y - box binding proteins are members of the cold shock domain ( csd ) super family of proteins .
they are involved in the regulation of transcription [ 2 , 3 ] , modulation of translation , dna repair , and drug resistance [ 68 ] , stress response to extracellular signals [ 9 , 10 ] and in an early stage of embryogenesis [ 11 , 12 ] .
several studies also showed up - regulation of yb-1 protein levels in proliferating cells in comparison to quiescent or non - proliferating cells [ 1 , 13 ] .
yb-1 activates many genes implicated in cell proliferation including dna polymerase  , proliferating cell nuclear antigen ( pcna ) [ 15 , 16 ] , thymidine kinase and topoisomerase ii [ 15 , 17 ] .
knock - out studies have been done to gain insight into the function of yb-1 in cell proliferation .
we showed previously that targeted disruption at the 5 end of one allele of chicken yb-1 gene in dt-40 cells resulted in major cell cycle defects , including a slower doubling time , increased genomic dna content , increased cell size and apoptosis in a fraction of the cell population .
in contrast , another group found that yb-1 heterozygous mutants did not show any apparent growth defects , whereas yb-1 cells exhibited a markedly reduced growth phenotype . targeted disruption at the 3 end of one allele of yb-1 rendered mouse embryonic stem cells more sensitive to cisplatin and mitomycin c without any apparent growth defects .
furthermore , down regulation of yb-1 by shrna resulted in a significant reduction in the rate of proliferation and increased rate of apoptosis .
these studies indicated that the amino terminus of yb-1 may be involved in cell proliferation . a definitive role for yb-1 in cell proliferation
these mice are embryonic lethal , indicating a non - redundant role for yb-1 in early embryonic development .
further studies with yb1 fibroblasts showed greatly reduced cell proliferation and altered cell morphology , demonstrating a critical role for yb-1 in cell proliferation .            in our earlier studies   we speculated that the altered phenotypes we observed in dt-40 cells might be due to a dominant negative effect exerted by a putative truncated protein encoded by the disrupted yb-1 allele .
if this assumption is correct , then introduction of the n - terminal domain of yb-1 into cells should mimic the phenotypic changes observed in the mutant dt40 cells .
therefore , we constructed clones expressing either the 26 or 77 amino acid polypeptide sequence corresponding to the n - terminus of yb-1 .
we also made an internal deletion which removed the alanine and proline - rich sequence of the n - terminal 77 amino acids of yb-1 .
these polypeptides were fused with the antennapedia homeodomain , which facilitates receptor independent uptake of the proteins into cells and a reporter green fluorescent protein ( gfp ) to monitor the uptake and cellular localization of the proteins .
these fusion proteins were introduced into rat hepatoma cells and their effect on cell growth studied .
our results clearly indicated a role for yb-1 in cell proliferation which is mediated by the n - terminal domain of yb-1 probably by sequestering cyclin d1 in the cytoplasm , thus blocking cell cycle progression from g2/m .

 antibodies and reagentsall the chemical reagents , unless otherwise specified were from sigma - aldrich ( st .
primary antibodies and fluorescent - labeled secondary antibodies were obtained from invitrogen - molecular probes ( carlsbad , ca ) . 
  all the chemical reagents , unless otherwise specified were from sigma - aldrich ( st .
louis , mo ) .   cell culture media were from invitrogen - gibco ( carlsbad , ca ) .
primary antibodies and fluorescent - labeled secondary antibodies were obtained from invitrogen - molecular probes ( carlsbad , ca ) . 

cloning and expression of apgfp - fusion proteinsthe puc9 vector was modified in our lab and the three sequences of yb-1 were cloned in frame with an upstream sequence coding for the 16 amino acid long antennapedia homeodomain ( ap ) and downstream sequence coding for gfp ( figure 2(a)).the three clones apyb77gfp , apyb36gfp , apyb26gfp and the control clone apgfp , which lacked the yb-1 sequence , were transformed into e. coli bl21de3 cells .
log phase cultures were induced overnight with 1  mm iptg and proteins were purified . 
  the puc9 vector was modified in our lab and the three sequences of yb-1 were cloned in frame with an upstream sequence coding for the 16 amino acid long antennapedia homeodomain ( ap ) and downstream sequence coding for gfp ( figure 2(a)).the three clones apyb77gfp , apyb36gfp , apyb26gfp and the control clone apgfp , which lacked the yb-1 sequence , were transformed into e. coli bl21de3 cells .
log phase cultures were induced overnight with 1  mm iptg and proteins were purified . 

