we investigated the rescue of f-box proteins by the proteasome inhibitor mg <dig>  and measured the levels of f-box mrna over time .
treatment of both csn <dig> and csn <dig> knockdown cells with mg <dig> resulted in skp <dig>  cdc <dig> and β-trcp protein rescue , indicating involvement of the proteasome in the loss of these f-box proteins.
however the strength of the observed rescue varied.
importantly, csn subunit depletion also affected levels of f-box mrna.
csn <dig> knockdown resulted in a significant reduction of skp <dig> mrna by day  <dig> post transfection.
thus it is likely that transcriptional changes also contribute to loss of skp <dig> protein in these cells.
in contrast cdc <dig> and β-trcp mrnas were significantly increased following csn <dig> knockdown despite clear loss of protein .
csn <dig> depletion resulted in a modest reduction of all three f-box protein mrnas but not enough to account for the observed loss of protein .
collectively these data indicate that, with the possible exception of skp <dig> in csn <dig> knockdown cells, proteasomal degradation rather than transcriptional repression was the main driver of f-box protein loss in csn <dig> and csn <dig> knockdown k <dig> cells.
both csn <dig> and csn <dig> knockdowns result in reduced cell growth and cell death
knockdown of either csn <dig> or csn <dig> dramatically diminished cell proliferation, followed by loss of cell viability .
analysis of cumulative growth demonstrated that proliferation of cells lacking csn <dig> was significantly reduced when compared to that of shvc cells by day  <dig> post transfection , whilst loss of proliferation following csn <dig> knockdown was even more marked .
this difference in diminished proliferation was corroborated by measurement of thymidine incorporation.
at day  <dig> post-transfection, there was no significant difference in the incorporation of tritiated thymidine into cellular dna between shvc and csn <dig> knockdown  whereas the csn <dig> knockdown cells already had markedly reduced thymidine incorporation .
however, by day  <dig> the csn <dig> knockdown cells also had a significant decrease in thymidine incorporation  which decreased even further by day  <dig> .
thus, although the changes in cul <dig> neddylation and f-box protein levels are very similar between the csn <dig> and csn <dig> knockdowns, differences were apparent in the growth kinetics of the cells.
knockdown of csn <dig> but not csn <dig> is associated with cell cycle arrest and defects in mitotic spindle formation
cell cycle analyses revealed striking differences between the profiles of csn <dig> and csn <dig> knockdown cells.
despite leading to significantly reduced cell proliferation, loss of csn <dig> did not affect the cell cycle distribution of k <dig> cells as compared to shvc k <dig> cells by day  <dig> post transfection .
given the significantly reduced cell numbers in csn <dig> knockdown by day  <dig>  this would suggest that these cells are growing more slowly, rather than arresting at any stage in the cell cycle.
in contrast, csn <dig> knockdown resulted in a reduction in cells in g <dig> , loss of cells from s phase  and accumulation in g2/m  by day  <dig> post transfection .
morphological analysis of jenner-giemsa stained csn <dig> knockdown cells identified a significant number of large cells which appeared to be arrested in late mitosis with chaotically organized condensed chromosomes .
cells of these morphologies were not observed in either shvc  or csn <dig> knockdown  cells.
to investigate this morphology further, shcsn <dig> cells were immunostained for tubulin.
as can be seen in fig.
4c, shvc cells retained the ability to form a mitotic spindle and correctly aligned chromosomes at various stages of mitosis were observed.
in striking contrast, cells lacking csn <dig> demonstrated either aberrant or absent microtubule structures.
in these cells, the condensed chromatids appeared to be either misaligned or indeed not associated with the spindle structures at all .
this aberrant cellular phenotype is in accordance with cell cycle arrest at g2/m.
knockdown of csn <dig> but not csn <dig> results in apoptotic cell death
in order to determine whether the subsequent loss of viability in csn <dig> and csn <dig> knockdown cells was due to apoptosis, cells were co-analysed for annexin v staining and propidium iodide uptake day  <dig> and day  <dig> post transfection, respectively .
annexin v binds phosphatidylserine, a phospholipid which is translocated from the inner to the outer leaflet of the plasma membrane during early apoptosis.
propidium iodide is taken up by all cells but is efficiently effluxed by viable cells, whereas dead/dying cells remain pi-positive.
