BACKGROUND
cop <dig>  constitutively photomorphogenic  <dig>  is the ubiquitin ligase containing ring-finger, coiled-coil and wd <dig> domains  <cit> , and well conserved from plants to animals  <cit> .
in plants, cop <dig> was identified as one of the cop proteins that act as a repressor of photomorphogenesis  <cit> , and functions downstream of the cop <dig> signalosome complex  <cit>  as a component of a multimeric e <dig> ubiquitin ligase complex that includes cullin  <dig> , damaged dna-binding protein  <dig> , ring-box  <dig> , and suppressor of phya  proteins  <cit> .
in response to multiple plant photoreceptors, the cop1-cul4-ddb1-rbx1-spa complex controls many light-regulated transcription factors  <cit> .
in contrast to its specific role in plants, mammalian cop <dig> is involved in many biological responses such as tumorigenesis  <cit> , dna damage response  <cit> , lipid metabolism  <cit> , and gluconeogenesis  <cit>  by targeting different substrates for degradation, which include p <dig>  <cit> , c-jun  <cit> , ets1/ <dig>  <cit> , trb <dig>  <cit> , and torc <dig>  <cit> .
particularly, in a dna-damage responsive pathway, cop <dig> functions downstream of atm/atr kinases by direct phosphorylation  <cit> , but the precise mechanism remains to be determined.
considering a wide range of cop <dig> action in various biological responses, components and pathways downstream of cop <dig> are not fully understood yet.
to better understand the cop1-signaling pathway, we searched for novel cop1-interacting proteins by yeast two-hybrid screening and identified fip <dig> as one such candidate.
fip <dig>  was first reported as a regulator of the retinoblastoma  protein  <cit> , identified as a tumor suppressor in human breast cancer  <cit> , and recently rediscovered as a mammalian counterpart of atg <dig> in the yeast atg1-atg13-atg <dig> complex  <cit> .
the mammalian ulk1-atg13-fip <dig> complex functions downstream of mtor, and, together with the beclin 1-vps <dig> kinase pathway and the atg5-atg <dig> and lc <dig> conjugation systems, plays a key role in the induction of autophagy, an intracellular lysosomal degradation system for cytoplasmic proteins and organelles  <cit> .
in this study, we investigated the interaction between cop <dig> and fip <dig> by the yeast two-hybrid assay, the gst-pulldown assay, and the split-gfp assay.
proliferating mammalian cells expressed several different forms of fip <dig> protein, and one of them was downregulated by the ectopic overexpression of cop <dig> protein, suggesting that cop <dig> modulates fip200-associated biological activities in a certain occasion, which may contribute to the complexity of the cop1-associated function.
RESULTS
identification of fip <dig> as an interactor with cop1
to explore the novel signaling pathway mediated by cop <dig>  we sought a candidate for interactors with cop <dig> by yeast two-hybrid screening of the human k <dig> erythroleukemia cdna library.
out of  <dig>  ×  <dig> transformants, we chose  <dig> potential clones that repeatedly exhibited positive signals.
these clones contained part of two independent cdnas, one for jun d and one for fip <dig>  <cit> /rb1-inducible coiled-coil  <dig>  <cit>  .
the presence of the former cdna was anticipated given that c-jun is a substrate of cop <dig>  <cit>  and that jund is highly homologous to c-jun, both of which belong to the same family of ap <dig> transcription factors.
the latter component, fip <dig>  also termed rb1cc <dig>  was originally shown to control retinoblastoma protein  <cit>  and functions as a tumor suppressor in human breast cancer  <cit> .
fip <dig> was recently rediscovered as a component of the mammalian ulk <dig> -atg13-fip <dig>  complex and plays an important role in the induction of autophagy  <cit> .
therefore, we decided to investigate the cop1-fip <dig> interaction and the role of cop <dig> in terms of uv response and induction of autophagy.
a yeast two-hybrid analysis using deletion mutants of cop <dig>  indicated that the ring domain at the n-terminus of cop <dig>  <cit> , but not the wd <dig> domain, is required for interaction with fip <dig>  showing a clear difference from jund, which interacted with the wd <dig> domain as is the case with most substrates for ubiquitin ligases containing the wd <dig> motif  <cit> .
in vitro binding assays using gst-fused fip <dig> protein  and cell lysate containing the ectopically expressed ha-tagged cop <dig>  showed that cop <dig> and fip <dig> interacted in vitro .