purification of proteinsinclusion bodies were isolated and solubilized as described   and the fusion proteins were further purified from the supernatants using deae sephacel ion - exchange chromatography at 4c as described .
these fractions were dialyzed overnight with 4 changes of tng buffer ( 50  mm tris - hcl , ph 8.0 , 100  mm nacl and 5% glycerol ) .
purity of the proteins was determined by running triplicates of the protein fractions on a 15% sds - page gel followed by coomassie staining or silver staining and western blotting with gfp antibody .
protein concentration was determined using bca protein assay kit ( pierce- rockford , il ) . 
 
inclusion bodies were isolated and solubilized as described   and the fusion proteins were further purified from the supernatants using deae sephacel ion - exchange chromatography at 4c as described .
these fractions were dialyzed overnight with 4 changes of tng buffer ( 50  mm tris - hcl , ph 8.0 , 100  mm nacl and 5% glycerol ) .
purity of the proteins was determined by running triplicates of the protein fractions on a 15% sds - page gel followed by coomassie staining or silver staining and western blotting with gfp antibody .
protein concentration was determined using bca protein assay kit ( pierce- rockford , il ) . 

synchronization and treatment of cellsrat hepatoma cells ( atcc - h-411e ) were grown in minimum essential medium supplemented with 10% fbs , 100  u / ml penicillin and 100  g / ml streptomycin , in a humidified incubator maintained at 37c and 5%  co2 .
cells fed every day were subcultured 1  :  5 on confluency , using 0.05% trypsin - edta . for experimental analysis
, cells were incubated with 40  g / ml of the purified proteins for the indicated time periods .
double thymidine block was used for synchronization of cells in g1 as described . for g2/m phase synchronization cells
were incubated with 1 to 2.5  g / ml nocodazole for 14 to 24  hrs . 
 
rat hepatoma cells ( atcc - h-411e ) were grown in minimum essential medium supplemented with 10% fbs , 100  u / ml penicillin and 100  g / ml streptomycin , in a humidified incubator maintained at 37c and 5%  co2 .
cells fed every day were subcultured 1  :  5 on confluency , using 0.05% trypsin - edta . for experimental analysis
, cells were incubated with 40  g / ml of the purified proteins for the indicated time periods .
for g2/m phase synchronization cells were incubated with 1 to 2.5  g / ml nocodazole for 14 to 24  hrs . 

immunoblottingcells were washed and lysed with ripa buffer following the published protocol . for nuclear and cytoplasmic fractions ,
the cells were scraped in 100  l ice cold pbs and centrifuged at 500  g for 5  min .
the cell pellet was resuspended in 100  l buffer a ( 50  mm nacl , 10  mm hepes ph-8.0 , 500  mm sucrose , 1  mm edta , 0.2% triton - x-100 , freshly added protease inhibitors and 7  mm -mercaptoethanol ) , vortexed at high speed for 45  sec and centrifuged at 2000  g for 2  min at 4c .
the pellet was resuspended in 100  l buffer b ( buffer a with 25% glycerol and 0.1  mm edta ) and nuclei were pelleted at 2000  g for 2  min at 4c .
the pellet was dissolved in 50  l buffer c ( buffer b with 350  mm nacl ) , incubated on ice for 30  min with intermittent high speed vortexing and spun down at 11000  g for 15  min at 4c .
the supernatant was diluted to 100  l with pbs and used as nuclear extract .
equal amounts of samples were analyzed on sds - page gel . 1  : 
7500 dilution of antirabbit gfp antibody was used as primary antibody for overnight incubation at 4c . 1  :  15000 dilution of hrp - conjugated antirabbit igg was used as secondary antibody for 2  hr at room temperature .
the blot was developed using chemiluminescence kit ( pierce , rockford , il ) according to the manufacturer 's protocol . 
 