in accordance with the marked increase in the sub-g <dig> fraction of shcsn <dig> cells , knockdown of csn <dig> lead to significant increases in both early apoptotic   and late apoptotic  cells  compared to shvc cells .
furthermore, csn <dig> knockdown cells demonstrated cleavage of caspase  <dig> , consistent with death by apoptosis in these cells.
in marked contrast, csn <dig> knockdown showed only a relatively small increase in annexin v positive cells  and no caspase  <dig> activation .
instead, the csn <dig> knockdown cells demonstrated increased retention of pi in the annexin v-ve cell population .
this large shift in pi single-positivity relative to shvcs was not observed in csn <dig> knockdown cells .
thus knockdown of csn <dig> in k <dig> cells did not result in overt apoptosis.
taken together these data indicate that knockdown in k <dig> cells of csn <dig>  but not csn <dig>  resulted in apoptotic cell death and that loss of cell viability in the csn <dig> knockdowns was likely to be by a non-apoptotic mechanism.
csn <dig> but not csn <dig> knockdown is associated with autophagy
whilst culturing the transfectants, marked differences in cell morphology were noted in the csn <dig> knockdown cells.
jenner-giemsa staining of cytospins identified large vacuoles in a significant proportion of csn <dig> knockdown cells which were not present in either shvc or shcsn <dig> cells .
staining with the autophagosome/autophagic vacuole marker monodansylcadaverine  identified these large vacuoles as autophagic bodies .
moreover, western blotting identified a highly significant accumulation of the autophagy associated lc3-ii protein  <cit>  in csn <dig> knockdown cells relative to shvc cells .
in contrast, no increase of lc3-ii was detected in csn <dig> knockdown cells.
to demonstrate this we used a prolonged exposure  of autoradiographs that allowed detection of basal expression of lc3-ii in shvc cells and demonstrated that this was not increased in shcsn <dig> knockdown cells .
thus, knockdown of csn <dig>  but not csn <dig>  appeared strongly associated with autophagy.
however, lc3-ii accumulation can occur as a result of either enhanced autophagy or inhibition of autophagy dependant lc3-ii degradation  <cit> .
indeed, treatment of vector control cells with the autophagy inhibitor 3-methyladenine  resulted in a growth pattern almost identical to that of shcsn <dig> cells, whilst having a relatively small additional effect on shcsn <dig> cells .
furthermore, treatment of vector control cells with the late stage autophagy inhibitor bafilomycin a <dig> recapitulated both the vacuolar morphology  and the lc3-ii protein accumulation  observed in cells lacking csn <dig> .
these observations suggest that csn <dig> knockdown is associated with the inhibition of autophagy.
loss of csn <dig> results in the formation of an alternative csn <dig> containing complex
the above data indicate that knockdown of csn <dig> or csn <dig> in k <dig> cells results in common derangement of scf activity as measured by accumulation of hyperneddylated cul <dig>  accumulation of p <dig> and the sequential loss of skp- <dig>  cdc <dig> and β-trcp.
however, the cellular responses to csn <dig> or csn <dig> knockdown were markedly different.
since it has previously been shown that depletion of individual subunits can destabilize the csn holocomplex  <cit>  we reasoned that one explanation for the differences between csn <dig> and csn <dig> knockdown cellular responses may be due to the generation, in csn <dig> knockdowns, of csn <dig> containing csn subcomplexes with altered activities.
to investigate this, 2-dimensional native gel electrophoresis was performed on knockdown cells day  <dig> post transfection .
immunoblotting of extracts from shvc cells with either csn <dig> or csn <dig> antibody identified  <dig> major complexes, one of approximately  <dig> kda, correlating in size with the csn holocomplex, and the second of  <dig> kda .
a multitude of proteins have been shown to associate with csn subunits  <cit>  and larger complexes have previously been observed.
monomeric csn <dig> and csn <dig> were also observed in the shvc cell extracts .
following knockdown of csn <dig>  csn <dig> protein was no longer detectable .
csn <dig> protein, as also shown in fig.