different forms of fip <dig> protein were expressed in cultured mammalian cells
to analyze the function of fip <dig> in mammalian cells, we raised a rabbit polyclonal antibody to fip <dig> using a polypeptide corresponding to the region isolated by the yeast two-hybrid screening, which specifically reacted with endogenous fip <dig> as well as ectopically-expressed fip <dig> protein by wester blotting .
in the lysate isolated from proliferating mammalian cells, our antibody recognized two forms of fip <dig> , the slower migrating form being more readily extracted from the cells .
because we have previously showed that cop <dig> is involved in cellular response mediated by uv stimulation  <cit> , we examined whether uv might affect fip <dig>  interestingly, uv stimulation altered the ratio between these two forms ; proliferating cells contained the slower migrating form more, while uv treatment decreased the expression of the slower migrating form and, instead, increased that of the faster migrating form .
fip <dig> is known to be modified by phosphorylation  <cit> , which often affects mobility in sds-page.
to test this possibility, we extracted the protein from cells treated with uv and un-treated cells in an sds sample buffer, isolated fip <dig> by immunoprecipitation, and treated it with phosphatase in vitro.
the result  showed that the difference in mobility was not due to the level of phosphorylation although both forms were phosphorylated.
currently, we do not know the exact molecular identity of these two variants, which might be generated by alternative splicing or other post-translational modifications.
fip <dig> interacts with cop <dig> in the cytoplasm of proliferating cells in response to uv stimulation
we have so far not been successful in detecting the cop1-fip <dig> complex in cell lysate by immunoprecipitation/immunoblotting.
one possible explanation for this is that our antibody does not recognize the complex.
another possibility is that the cop1-fip <dig> complex may not be efficiently eluted from the cells in a buffer suitable for immunoprecipitation.
in fact, we identified different forms of fip <dig> by western blotting possibly due to alternative splicing  <cit>  and one of them was not efficiently extracted in a buffer for immunoprecipitation .
to overcome these problems and to further investigate the interaction between cop <dig> and fip <dig> in vivo, we performed a split-gfp analysis  <cit> , in which gfp  was split into two domains, n-terminal  and c-terminal , and fused to two molecules , respectively .
if these two molecules interact with each other in the cell, the gfp  signal will be restored.
in transfected cells, cop1-yn, cop1-yc  and fip200-yn together with yc and yn did not generate any significant signals above the background level, but we detected the gfp signal in the cells co-transfected with both wild-type cop1-yc and fip200-yn after uv exposure .
the restored signal was predominantly in the cytoplasm  with some in the nucleus too.
we also detected interaction between cop1-yn and cop1-yc as a control.
in this case, the signal was both in the nucleus and in the cytoplasm .
quantification by flow cytometric analysis showed that the cop1-cop <dig> interaction was constitutive, whereas the cop1-fip <dig> interaction was inducible in response to uv treatment .
importantly, interaction was diminished when we used a cop <dig> mutant , which contains a serine to alanine substitution at the conserved atm/atr phosphorylation site at the 389th codon  <cit>  , suggesting that uv-mediated phosphorylation of cop <dig> is required for the efficient formation of a complex between cop <dig> and fip <dig>  taken together, while cop <dig> stably forms a multimeric complex in the cell, its binding to fip <dig> in the cytoplasm is enhanced by uv stimulation.
ectopic expression of cop <dig> reduced the expression of a certain form of fip <dig> protein and exhibited tumorigenisity in response to uv
to examine the effect of cop <dig> on fip <dig>  we ectopically expressed gfp-tagged cop <dig> in nih3t <dig>  cells.
figure 4a shows that the level of ectopic expression was approximately the same as that of the endogenous protein.
interestingly, the faster-migrating form of fip <dig> was downregulated in nih/gfp-cop <dig> cells compared to that of the control  cells transfected with the control gfp-vector, whereas the slower-migrating form remained the same.
because it is known that fip <dig> forms a complex with ulk <dig>  atg <dig>  and atg <dig> to function downstream of mtor to induce autophagy  <cit> , we investigated their expression.
figure 4a shows that ectopic expression of cop <dig> affected differently; ulk <dig> was almost unaffected, atg <dig> was upregulated and atg <dig> was slightly downregulated.