cells were washed and lysed with ripa buffer following the published protocol . for nuclear and cytoplasmic fractions ,
the cells were scraped in 100  l ice cold pbs and centrifuged at 500  g for 5  min .
the cell pellet was resuspended in 100  l buffer a ( 50  mm nacl , 10  mm hepes ph-8.0 , 500  mm sucrose , 1  mm edta , 0.2% triton - x-100 , freshly added protease inhibitors and 7  mm -mercaptoethanol ) , vortexed at high speed for 45  sec and centrifuged at 2000  g for 2  min at 4c . the resulting supernatant was used as cytoplasmic extract .
the pellet was resuspended in 100  l buffer b ( buffer a with 25% glycerol and 0.1  mm edta ) and nuclei were pelleted at 2000  g for 2  min at 4c .
the pellet was dissolved in 50  l buffer c ( buffer b with 350  mm nacl ) , incubated on ice for 30  min with intermittent high speed vortexing and spun down at 11000  g for 15  min at 4c .
the supernatant was diluted to 100  l with pbs and used as nuclear extract .
equal amounts of samples were analyzed on sds - page gel . 1  : 
7500 dilution of antirabbit gfp antibody was used as primary antibody for overnight incubation at 4c . 1  : 
15000 dilution of hrp - conjugated antirabbit igg was used as secondary antibody for 2  hr at room temperature .
the blot was developed using chemiluminescence kit ( pierce , rockford , il ) according to the manufacturer 's protocol . 

immunoprecipitationequal amounts of whole cell lysates or nuclear and cytoplasmic extracts were diluted to 500  l with lysis buffer and incubated with 1  :  2000 dilution of cyclin a , b1 , d1 d2 and gfp antibodies overnight at 4c .
equal amount of protein a sepharose beads ( amersham biosciences  piscataway , nj ) was added to each sample and incubated for 2 
the beads were pelleted down at 8000  g for 4  min and the pellet was washed with lysis buffer twice .
the resulting pellet was resuspended in sample buffer and loaded onto sds - page gels . 
 
equal amounts of whole cell lysates or nuclear and cytoplasmic extracts were diluted to 500  l with lysis buffer and incubated with 1  :  2000 dilution of cyclin a , b1 , d1 d2 and gfp antibodies overnight at 4c .
piscataway , nj ) was added to each sample and incubated for 2  hr at room temperature .
the beads were pelleted down at 8000  g for 4  min and the pellet was washed with lysis buffer twice .
the resulting pellet was resuspended in sample buffer and loaded onto sds - page gels . 

immunocytochemistry and indirect immunofluorescencecells were rinsed twice with phosphate buffered saline ( pbs ) , fixed for 15  min . with 4% p - formaldehyde , washed twice with pbs and stored at 4c .
cells were blocked with 5% goat serum and 3% bsa in pbs for 30  min . and rinsed with pbs thrice .
then the cells were incubated with primary antibodies antirabbit gfp ( 1  :  500 ) or antirabbit yb-1 ( 1  :  300 ) and antimouse cyclin d1 ( 1  :  300 ) for 3  hrs followed by several washes with pbs . subsequently cells were incubated with secondary antibodies fitc conjugated antirabbit igg ( 1  :  500 ) and texas - red conjugated antimouse igg ( 1  :  500 ) for 1  hr at room temperature in dark room . coverslips were washed several times with pbs and once with water to remove any salts and left to air - dry for 45  min in absence of light .
the coverslips were mounted onto glass slides using vectashield mounting medium containing dapi ( vector laboratories 
cells were rinsed twice with phosphate buffered saline ( pbs ) , fixed for 15  min . with 4% p - formaldehyde , washed twice with pbs and stored at 4c .
cells were blocked with 5% goat serum and 3% bsa in pbs for 30  min . and rinsed with pbs thrice .
then the cells were incubated with primary antibodies antirabbit gfp ( 1  :  500 ) or antirabbit yb-1 ( 1  :  300 ) and antimouse cyclin d1 ( 1  :  300 ) for 3  hrs followed by several washes with pbs .
subsequently cells were incubated with secondary antibodies fitc conjugated antirabbit igg ( 1  :  500 ) and texas - red conjugated antimouse igg ( 1  :  500 ) for 1  hr at room temperature in dark room . coverslips were washed several times with pbs and once with water to remove any salts and left to air - dry for 45  min in absence of light .
the coverslips were mounted onto glass slides using vectashield mounting medium containing dapi ( vector laboratories 
confocal microscopyconfocal images were captured on lsm 510 laser - scanning three - color microscope ( carl zeiss , jena , germany ) using argon laser at 75% output and hene1 laser at excitation wavelengths 488  nm and 543  nm , respectively .
optical slice thickness was set to 0.3  m , magnification to 40 and sections presented were taken approximately at the mid height level of cells . photomultiplier gain and laser power were identical within each experiment .
all images were analyzed and adjusted for contrast in adobe photoshop 5.0 ( san jose , ca ) . 
 