1c, was greatly reduced in cells lacking csn <dig>  with significant loss of the csn complex relative to vector controls .
interestingly, a longer exposure of the autoradiograph identified a csn <dig> containing subcomplex of ~ <dig> kda in cells lacking csn <dig> .
discussion
the achievement of almost complete csn <dig> and csn <dig> knockdown in this study has provided a powerful tool to study the function of these csn subunits more closely.
at the molecular level, csn <dig> and csn <dig> knockdowns resulted in aberrant scf activity, with the accumulation of neddylated cul- <dig>  loss of the f-box protein skp <dig> and an increase in the skp <dig> target protein, p <dig>  this complements another report in which csn <dig> and csn <dig> knockdown also resulted in increased neddylation of cul- <dig> with a concomitant loss of skp <dig> and increase in p <dig> protein in human epithelial cell lines rather than haemopoietic cells  <cit> .
thus it appears that the csn complex has highly conserved activities across cells from different cell lineages and that disruption of the complex by loss of any subunit causes derangement of these activities.
it was also observed that csn <dig> knockdown not only results in loss of csn <dig> protein but also results in a significant reduction of csn <dig> mrna.
moreover, both csn <dig> and csn <dig> knockdown resulted in temporal alterations of f-box protein mrna.
together with other recent reports  <cit> , this data suggests that csn subunits or the csn complex as a whole may have a direct role in transcriptional regulation of csn subunits and f-box proteins.
however, it is also possible that the altered mrna levels observed are due to secondary effects of aberrant crl mediated protein degradation such as accumulation of proteins involved in transcriptional regulation.
here we show for the first time sequential loss of f-box proteins following knockdown of csn subunits.
moreover, protein levels were at least partially restored in both knockdowns upon treatment with the proteasome inhibitor mg <dig>  these observations are in accordance with the finding that f-box proteins are autocatalytically degraded in the presence of hyperneddylated cul- <dig>  <cit> .
however, the three f-box proteins studied were each lost at a different rate, with the loss of skp <dig> protein being the most rapid.
the sequential loss of f-box proteins is of great interest as it may explain published results which document the loss of particular f-box proteins at a specific time point post csn manipulation, but no reduction in other f-box proteins  <cit> .
the csn <dig> knockdown cultures contained aberrantly large cells and were associated with g2/m arrest and apoptosis.
this data complements previous studies demonstrating that csn <dig> loss inhibits proliferation and induces apoptosis  <cit> .
closer analysis of csn <dig> knockdown cells identified disorganized condensed chromatids and abnormal mitotic spindles in the large cells.
a recent report described stabilization of the microtubule end-binding protein  <dig>  by the csn complex in human cells  <cit> .
eb <dig>  which is a master regulator of microtubule dynamics, was shown to bind the csn via csn <dig>  and was also shown to be reduced in cells lacking csn <dig> or csn <dig>  <cit> .
eb <dig> has recently been shown to directly interact with and regulate the activity of aurora b, one essential component of the chromosomal passenger complex that is required for correct chromosomal alignment and spindle assembly checkpoint  <cit> .
furthermore, the dynamic behaviour of aurora b on mitotic chromosomes has been shown to be regulated by a cul <dig> e <dig> ligase  <cit> .
given that we observed hyperneddylated cul <dig> in the csn <dig> knockdown cells , our data suggests that csn5/the csn complex is integral to the regulation of multiple components of the mitotic machinery.
k <dig> cells in which csn <dig> had been knocked down did not display apoptosis markers as in csn <dig> knockdown cells, but were instead associated with features of autophagy.
interestingly, autophagy inhibitors recapitulated the cell growth kinetics, vacuolar morphology and lc3-ii accumulation of cells lacking csn <dig>  whilst treatment of csn <dig> knockdown cells with one of these inhibitors  had a comparatively mild effect on cell growth.
these findings suggest that csn <dig> knockdown k <dig> cells undergo autophagy inhibition resulting in non-apoptotic cell death.
this is the first data to show an association between the csn complex and autophagy.
the distinct phenotypes observed between csn <dig> and csn <dig> knockdowns may arise as a result of aberrant csn <dig> activity within the observed csn subcomplex in cells lacking csn <dig>  however, it is important to note that both the csn subunits studied here have csn independent functions  <cit> , and that csn <dig> has been shown to function within a csn subcomplex in k <dig>  <cit> .
therefore, we cannot rule out the possible contribution of the independent functions of csn <dig> and csn <dig> to the phenotypic differences observed between the knockdowns.
it is also noteworthy that the subcomplex observed in this study may be a result of csn complex breakdown in the absence of csn <dig>  <cit> , rather than a functional complex contributing to the observed phenotypic differences between knockdowns.
moreover, as we see no effect of csn <dig> loss on the level of csn <dig> protein, one possibility not investigated here is the formation of a csn <dig> containing subcomplex in the absence of csn <dig>  this is an intriguing possibility, particularly given the recent findings of su et al who demonstrated an increase in the proportion of csn <dig> residing in mini-complexes upon csn <dig> knockdown  <cit> .
it will be of great interest to determine the precise mechanism accounting for the divergent phenotypes encountered here, and is something which is currently under investigation.