we did not detect any direct binding of cop <dig> with ulk <dig>  atg <dig>  and atg <dig> , suggesting that cop <dig> affects these components through interaction with fip <dig>  interestingly, treatment of cells with an inhibitor to proteasome, mg <dig>  reversed the effect of cop <dig> overexpression.
when we investigated autophagy in these cells, however, autophagy was fully induced in response to amino acid starvation .
cop <dig> may affect other activities associated with fip <dig>
because interaction between cop <dig> and fip <dig> was enhanced by uv stimulation and sa mutation diminished the interaction, we ectopically expressed wild type and sa mutant form of cop <dig> in nih3t <dig> cells, and examined the effect of uv on fip <dig>  figure 4b, left panel shows that both wild type and sa mutant cop <dig> were successfully overexpressed.
immunoprecipitation of the ha-tagged exogenous cop <dig> protein with the antibody to the ha epitope brought down the endogenous cop <dig> protein , indicating that cop <dig> formed a dimer or a larger multimeric complex, which was expected from the split-gfp analysis .
the sa mutant retained the ability to interact with the endogenous cop <dig>  upon stimulation with uv , the slower migrating form of fip <dig> decreased and the faster migrating form increased in control cells.
overexpression of wild type cop <dig> reduced the level of faster migrating form at time  <dig> and blocked its induction by uv stimulation, whereas overexpression of sa mutant did not affect the band shift of fip <dig> upon uv stimulation.
fip <dig> was identified as a tumor suppressor  <cit> .
if cop <dig> negatively regulates fip <dig>  one might expect that cop <dig> act as an oncogene.
in addition, cop <dig> responds to uv stimulation and becomes a substrate of atm/atr kinases  <cit> .
we, therefore, tested whether the overexpression of cop <dig> facilitates cellular transformation in response to uv irradiation.
we treated nih3t <dig> mouse fibroblasts expressing cop <dig> with uv, let them recover for passaging, and subcutaneously injected them into nod-scid mice .
ectopic expression of the cop <dig> protein itself was not tumorigenic because no trace of cells was detected  <dig> months after injection.
however, after treatment with uv and successive passages in a recovery culture, cells ectopically expressing cop <dig> formed a tumor of significant size in mice.
importantly, cells transfected with sa mutant of cop <dig>  which did not interact with fip <dig> , failed to form tumors even after uv stimulation, suggesting that cop <dig> requires interaction with fip <dig> to exhibit its oncogenic properties.
we currently do not know the physiological significance of the impact of cop <dig> overexpression on autophagy.
however, considering the differential effect on the expression of the components of the fip <dig> complex and the fip <dig> subtypes and that tumorigenic function of cop <dig> requires interaction with fip <dig>  it is feasible to say that cop <dig> may regulate biological activities associated with fip <dig> in a certain occasion.
discussion
as distinct from its plant counterpart  <cit> , mammalian cop <dig> is involved in many biological occasions  <cit> .
multi-functionality of cop <dig> partly stems from its variety of substrates and various adaptor or accessory proteins to interact with.
although several proteins have been identified as the target of cop <dig>  <cit> , it is reasonable to speculate that more substrates and downstream pathways are yet to be found.
our findings imply that autophagy may situate downstream of the signaling pathway mediated by cop <dig>  which may partly explain the multifunction of cop <dig> because autophagy is reported to be involved in many biological occasions  <cit> .
by yeast-two hybrid screening, we identified c-terminal polypeptide of fip <dig> as the interactor of cop <dig>  and raised antibody against this portion of the protein.
using this antibody, we detected at least two different forms of fip <dig> in proliferating mammalian cells, both of which should, therefore, share the epitope in the c-terminus of fip <dig>  currently, we do not know the identity of these two forms of fip <dig> recognized by our antibody because phosphorylation  and ubiquitination  do not seem to account for the mobility difference of these two forms.
it is feasible to suspect that alternative splicing generated these two forms, but the extensive rt-pcr analysis using a series of primers designed within different exons failed to identify the alternative transcripts .
therefore we do not exclude the possibility that the faster-migrating form is the product of other post-translational modifications.
whatever the mechanism, it is important to emphasize that these two forms were extracted in different conditions and that only the faster-migrating form was downregulated by the ectopic expression of cop <dig>  suggesting that they locate in different compartments within the cell and that one of the two is the possible target of cop <dig>
we do not yet know whether fip <dig> is a substrate for the cop <dig> ligase.