confocal images were captured on lsm 510 laser - scanning three - color microscope ( carl zeiss , jena , germany ) using argon laser at 75% output and hene1 laser at excitation wavelengths 488  nm and 543  nm , respectively .
optical slice thickness was set to 0.3  m , magnification to 40 and sections presented were taken approximately at the mid height level of cells . photomultiplier gain and laser power were identical within each experiment .
all images were analyzed and adjusted for contrast in adobe photoshop 5.0 ( san jose , ca ) . 

dna fragmentationexponentially growing cells were incubated with the fusion proteins for 24 , 48 and 72  hr .
exponentially growing cells were incubated with the fusion proteins for 24 , 48 and 72  hr .
facs analysiscells were harvested by trypsinization or scraping on ice and pelleted at 500  g for 5  min .
they were washed with pbs , fixed with ice cold 70% etoh , incubated on ice for 45  min and centrifuged at 500  g for 5  min .
the pellet was washed with hanks balanced salt solution ( hbss ) containing 1% bsa , resuspended in propidium iodide buffer ( 1  :  1 dilution of 20  ug / ml of pi in hbss and 1  mg / ml rnase a ) and incubated for 30  min in a 37c water bath .
for analysis of apoptotic cells , aposcreen annexin v - fitc kit ( southern biotechnology associates inc .
cell debris was excluded from the facs analysis by appropriate forward and light scatter gating .
cells were harvested by trypsinization or scraping on ice and pelleted at 500  g for 5  min .
they were washed with pbs , fixed with ice cold 70% etoh , incubated on ice for 45  min and centrifuged at 500  g for 5  min .
the pellet was washed with hanks balanced salt solution ( hbss ) containing 1% bsa , resuspended in propidium iodide buffer ( 1  :  1 dilution of 20  ug / ml of pi in hbss and 1  mg / ml rnase a ) and incubated for 30  min in a 37c water bath .
for analysis of apoptotic cells , aposcreen annexin v - fitc kit ( southern biotechnology associates inc
cell debris was excluded from the facs analysis by appropriate forward and light scatter gating .
earlier we disrupted one allele of the yb-1 gene in dt-40 cells and observed profound cell cycle defects .
we proposed that this effect could be due to truncated proline - rich polypeptide encoded within exon 1 and produced by the disrupted allele . in order to test whether the putative truncated protein could be responsible for the previously observed results
, we developed an expression cassette containing the amino - terminal 77 amino acids of yb-1 inserted between the 16 amino acid antennapedia peptide and a gfp reporter .
two other fusions containing the 26 n - terminal amino acids of yb-1 ( apyb26gfp ) and a clone with an internal deletion that removed the alanine - proline - rich sequence from the n - terminal 77 amino acids of yb-1 ( apyb36gfp ) were also generated ( figure 1(b ) line drawings ) .  
these fusion proteins were purified to more than 80% homogeneity as assessed by polyacrylamide gel electrophoresis and their sizes were confirmed by western blotting ( figure 1(b ) ) .         in order to show that the antennapedia peptide can direct the cellular uptake of the yb-1 fusion proteins , we incubated rat hepatoma cells with 40  g / ml of each protein for 3 and 18  hrs .
results presented in figure 2 indicate that that the fusion proteins are internalized as early as 3  hrs ( figure 2(a ) , lanes marked l ) and continued to accumulate for at least 18  hr ( figure 2 ) . at this time
more than 80% of cells have taken up the fusion proteins indicating that the antennapedia peptide allows the cells to take up the proteins efficiently ( see figure 3 ) similar results were obtained with primary cells isolated from rat aortic smooth muscle ( rasm ) ( data not shown ) .
these results indicate that the fusion proteins are readily internalized by tumor and primary cells .
having demonstrated internalization of all the proteins , we then studied the localization of these proteins within cells .
cells were incubated with the fusion proteins for 3 or 18  hr and prepared for confocal microscopy .
results indicated that more than 80% of the cells had taken up the proteins by 18  hrs .
apgfp , which did not contain any yb-1 sequences , was distributed throughout the cell , that is , in both nucleus and cytoplasm ( figure 3(a ) panels a and e ) .