CONCLUSIONS
in conclusion, we have shown that loss of either csn <dig> or csn <dig> in human k <dig> cells results in significant loss of viability but by very different mechanisms, potentially attributable to the formation of a csn <dig> containing subcomplex in the absence of csn <dig>  furthermore, we have provided data to suggest a possible function of the csn complex in the transcriptional regulation of both its own components and crl subunits.
finally, we have demonstrated here for the first time the sequential loss of f-box proteins in the absence of the csn complex and have provided the first evidence of a link between the csn complex and autophagy.

 the regulated expression and degradation of proteins are critical to all aspects of cell development and proliferation. the two main routes for eukaryotic intracellular protein clearance are the ubiquitin-proteasome system and the autophagy-lysosome pathway. a key component involved in regulating degradation of proteins by the ups is the cop signalosome. the csn is an eight-subunit protein complex, highly conserved amongst eukaryotes originally identified in arabidopsis as a negative regulator of photomorphogenesis. through its function in the regulation of the ups, the csn has been implicated in the regulation of biological processes as diverse as dna replication and repair, cell-cycle progression and cell development. degradation of cellular proteins by the 26s proteasome is preceded by ubiquitination of target proteins, a process mediated by three enzyme complexes; a ubiquitin activating enzyme, a ubiquitin conjugating enzyme and a ubiquitin ligase. the e ligase interacts with the protein substrate and thus confers the specificity of the ups. the largest known class of e ubiquitin ligases comprises the cullin-ring ligases of which the best studied is the scf complex. the cullin subunit of the scf forms a scaffold to recruit and bring into close proximity the e and its substrate, thereby facilitating ubiquitin transfer from the e to target proteins. the ring protein is the fourth subunit of the scf and is responsible for e recruitment, whilst the variable f-box protein subunit, recruited to the scf complex via the adaptor protein skp binds substrates selectively. in yeast, over f-box proteins are known, over in a. thaliana, and ~ in humans. since each cullin forms complexes with a variable substrate recognition subunit specificity in crl target protein recruitment is achieved by the large number of variable srs containing crls. it is thought that, altogether, the human genome may have the capacity to code for as many as different crls. given the potential number and diversity of target proteins requiring crl mediated ubiquitination for degradation, dynamic regulation of the crl complex repertoire in a cell at any given time is essential. all cullins studied have been shown to be modified by neddylation, which facilitates their ubiquitin ligase activity possibly via increased e affinity. the deneddylation of cullins is mediated by the csn complex. although initial studies indicated a negative role for deneddylation, further studies have implicated deneddylation in the positive regulation of crl activity. it has since been proposed by several groups that optimal crl activity requires the cyclic neddylation and deneddylation of the cullin subunit. although the exact mechanisms are not fully understood, it is thought that f-box proteins themselves are targeted for degradation in part by autoubiquitination within the scf complex. the deneddylation of cullins by the csn is believed to regulate the autoubiquitination of srss, thereby modulating crl composition and activity. furthermore, the csn has been shown to be associated with a deubiquitinase activity which may further stabilize autoubiquitinated srss. the csn complex is therefore an integral regulator of crl activity and subsequent protein degradation. in this present study we have investigated the effects of knocking down csn and csn in the model k cell line, a model of human erythrocyte and megakaryocyte progenitors. whilst knockdown of either csn or csn resulted in common changes including the sequential loss of f-box proteins skp cdc and β-trcp other important differences occurred. for example csn knockdown resulted in apoptotic cell death associated with aberrant mitosis, whereas csn knockdown cells underwent non-apoptotic cell death that was associated with both inhibition of autophagy and the formation of a novel csn containing csn subcomplex.  csn and csn knockdown and resulting aberrant scf activity plasmids encoding short hairpin rna to either csn or csn were electroporated into k cells together with h-2kk plasmid and transfected cells sorted by anti- h-2kk magnetic beads. three days post transfection, neither csn nor csn protein could be detected by western blot analysis in their respective knockdown protein extracts. furthermore, csn knockdown of over 99% and csn knockdown of over 95% was achieved at the mrna level relative to mock transfectants as measured by quantitative real-time pcr. this level of knockdown was maintained throughout the time course of the experiments. transfection with either the h-2kk plasmid alone or co-transfection with h-2kk plasmid and empty vector had no effect on the level of csn or csn mrna or protein relative to mock transfected cells. co-transfections with h-2kk and a vector encoding a control scrambled shrna sequence had no effect on the level of csn or csn mrna or protein relative to cells co-transfected with empty vector, demonstrating