fip <dig> bound to the ring domain, but not the wd <dig> domain, of cop <dig>  which makes a clear difference from other substrates such as jund.
we, so far, did not see the ubiquitinated fip <dig> protein in cop1-overexpressing cells.
however, we did observe the downregulation of faster-migrating form of fip <dig> in cop1-overexpressing cells in an mg132-sensitive manner, suggesting that cop <dig> somehow induced proteasome-mediated degradation of fip <dig>  at present, we do not exclude the possibility that cop <dig> altered the level of fip <dig> expression through mechanisms other than direct ubiquitination.
cop <dig> might affect alternative-splicing to affect the expression of faster-migrating form, which step is sensitive to the action of proteasome.
cells with ectopic overexpression of cop <dig> still underwent autophagy in response to amino acid starvation even though the faster-migrating form of fip <dig> was efficiently downregulated, and the expression of atg <dig> and atg <dig> was modulated .
it could be that the remaining components of the fip <dig> complex were sufficient to the initiate autophagic program or alternative form of fip <dig> may respond to different inducers of autophagy such as uv.
to answer this question, molecular identification of two forms of fip <dig> is the urgent matter.
knowing the difference between the two, we could compare the composition of the different complexes and examine the role of each form in response to various stimuli, and the potential functions associated with fip <dig> in the cell cycle control  <cit> , p <dig> regulation  <cit>  and dna damage repair  <cit>  as well as autophagy  <cit> .
we have tried to establish the in vitro ubiquitination assay for cop <dig> and fip <dig> using recombinant proteins without success .
this could be due to the lack of the cop1-accessory proteins or, alternatively, cop <dig> may favor the fip200-containing complex rather than a single polypeptide.
in addition to the identity of fip <dig> variants, an adaptor protein of cop <dig> specific to fip <dig> will be required for establishment of the in vitro reconstitution system, which will give us many clues to biochemically understand the nature of cop1-associated activities in mammals.
methods
yeast two-hybrid screening
the entire coding sequence  and deletion mutants  of mouse cop <dig> were fused in-frame to the gal <dig> dna-binding domain of the pas <dig> vector  <cit> .
the resulting ‘bait’ plasmid  was used to screen pact- human k <dig> erythroleukemia libraries  by the yeast two-hybrid method in y <dig> yeast cells  <cit> .
in vitro binding assay
a cdna fragment containing the c-terminal domain of fip <dig>  was inserted into the pgex vector  in-frame with glutathione s-transferase .
expression and purification of gst-fused proteins and the binding conditions were as described  <cit> .
cell culture, transfection, retroviral infection, and treatment with uv
nih3t <dig>  mouse fibroblasts , mouse embryonic fibroblasts , and 293t human embryonic kidney  cells were cultured, transfected via the calcium phosphate-dna precipitation method  <cit> , and infected with retroviral vectors as described  <cit> .
for treatment with uv, cells were washed with pbs twice, exposed to uv light in a uv crosslinker  , and incubated in a serum-containing complete medium.
in some cases, cells were treated with  <dig> μm mg <dig>  before harvest.
plasmid construction
the gfp-fused protein expression vector , into which cop <dig> cdnas were subcloned, was described previously  <cit> .
cop <dig> mutants   <cit>  were generated by pcr.
protein analyses
cell lysis, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis , and immunoblotting were performed as described  <cit> .
two types of lysis buffer used in this study were ebc buffer  containing  <dig> kiu/ml of aprotinin, 1mm pmsf,  <dig>  mm naf,  <dig>  mm na3vo <dig>  and  <dig> mm β-glycerophosphate, and sds-sample buffer .
in some cases, immunoprecipitated proteins were treated with phosphatase before immunoblotting  <cit> .
a rabbit polyclonal antibody to an ha-epitope  was obtained from santa cruz.
a mouse monoclonal antibody to an ha-epitope  was purchased from boehringer mannheim.
rabbit polyclonal antibodies to ulk <dig>  and atg <dig>  were from sigma.
rabbit polyclonal antibodies to lc <dig>  and p <dig>  were acquired from medical & biological laboratories .
rabbit polyclonal antibodies to fip <dig>  p <dig>  and cop <dig> were generated using bacterially produced polypeptides in our laboratory.
a rabbit polyclonal antibody to atg <dig> was provided by dr. noboru mizushima.
split gfp assay
gfp  was split into two domains, n-terminal  and c-terminal .
each domain was fused to two molecules , and transfected into cells as described above.
gfp signals were observed using phase-contrast or fluorescence microscopy and measured with a flow cytometer.
a human cdna clone containing entire coding sequence of fip <dig> was obtained from kazasa dna research institute .
tumorigenicity assay
cells  were subcutaneously injected into nod-scid mice.
after  <dig> weeks or  <dig>  months, mice were sacrificed and the size of the tumor was measured.