this is probably due to the fact that gfp alone can distribute all over the cell unless specific targeting signals are included ( reviewed in ) or   that the cell penetrating antennapedia moiety may be responsible for the translocation of gfp through both cytoplasmic and nuclear membranes .  
in contrast , incubation of the three yb-1 fusion proteins indicated that both at 3 and 18 hours , most of the protein was localized only in the cytoplasm ( figure 3(a ) , panels b and d , c and e , and f and h ) .
these results suggest that the amino terminus of yb-1 restricted the fusion proteins to the cytoplasm of cells .        
nuclear and cytoplasmic extracts of rat hepatoma cells were analyzed by immunoblotting analysis using anti - gfp antibodies .  
these studies indicated that the apybgfp fusion proteins were detected only in the cytoplasmic extracts of cells even after 18  hr , whereas the apgfp was detected both in nuclear and cytoplasmic extracts ( figure 3(b ) ) .
less than 2 to 3% of the internalized apybgfp proteins was found in the nuclei where as in the control apgfp as much as 30 to 40% was detected in the nuclei by 18 hours .
we conclude that the presence of n - terminal amino acid sequence of yb-1 in the fusion protein is responsible for their restricted localization to the cytoplasm of cells .
having demonstrated the delivery of the amino - terminal yb-1 peptides into cells , we wondered whether these yb-1 peptides induce cell cycle defects similar to those we previously observed in yb-1-disrupted heterozygous mutant dt-40 cells .  
cells were incubated with each one of these fusion proteins for 24 , 48 and 72  hrs .
half of the cells were stained with propidium iodide and subjected to facs analysis to determine the dna content .
our results indicate that ~32% of cells were arrested in g2/m phase of the cell cycle following 72 hr incubation with apyb77gfp ( figure 4 ) while apyb26gfp induced more than 24% of the cells to accumulate in g2/m following the same 72  hr incubation ( figure 4 ) .
in contrast , only about 10% apyb36gfp - incubated cells were in g2/m ( figure 5 ) , which is very similar to the apgfp - incubated cells where less than 6%8% of the cells were in the g2/m phase ( figure 4 ) .
these results suggest that the n - terminal sequence of yb-1 , containing the alanine - proline - rich domain , may be involved in regulating the progression of cells from g2/m to g1 .      in order to show that the block in g2/m phase was not due to differences in the uptake of the fusion proteins , the remaining half of the samples was used to quantify the mean gfp fluorescence in the gfp positive cells .
the results presented in figure 4(b ) indicated that the percentage of gfp - positive cells was similar for all the fusions .
in addition , no difference was observed in the mean gfp fluorescence of different fusion proteins .  
these results suggest that the cell cycle arrest in apybgfp - incubated cells is not due to variation in the number of fusion protein - positive cells or amount of the internalized proteins , but is due to the presence of yb-1 n - terminal sequence .
further , this data clearly implicate the n - terminus of yb-1 in the arrest of cells at the g2/m phase of the cell cycle .
earlier we demonstrated apoptosis in heterozygous mutant dt-40 cells and speculated that this might be due to a dominant negative effect of the potential truncated protein encoded by the defective allele .
subsequently another group reported that down - regulation of yb-1 resulted in an increased rate of apoptotic cell death , demonstrating a direct relationship between yb-1 levels and apoptosis .
based on these observations we hypothesized that the n - terminus of yb-1 is involved in regulating apoptotis .             to test the possibility that the n - terminus of yb-1 regulates apoptosis , we carried out experiments that detect the presence of phosphatidylserine on the cell surface using fluorescein - conjugated annexin v and dna fragmentation , both of which are hallmarks of apoptosis .
cells were incubated with the fusion proteins for 24 , 48 and 72  hr and half of the cells were stained with annexin v and analyzed by facs .
our results indicated that in apgfp - incubated cells less than 1 to 2% of cells were apoptotic after 72  hr incubation ( figure 5(a ) ) .
in contrast , incubation with apyb77gfp stimulated 22 - 23% of cells to become annexin v - positive by 72  hrs .