that the results were specific and not due to off-target effects. the instability of particular csn subunits in the absence of another subunit has been reported previously. for example, csn knockdown has been shown to result in the loss of csn csn and csn protein. we therefore determined csn and csn protein levels in csn and csn knockdown cells, respectively, by western blotting. a significant reduction in csn protein was observed in cells lacking csn whilst depletion of csn had no effect on csn protein or mrna levels relative to vector control cells. interestingly, csn knockdown not only resulted in loss of csn protein, but also in the significant reduction of csn mrna as determined by qrt-pcr. consistent with previous studies of csn deregulation, both csn and csn knockdowns were associated with accumulation of neddylated cul indicating functional dysregulation of the csn following knockdown of either subunit. studies demonstrating cul hyperneddylation in csn deficient cells have also demonstrated degradation of the f-box protein skp. in agreement with these observations, complete loss of skp protein was observed in both the csn and csn knockdown cells. skp binds to and mediates the ubiquitination of multiple proteins, including the cyclin dependent kinase inhibitor p27kip. consistent with loss of skp knockdown of csn and csn resulted in the accumulation of p. loss of csn and csn results in the sequential loss of f-box proteins skp is one of many f-box proteins that can bind to the cul1/roc1/skp complex, thereby altering the substrate specificity of the scf. analysis of three such f-box proteins, skp cdc and β-trcp revealed a sequential loss of protein in csn and csn knockdown cells. in csn knockdown cells, skp protein was reduced by ~60% by day and over 90% by day whereas cdc was lost at a slower rate with 30–40% lost by day increasing to ~70% by day. loss of β-trcp was still more retarded with ~70% protein remaining at day. by day all three f-box proteins were undetectable in csn knockdown cells. similar sequential loss of f-box proteins was observed in csn knockdown cells. at day post transfection skp protein could not be detected, cdc protein was greatly reduced but remained detectable, and β-trcp was largely unaffected. in order to determine the mechanism of f-box protein loss in both csn and csn knockdown cells, we investigated the rescue of f-box proteins by the proteasome inhibitor mg and measured the levels of f-box mrna over time. treatment of both csn and csn knockdown cells with mg resulted in skp cdc and β-trcp protein rescue, indicating involvement of the proteasome in the loss of these f-box proteins. however the strength of the observed rescue varied. importantly, csn subunit depletion also affected levels of f-box mrna. csn knockdown resulted in a significant reduction of skp mrna by day post transfection. thus it is likely that transcriptional changes also contribute to loss of skp protein in these cells. in contrast cdc and β-trcp mrnas were significantly increased following csn knockdown despite clear loss of protein. csn depletion resulted in a modest reduction of all three f-box protein mrnas but not enough to account for the observed loss of protein. collectively these data indicate that, with the possible exception of skp in csn knockdown cells, proteasomal degradation rather than transcriptional repression was the main driver of f-box protein loss in csn and csn knockdown k cells. both csn and csn knockdowns result in reduced cell growth and cell death knockdown of either csn or csn dramatically diminished cell proliferation, followed by loss of cell viability. analysis of cumulative growth demonstrated that proliferation of cells lacking csn was significantly reduced when compared to that of shvc cells by day post transfection, whilst loss of proliferation following csn knockdown was even more marked. this difference in diminished proliferation was corroborated by measurement of thymidine incorporation. at day post-transfection, there was no significant difference in the incorporation of tritiated thymidine into cellular dna between shvc and csn knockdown whereas the csn knockdown cells already had markedly reduced thymidine incorporation. however, by day the csn knockdown cells also had a significant decrease in thymidine incorporation which decreased even further by day. thus, although the changes in cul neddylation and f-box protein levels are very similar between the csn and csn knockdowns, differences were apparent in the growth kinetics of the cells. knockdown of csn but not csn is associated with cell cycle arrest and defects in mitotic spindle formation cell cycle analyses revealed striking differences between the profiles of csn and csn knockdown cells. despite leading to significantly reduced cell proliferation, loss of csn did not affect the cell cycle distribution of k cells as compared to shvc k cells by day post transfection. given the significantly reduced cell numbers in csn knockdown by day this would suggest that these cells are growing more slowly, rather than arresting at any stage in the cell cycle. in contrast, csn knockdown resulted in a reduction in cells in g, loss of cells from s phase and accumulation in g2/m by day post transfection. morphological analysis of jenner-giemsa stained csn knockdown cells identified a significant number of large cells which appeared to be arrested in late mitosis with chaotically organized condensed chromosomes. cells of these morphologies were not observed in either shvc or csn knockdown cells. to