CONCLUSIONS
in this study, we found the interaction between fip <dig> and cop <dig>  ectopic expression of cop <dig> reduced one of the different forms of fip <dig>  suggesting that cop <dig> modulates fip200-associated activities, which may contribute to a variety of cellular functions that cop <dig> is involved in.
authors' contributions
sk carried out the molecular biological studies.
ni carried out the molecular cellular studies.
ay performed the cellular analysis.
ny-k and j-yk conceived of the study and participated in its design and coordination and drafted the manuscript.
all authors read and approved the final manuscript.

 cop constitutively photomorphogenic is the ubiquitin ligase containing ring-finger, coiled-coil and wd domains, and well conserved from plants to animals. in plants, cop was identified as one of the cop proteins that act as a repressor of photomorphogenesis, and functions downstream of the cop signalosome complex as a component of a multimeric e ubiquitin ligase complex that includes cullin, damaged dna-binding protein, ring-box, and suppressor of phya proteins. in response to multiple plant photoreceptors, the cop1-cul4-ddb1-rbx1-spa complex controls many light-regulated transcription factors. in contrast to its specific role in plants, mammalian cop is involved in many biological responses such as tumorigenesis, dna damage response, lipid metabolism, and gluconeogenesis by targeting different substrates for degradation, which include p, c-jun, ets1/, trb, and torc. particularly, in a dna-damage responsive pathway, cop functions downstream of atm/atr kinases by direct phosphorylation, but the precise mechanism remains to be determined. considering a wide range of cop action in various biological responses, components and pathways downstream of cop are not fully understood yet. to better understand the cop1-signaling pathway, we searched for novel cop1-interacting proteins by yeast two-hybrid screening and identified fip as one such candidate. fip was first reported as a regulator of the retinoblastoma protein, identified as a tumor suppressor in human breast cancer, and recently rediscovered as a mammalian counterpart of atg in the yeast atg1-atg13-atg complex. the mammalian ulk1-atg13-fip complex functions downstream of mtor, and, together with the beclin 1-vps kinase pathway and the atg5-atg and lc conjugation systems, plays a key role in the induction of autophagy, an intracellular lysosomal degradation system for cytoplasmic proteins and organelles. in this study, we investigated the interaction between cop and fip by the yeast two-hybrid assay, the gst-pulldown assay, and the split-gfp assay. proliferating mammalian cells expressed several different forms of fip protein, and one of them was downregulated by the ectopic overexpression of cop protein, suggesting that cop modulates fip200-associated biological activities in a certain occasion, which may contribute to the complexity of the cop1-associated function.  identification of fip as an interactor with cop1 to explore the novel signaling pathway mediated by cop we sought a candidate for interactors with cop by yeast two-hybrid screening of the human k erythroleukemia cdna library. out of  ×  transformants, we chose potential clones that repeatedly exhibited positive signals. these clones contained part of two independent cdnas, one for jun d and one for fip /rb1-inducible coiled-coil. the presence of the former cdna was anticipated given that c-jun is a substrate of cop and that jund is highly homologous to c-jun, both of which belong to the same family of ap transcription factors. the latter component, fip also termed rb1cc was originally shown to control retinoblastoma protein and functions as a tumor suppressor in human breast cancer. fip was recently rediscovered as a component of the mammalian ulk -atg13-fip complex and plays an important role in the induction of autophagy. therefore, we decided to investigate the cop1-fip interaction and the role of cop in terms of uv response and induction of autophagy. a yeast two-hybrid analysis using deletion mutants of cop indicated that the ring domain at the n-terminus of cop, but not the wd domain, is required for interaction with fip showing a clear difference from jund, which interacted with the wd domain as is the case with most substrates for ubiquitin ligases containing the wd motif. in vitro binding assays using gst-fused fip protein and cell lysate containing the ectopically expressed ha-tagged cop showed that cop and fip interacted in vitro. different forms of fip protein were expressed in cultured mammalian cells to analyze the function of fip in mammalian cells, we raised a rabbit polyclonal antibody to fip using a polypeptide corresponding to the region isolated by the yeast two-hybrid screening, which specifically reacted with endogenous fip as well as ectopically-expressed fip protein by wester blotting. in the lysate isolated from proliferating mammalian cells, our antibody recognized two forms of fip, the slower migrating form being more readily extracted from the cells. because we have previously showed that cop is involved in