again , the uptake of these proteins was comparable as evidenced by the number of gfp positive cells and their mean gfp fluorescence ( figure 5(c ) ) .
these results clearly suggest that the n - terminal alanine - proline - rich 26 amino acids is part of a major death domain of yb-1 .      
dna from cells incubated with the fusion proteins for various time periods was analyzed by agarose gel electrophoresis .
it can be seen that the pattern of dna fragmentation , as evidenced by the appearance of the 180200  bp ladder , was clearly seen with the dna from cells treated with yb-1 containing fusion proteins ( figure 5(b ) ) but not with cells incubated with the control fusion protein , apgfp .
futher , staining of the nuclei with dapi indicated chromatin condensation and broken nuclei in cells treated with the apybgfp proteins for 72 hr but not in apgfp - incubated cell nuclei which showed normal appearance(see in supplementary material figure s1 available online at doi : 10.155/2009/243532).based on these results we conclude that the amino terminal region of yb-1 is involved in apoptosis .
it has been shown that yb-1 regulates transcription of g2/m phase cyclins a and b1 .
since cyclin d1 is upregulated during g2 phase of cell cycle by ras - mediated translational control of its mrna , and since it is localization within the nucleus is very important for cell cycle progression , we wondered whether yb-1 interacts with any of these cell cycle - specific cyclins . in order to study this interaction , whole cell extracts of rat hepatoma cells
were immunoprecipitated with antibodies for cyclins a , b1 , d1 and d2 and examined for the presence of yb-1 .
our results clearly indicate that yb-1 predominantly interacts with cyclin d1 as this is the major band observed in the pull - down assays ( figure 6(a ) ) .
we observed a minor band with cyclins b and d2 which are also upregulated in g2 . in order to show that this interaction is not due to an artifact of protein extraction methods used , we carried out immunoprecipitations of extracts isolated from asynchronous as well as from nocodazole - treated cultures .
nocodazole , as expected , blocked cells in the g2/m ( supplementary figure s2 ) . since anticyclin d1
antibody pulled - down yb-1 from nocodazole - treated cell extracts , it indicates that yb-1 interacts with cyclin d1 .
reciprocal experiments using anti - yb-1 antibody confirmed that yb-1 indeed binds to cyclin d1 ( supplementary figure s3 ) .           in order to investigate
if yb-1 and cyclin d1 co - localize in cells in different phases of the cell cycle , we analyzed their distribution in g1/s and g2/m phase synchronized cells using confocal microscopy .
we observed that in the asynchronized and g1/s phase synchronized cells yb-1 was predominantly cytoplasmic ( figure 6(b ) , panels a and d ) whereas cyclin d1 was predominantly nuclear ( figure 6(b ) , panels c and f ) with minimal co - localization ( figure 6(b ) , panels b and e ) .
in the g2/m phase - blocked cells yb-1 and cyclin d1 were partially co - localized mainly in the cytoplasm ( figure 6(b ) , panels g , h and i ) .         in order to show that this colocalization is due to specific interaction between yb-1 and cyclin d1
, we carried out immunoprecipitations with isolated nuclear and cytoplasmic extracts using specific antisera ( supplementary figures s3a and s3b ) .
anticyclin d1 antibody pulled down only cyclin d1 and anti - yb-1 antibody brought down only yb-1 from the cytoplasm indicating that these proteins are indeed localized in the nuclei and cytoplasm , respectively , of untreated cells .
however , in the nocodazole - treated cells , which are arrested in g2 phase , cyclin d1 antibody pulled down yb-1 from cytoplasm and vice versa .
as expected , nocodazole blocked a large proportion of cells in g2/m phase of cell cycle ( supplementary figure s2 ) .  
we conclude that yb-1 and cyclin d1 interact with each other in the cytoplasm of g2 phase cells .
interestingly when wortmannin , an inhibitor of pi-3 kinase and akt pathway was used , a significant amount of cyclin d1 could be detected in the nuclei ( supplementary figure s4 ) .
this indicates that the interaction between yb-1 and cyclin d1 depends on phosphorylation of one or both proteins .
if the amino terminus of yb-1 interacts with cyclin d1 , whole cell extracts of asynchronized and cells synchronized in g2/m phase using nocodozole were incubated with gfp antibody and probed for cyclin d1 .