investigate this morphology further, shcsn cells were immunostained for tubulin. as can be seen in fig. 4c, shvc cells retained the ability to form a mitotic spindle and correctly aligned chromosomes at various stages of mitosis were observed. in striking contrast, cells lacking csn demonstrated either aberrant or absent microtubule structures. in these cells, the condensed chromatids appeared to be either misaligned or indeed not associated with the spindle structures at all. this aberrant cellular phenotype is in accordance with cell cycle arrest at g2/m. knockdown of csn but not csn results in apoptotic cell death in order to determine whether the subsequent loss of viability in csn and csn knockdown cells was due to apoptosis, cells were co-analysed for annexin v staining and propidium iodide uptake day and day post transfection, respectively. annexin v binds phosphatidylserine, a phospholipid which is translocated from the inner to the outer leaflet of the plasma membrane during early apoptosis. propidium iodide is taken up by all cells but is efficiently effluxed by viable cells, whereas dead/dying cells remain pi-positive. in accordance with the marked increase in the sub-g fraction of shcsn cells, knockdown of csn lead to significant increases in both early apoptotic and late apoptotic cells compared to shvc cells. furthermore, csn knockdown cells demonstrated cleavage of caspase, consistent with death by apoptosis in these cells. in marked contrast, csn knockdown showed only a relatively small increase in annexin v positive cells and no caspase activation. instead, the csn knockdown cells demonstrated increased retention of pi in the annexin v-ve cell population. this large shift in pi single-positivity relative to shvcs was not observed in csn knockdown cells. thus knockdown of csn in k cells did not result in overt apoptosis. taken together these data indicate that knockdown in k cells of csn but not csn resulted in apoptotic cell death and that loss of cell viability in the csn knockdowns was likely to be by a non-apoptotic mechanism. csn but not csn knockdown is associated with autophagy whilst culturing the transfectants, marked differences in cell morphology were noted in the csn knockdown cells. jenner-giemsa staining of cytospins identified large vacuoles in a significant proportion of csn knockdown cells which were not present in either shvc or shcsn cells. staining with the autophagosome/autophagic vacuole marker monodansylcadaverine identified these large vacuoles as autophagic bodies. moreover, western blotting identified a highly significant accumulation of the autophagy associated lc3-ii protein in csn knockdown cells relative to shvc cells. in contrast, no increase of lc3-ii was detected in csn knockdown cells. to demonstrate this we used a prolonged exposure of autoradiographs that allowed detection of basal expression of lc3-ii in shvc cells and demonstrated that this was not increased in shcsn knockdown cells. thus, knockdown of csn but not csn appeared strongly associated with autophagy. however, lc3-ii accumulation can occur as a result of either enhanced autophagy or inhibition of autophagy dependant lc3-ii degradation. indeed, treatment of vector control cells with the autophagy inhibitor 3-methyladenine resulted in a growth pattern almost identical to that of shcsn cells, whilst having a relatively small additional effect on shcsn cells. furthermore, treatment of vector control cells with the late stage autophagy inhibitor bafilomycin a recapitulated both the vacuolar morphology and the lc3-ii protein accumulation observed in cells lacking csn. these observations suggest that csn knockdown is associated with the inhibition of autophagy. loss of csn results in the formation of an alternative csn containing complex the above data indicate that knockdown of csn or csn in k cells results in common derangement of scf activity as measured by accumulation of hyperneddylated cul accumulation of p and the sequential loss of skp- cdc and β-trcp. however, the cellular responses to csn or csn knockdown were markedly different. since it has previously been shown that depletion of individual subunits can destabilize the csn holocomplex we reasoned that one explanation for the differences between csn and csn knockdown cellular responses may be due to the generation, in csn knockdowns, of csn containing csn subcomplexes with altered activities. to investigate this, 2-dimensional native gel electrophoresis was performed on knockdown cells day post transfection. immunoblotting of extracts from shvc cells with either csn or csn antibody identified major complexes, one of approximately kda, correlating in size with the csn holocomplex, and the second of kda. a multitude of proteins have been shown to associate with csn subunits and larger complexes have previously been observed. monomeric csn and csn were also observed in the shvc cell extracts. following knockdown of csn csn protein was no longer detectable. csn protein, as also shown in fig. 1c, was greatly reduced in cells lacking csn with significant loss of the csn complex relative to vector controls. interestingly, a longer exposure of the autoradiograph identified a csn containing subcomplex of ~ kda in cells lacking csn. discussion the achievement of almost complete csn and csn knockdown in this study has provided a powerful tool to study the function of these csn subunits more closely. at the molecular level, csn and csn knockdowns resulted in aberrant scf activity, with the accumulation of neddylated cul- loss of