cellular response mediated by uv stimulation, we examined whether uv might affect fip interestingly, uv stimulation altered the ratio between these two forms; proliferating cells contained the slower migrating form more, while uv treatment decreased the expression of the slower migrating form and, instead, increased that of the faster migrating form. fip is known to be modified by phosphorylation, which often affects mobility in sds-page. to test this possibility, we extracted the protein from cells treated with uv and un-treated cells in an sds sample buffer, isolated fip by immunoprecipitation, and treated it with phosphatase in vitro. the result showed that the difference in mobility was not due to the level of phosphorylation although both forms were phosphorylated. currently, we do not know the exact molecular identity of these two variants, which might be generated by alternative splicing or other post-translational modifications. fip interacts with cop in the cytoplasm of proliferating cells in response to uv stimulation we have so far not been successful in detecting the cop1-fip complex in cell lysate by immunoprecipitation/immunoblotting. one possible explanation for this is that our antibody does not recognize the complex. another possibility is that the cop1-fip complex may not be efficiently eluted from the cells in a buffer suitable for immunoprecipitation. in fact, we identified different forms of fip by western blotting possibly due to alternative splicing and one of them was not efficiently extracted in a buffer for immunoprecipitation. to overcome these problems and to further investigate the interaction between cop and fip in vivo, we performed a split-gfp analysis, in which gfp was split into two domains, n-terminal and c-terminal, and fused to two molecules, respectively. if these two molecules interact with each other in the cell, the gfp signal will be restored. in transfected cells, cop1-yn, cop1-yc and fip200-yn together with yc and yn did not generate any significant signals above the background level, but we detected the gfp signal in the cells co-transfected with both wild-type cop1-yc and fip200-yn after uv exposure. the restored signal was predominantly in the cytoplasm with some in the nucleus too. we also detected interaction between cop1-yn and cop1-yc as a control. in this case, the signal was both in the nucleus and in the cytoplasm. quantification by flow cytometric analysis showed that the cop1-cop interaction was constitutive, whereas the cop1-fip interaction was inducible in response to uv treatment. importantly, interaction was diminished when we used a cop mutant, which contains a serine to alanine substitution at the conserved atm/atr phosphorylation site at the 389th codon, suggesting that uv-mediated phosphorylation of cop is required for the efficient formation of a complex between cop and fip taken together, while cop stably forms a multimeric complex in the cell, its binding to fip in the cytoplasm is enhanced by uv stimulation. ectopic expression of cop reduced the expression of a certain form of fip protein and exhibited tumorigenisity in response to uv to examine the effect of cop on fip we ectopically expressed gfp-tagged cop in nih3t cells. figure 4a shows that the level of ectopic expression was approximately the same as that of the endogenous protein. interestingly, the faster-migrating form of fip was downregulated in nih/gfp-cop cells compared to that of the control cells transfected with the control gfp-vector, whereas the slower-migrating form remained the same. because it is known that fip forms a complex with ulk atg and atg to function downstream of mtor to induce autophagy, we investigated their expression. figure 4a shows that ectopic expression of cop affected differently; ulk was almost unaffected, atg was upregulated and atg was slightly downregulated. we did not detect any direct binding of cop with ulk atg and atg, suggesting that cop affects these components through interaction with fip interestingly, treatment of cells with an inhibitor to proteasome, mg reversed the effect of cop overexpression. when we investigated autophagy in these cells, however, autophagy was fully induced in response to amino acid starvation. cop may affect other activities associated with fip because interaction between cop and fip was enhanced by uv stimulation and sa mutation diminished the interaction, we ectopically expressed wild type and sa mutant form of cop in nih3t cells, and examined the effect of uv on fip figure 4b, left panel shows that both wild type and sa mutant cop were successfully overexpressed. immunoprecipitation of the ha-tagged exogenous cop protein with the antibody to the ha epitope brought down the endogenous cop protein, indicating that cop formed a dimer or a larger multimeric complex, which was expected from the split-gfp analysis. the sa mutant retained the ability to interact with the endogenous cop upon stimulation with uv, the slower migrating form of fip decreased and the faster migrating form increased in control cells. overexpression of wild type cop reduced the level of faster migrating form at time and blocked its induction by uv stimulation, whereas overexpression of sa mutant did not affect the band shift of fip upon uv stimulation. fip was identified as a tumor suppressor. if cop negatively regulates fip one might expect that cop act as an oncogene. in addition, cop responds to uv stimulation and becomes a substrate of atm/atr kinases. we, therefore, tested whether the overexpression of cop facilitates cellular transformation in response to uv irradiation. we treated nih3t mouse fibroblasts expressing cop with uv, let them recover for passaging, and subcutaneously injected them into nod-scid mice. ectopic expression of the cop protein itself was not tumorigenic because no trace of cells was detected months after injection. however, after treatment with uv and successive passages in a recovery culture, cells ectopically expressing cop formed a tumor of significant size in mice. importantly, cells transfected with sa mutant of cop which did not interact with fip, failed to form tumors even after uv stimulation, suggesting that cop requires interaction with fip to exhibit its oncogenic properties. we currently do not know the physiological significance of the impact of cop overexpression on autophagy. however, considering the differential effect on the expression of the components of the fip complex and the fip subtypes and that tumorigenic function of cop requires interaction with fip it is feasible to say that cop may regulate biological activities associated with fip in a certain occasion. discussion as distinct from its plant counterpart, mammalian cop is involved in many biological occasions. multi-functionality of cop partly stems from its variety of substrates and various adaptor or accessory proteins to interact with. although several proteins have been identified as the target of cop, it is reasonable to speculate that more substrates and downstream pathways are yet to be found. our findings imply that autophagy may situate downstream of the signaling pathway mediated by cop which may partly explain the multifunction of cop because autophagy is reported to be involved in many biological occasions. by yeast-two hybrid screening, we identified c-terminal polypeptide of fip as the interactor of cop and raised antibody against this portion of the protein. using this antibody, we detected at least two different forms of fip in proliferating mammalian cells, both of which should, therefore, share the epitope in the c-terminus of fip currently, we do not know the identity of these two forms of fip recognized by our antibody because phosphorylation and ubiquitination do not seem to account for the mobility difference of these two forms. it is feasible to suspect that alternative splicing generated these two forms, but the extensive rt-pcr analysis using a series of primers designed within different exons failed to identify the alternative transcripts. therefore we do not exclude the possibility that the faster-migrating form is the product of other post-translational modifications. whatever the mechanism, it is important to emphasize that these two forms were extracted in different conditions and that only the faster-migrating form was downregulated by the ectopic expression of cop suggesting that they locate in different compartments within the cell and that one of the two is the possible target of cop we do not yet know whether fip is a substrate for the cop ligase. fip bound to the ring domain, but not the wd domain, of cop which makes a clear difference from other substrates such as jund. we, so far, did not see the ubiquitinated fip protein in cop1-overexpressing cells. however, we did observe the downregulation of faster-migrating form of fip in cop1-overexpressing cells in an mg132-sensitive manner, suggesting that cop somehow induced proteasome-mediated degradation of fip at present, we do not exclude the possibility that cop altered the level of fip expression through mechanisms other than direct ubiquitination. cop might affect alternative-splicing to affect the expression of faster-migrating form, which step is sensitive to the action of proteasome. cells with ectopic overexpression of cop still underwent autophagy in response to amino acid starvation even though the faster-migrating form of fip was efficiently downregulated, and the expression of atg and atg was modulated. it could be that the remaining components of the fip complex were sufficient to the initiate autophagic program or alternative form of fip may respond to different inducers of autophagy such as uv. to answer this question, molecular identification of two forms of fip is the urgent matter. knowing the difference between the two, we could compare the composition of the different complexes and examine the role of each form in response to various stimuli, and the potential functions associated with fip in the cell cycle control, p regulation and dna damage repair as well as autophagy. we have tried to establish the in vitro ubiquitination assay for cop and fip using recombinant proteins without success. this could be due to the lack of the cop1-accessory proteins or, alternatively, cop may favor