the f-box protein skp and an increase in the skp target protein, p this complements another report in which csn and csn knockdown also resulted in increased neddylation of cul- with a concomitant loss of skp and increase in p protein in human epithelial cell lines rather than haemopoietic cells. thus it appears that the csn complex has highly conserved activities across cells from different cell lineages and that disruption of the complex by loss of any subunit causes derangement of these activities. it was also observed that csn knockdown not only results in loss of csn protein but also results in a significant reduction of csn mrna. moreover, both csn and csn knockdown resulted in temporal alterations of f-box protein mrna. together with other recent reports, this data suggests that csn subunits or the csn complex as a whole may have a direct role in transcriptional regulation of csn subunits and f-box proteins. however, it is also possible that the altered mrna levels observed are due to secondary effects of aberrant crl mediated protein degradation such as accumulation of proteins involved in transcriptional regulation. here we show for the first time sequential loss of f-box proteins following knockdown of csn subunits. moreover, protein levels were at least partially restored in both knockdowns upon treatment with the proteasome inhibitor mg these observations are in accordance with the finding that f-box proteins are autocatalytically degraded in the presence of hyperneddylated cul-. however, the three f-box proteins studied were each lost at a different rate, with the loss of skp protein being the most rapid. the sequential loss of f-box proteins is of great interest as it may explain published results which document the loss of particular f-box proteins at a specific time point post csn manipulation, but no reduction in other f-box proteins. the csn knockdown cultures contained aberrantly large cells and were associated with g2/m arrest and apoptosis. this data complements previous studies demonstrating that csn loss inhibits proliferation and induces apoptosis. closer analysis of csn knockdown cells identified disorganized condensed chromatids and abnormal mitotic spindles in the large cells. a recent report described stabilization of the microtubule end-binding protein by the csn complex in human cells. eb which is a master regulator of microtubule dynamics, was shown to bind the csn via csn and was also shown to be reduced in cells lacking csn or csn. eb has recently been shown to directly interact with and regulate the activity of aurora b, one essential component of the chromosomal passenger complex that is required for correct chromosomal alignment and spindle assembly checkpoint. furthermore, the dynamic behaviour of aurora b on mitotic chromosomes has been shown to be regulated by a cul e ligase. given that we observed hyperneddylated cul in the csn knockdown cells, our data suggests that csn5/the csn complex is integral to the regulation of multiple components of the mitotic machinery. k cells in which csn had been knocked down did not display apoptosis markers as in csn knockdown cells, but were instead associated with features of autophagy. interestingly, autophagy inhibitors recapitulated the cell growth kinetics, vacuolar morphology and lc3-ii accumulation of cells lacking csn whilst treatment of csn knockdown cells with one of these inhibitors had a comparatively mild effect on cell growth. these findings suggest that csn knockdown k cells undergo autophagy inhibition resulting in non-apoptotic cell death. this is the first data to show an association between the csn complex and autophagy. the distinct phenotypes observed between csn and csn knockdowns may arise as a result of aberrant csn activity within the observed csn subcomplex in cells lacking csn however, it is important to note that both the csn subunits studied here have csn independent functions, and that csn has been shown to function within a csn subcomplex in k. therefore, we cannot rule out the possible contribution of the independent functions of csn and csn to the phenotypic differences observed between the knockdowns. it is also noteworthy that the subcomplex observed in this study may be a result of csn complex breakdown in the absence of csn, rather than a functional complex contributing to the observed phenotypic differences between knockdowns. moreover, as we see no effect of csn loss on the level of csn protein, one possibility not investigated here is the formation of a csn containing subcomplex in the absence of csn this is an intriguing possibility, particularly given the recent findings of su et al who demonstrated an increase in the proportion of csn residing in mini-complexes upon csn knockdown. it will be of great interest to determine the precise mechanism accounting for the divergent phenotypes encountered here, and is something which is currently under investigation.  in conclusion, we have shown that loss of either csn or csn in human k cells results in significant loss of viability but by very different mechanisms, potentially attributable to the formation of a csn containing subcomplex in the absence of csn furthermore, we have provided data to suggest a possible function of the csn complex in the transcriptional regulation of both its own components and crl subunits. finally, we have demonstrated here for the first time the sequential loss of f-box proteins in the absence of the csn complex and have provided the first evidence of a link between the csn complex and autophagy. 
greater than 95% knockdown of the non-catalytic subunit csn and the deneddylating subunit csn of the csn was achieved in the human myeloid progenitor cell line k csn knockdown led to a reduction of both csn protein and mrna whilst csn knockdown had little effect on csn both knockdowns inhibited csn deneddylase function as demonstrated by accumulation of neddylated cul furthermore, both knockdowns resulted in the sequential loss of skp cdc and β-trcp f-box proteins. these proteins were rescued by the proteasome inhibitor mg indicating the autocatalytic degradation of f-box proteins upon loss of csn or csn interestingly, altered f-box protein gene expression was also observed in csn and csn knockdowns, suggesting a potential role of the csn in regulating f-box protein transcription. our data present the first evidence for the sequential loss of f-box proteins upon csn manipulation and are the first to identify a potential link between csn function and autophagy. treatment of vector control k cells with the autophagy inhibitors 3-methyladenine and bafilomycin a recapitulated the growth kinetics, vacuolar morphology and lc3-ii accumulation of csn knockdown cells indicating that the cellular phenotype of csn cells arises from autophagy inhibition. loss of either csn subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. csn knockdown caused mitotic defects, g2/m arrest and apoptotic cell death. csn knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker lc3-ii and autophagic vacuoles. we conclude that csn is required for csn integrity and the stability of individual csn subunits, and postulate that csn loss results in a phenotype distinct from that of cells lacking csn possibly as a consequence of altered csn activity within a resultant csn subcomplex.
 the cop9/signalosome is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based e ubiquitin ligases, regulates protein degradation. although studied in model human cell lines such as hela, very little is known about the role of the csn in haemopoietic cells.  greater than 95% knockdown of the non-catalytic subunit csn and the deneddylating subunit csn of the csn was achieved in the human myeloid progenitor cell line k csn knockdown led to a reduction of both csn protein and mrna whilst csn knockdown had little effect on csn both knockdowns inhibited csn deneddylase function as demonstrated by accumulation of neddylated cul furthermore, both knockdowns resulted in the sequential loss of skp cdc and β-trcp f-box proteins. these proteins were rescued by the proteasome inhibitor mg indicating the autocatalytic degradation of f-box proteins upon loss of csn or csn interestingly, altered f-box protein gene expression was also observed in csn and csn knockdowns, suggesting a potential role of the csn in regulating f-box protein transcription. loss of either csn subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. csn knockdown caused mitotic defects, g2/m arrest and apoptotic cell death. csn knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker lc3-ii and autophagic vacuoles. treatment of vector control k cells with the autophagy inhibitors 3-methyladenine and bafilomycin a recapitulated the growth kinetics, vacuolar morphology and lc3-ii accumulation of csn knockdown cells indicating that the cellular phenotype of csn cells arises from autophagy inhibition. finally, loss of csn was associated with the formation of a csn containing subcomplex.  we conclude that csn is required for csn integrity and the stability of individual csn subunits, and postulate that csn loss results in a phenotype distinct from that of cells lacking csn possibly as a consequence of altered csn activity within a resultant csn subcomplex. our data present the first evidence for the sequential loss of f-box proteins upon csn manipulation and are the first to identify a potential link between csn function and autophagy. 