the fip200-containing complex rather than a single polypeptide. in addition to the identity of fip variants, an adaptor protein of cop specific to fip will be required for establishment of the in vitro reconstitution system, which will give us many clues to biochemically understand the nature of cop1-associated activities in mammals. methods yeast two-hybrid screening the entire coding sequence and deletion mutants of mouse cop were fused in-frame to the gal dna-binding domain of the pas vector. the resulting ‘bait’ plasmid was used to screen pact- human k erythroleukemia libraries by the yeast two-hybrid method in y yeast cells. in vitro binding assay a cdna fragment containing the c-terminal domain of fip was inserted into the pgex vector in-frame with glutathione s-transferase. expression and purification of gst-fused proteins and the binding conditions were as described. cell culture, transfection, retroviral infection, and treatment with uv nih3t mouse fibroblasts, mouse embryonic fibroblasts, and 293t human embryonic kidney cells were cultured, transfected via the calcium phosphate-dna precipitation method, and infected with retroviral vectors as described. for treatment with uv, cells were washed with pbs twice, exposed to uv light in a uv crosslinker, and incubated in a serum-containing complete medium. in some cases, cells were treated with μm mg before harvest. plasmid construction the gfp-fused protein expression vector, into which cop cdnas were subcloned, was described previously. cop mutants were generated by pcr. protein analyses cell lysis, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting were performed as described. two types of lysis buffer used in this study were ebc buffer containing kiu/ml of aprotinin, 1mm pmsf, mm naf, mm na3vo and mm β-glycerophosphate, and sds-sample buffer. in some cases, immunoprecipitated proteins were treated with phosphatase before immunoblotting. a rabbit polyclonal antibody to an ha-epitope was obtained from santa cruz. a mouse monoclonal antibody to an ha-epitope was purchased from boehringer mannheim. rabbit polyclonal antibodies to ulk and atg were from sigma. rabbit polyclonal antibodies to lc and p were acquired from medical & biological laboratories. rabbit polyclonal antibodies to fip p and cop were generated using bacterially produced polypeptides in our laboratory. a rabbit polyclonal antibody to atg was provided by dr. noboru mizushima. split gfp assay gfp was split into two domains, n-terminal and c-terminal. each domain was fused to two molecules, and transfected into cells as described above. gfp signals were observed using phase-contrast or fluorescence microscopy and measured with a flow cytometer. a human cdna clone containing entire coding sequence of fip was obtained from kazasa dna research institute. tumorigenicity assay cells were subcutaneously injected into nod-scid mice. after weeks or months, mice were sacrificed and the size of the tumor was measured.  in this study, we found the interaction between fip and cop ectopic expression of cop reduced one of the different forms of fip suggesting that cop modulates fip200-associated activities, which may contribute to a variety of cellular functions that cop is involved in. authors' contributions sk carried out the molecular biological studies. ni carried out the molecular cellular studies. ay performed the cellular analysis. ny-k and j-yk conceived of the study and participated in its design and coordination and drafted the manuscript. all authors read and approved the final manuscript. 
here, we identified fip a key regulator of mammalian autophagy, as a novel cop1-interacting protein by yeast two-hybrid screening. split-gfp analysis revealed that interaction between cop and fip predominantly occurred in the cytoplasm and was enhanced in cells treated with uv irradiation. different forms of fip protein were expressed in cultured mammalian cells, and ectopic expression of cop reduced one of such forms. the interaction was confirmed by a gst-pulldown assay. these data suggest that cop modulates fip200-associated activities, which may contribute to a variety of cellular functions that cop is involved in. the ubiquitin ligase cop constitutively photomorphogenic functions in many biological responses in mammalian cells, but its downstream pathway remains unclear.
 the ubiquitin ligase cop constitutively photomorphogenic functions in many biological responses in mammalian cells, but its downstream pathway remains unclear.  here, we identified fip a key regulator of mammalian autophagy, as a novel cop1-interacting protein by yeast two-hybrid screening. the interaction was confirmed by a gst-pulldown assay. split-gfp analysis revealed that interaction between cop and fip predominantly occurred in the cytoplasm and was enhanced in cells treated with uv irradiation. different forms of fip protein were expressed in cultured mammalian cells, and ectopic expression of cop reduced one of such forms.  these data suggest that cop modulates fip200-associated activities, which may contribute to a variety of cellular functions that cop is involved in. cop1fip200uvautophagy 