however , in nocodazole - blocked cells , cyclin d1 co - precipitated with both apyb26gfp and apyb77gfp at the 3  hr and 18  hr time points ( figure 6(c ) ) , while it was absent in the immunoprecipitates prepared from apyb36gfp - incubated cells . since asynchronous cultures also contain some cells in g2/m we expect some interaction between cyclin d1 and yb-1 , as shown in supplementary figure s3b .
this may be due to sensitivity of the technique or that the interaction in the presence of endogenous yb-1 may be masking detection . nevertheless , these results suggest that the alanine - proline - rich n - terminal sequence of yb-1 may be involved in sequestering cyclin d1 in the cytoplasm when the cells are blocked in the g2/m phase of cell cycle .  
retention of cyclin d1 in the cytoplasm of g2/m phase cells by the yb-1 fusion proteins may be responsible for the observed effects of these fusions on cell cycle progression and apoptosis .
yb-1 expression is closely associated with cell proliferation . for example , yb-1 is highly expressed in regenerating liver and liver cancers but barely detectable in normal adult liver where cells are quiescent [ 28 , 29 ] .
our earlier studies showed that disruption of one allele of chk - yb-1b gene resulted in abnormalities in the heterozygous dt-40 mutants , including slower growth rate and increased genomic dna content that is associated with apoptosis .
we speculated that the defects seen may be due to a dominant negative effect of the putative n - terminal truncated protein encoded by the disrupted yb-1 allele .
if our assumption was correct , then introduction of peptides corresponding to the amino terminus of yb-1 should mimic the defects that were observed in the mutant dt40 cells .           
the antennapedia peptide was shown to be the most efficient and least cytotoxic of all cell penetrating peptides .
therefore the yb-1 sequences were cloned in such a way that the fusion proteins carried the antennapedia peptide at the amino end and a reporter gfp sequence at the carboxyl end .
we report here that internalization of the four apgfp fusion proteins occurs at high efficiency in exponentially growing rat hepatoma cells . the control protein , apgfp , which does not have any yb-1 sequence , is distributed throughout the cell  both nuclei and cytoplasm .
in contrast , yb-1 fusion proteins are restricted to the cytoplasm of cells . since apgfp is distributed all over the cell
the ability of the yb-1 n - terminus to associate with cyclin d1 and potentially retain it in the cytoplasm of cells may account for the effect of the yb-1 fusions on cell cycle progression and apoptsis .   cyclin d1 , encoded by the bcl-1 gene , is a putative protooncogene , and previous studies have shown that both yb-1 and cyclin d1 are upregulated in many neoplasms [ 1 , 31 , 32 ] .
although yb-1 is generally a cytoplasmic protein and cyclin d1 a nuclear protein , yb-1 is found in the nucleus in mid g1 to s phase   at the same time as cyclin d1 .
cyclin d1 relocates to the cytoplasm at the end of s phase and into the g2 phase [ 33 , 34 ] , when yb-1 is also concentrated in the cytoplasm .
the levels of cyclin d1 in the g2/m phase of cell cycle are crucial for the cells to decide if they have to undergo another round of replication .            
it has been shown that cyclin d1-knockout mouse embryos survive for about 13 days and then die .   
however , in epithelial cells cyclin d2 compensates for the loss of cyclin d1 indicating the importance of these cyclins in cell proliferation .
cyclin d1 is considered to be a g1 cyclin , as its interaction with cdk4/6 contributes to the phosphorylation of rb protein and also sequesters the cdk inhibitors , which is required for the cell cycle progression to the s phase .            
cyclin d1 levels also increase in g2 and are maintained throughout mitosis and g1 [ 10 , 34 , 38 ] .
the regulation appears to be at posttranscriptional level , indicating that de novo synthesis of cyclin d1 occurs during g2 phase .
its fate is determined through receptor - coupled ras signaling [ 34 , 37 ] .
it is likely that when cyclin d1 is present in cytoplasm in g2 that the amino end domain of yb-1 sequesters cyclin d1 and prevents its import into nucleus .
nuclear translocation of yb-1 requires phosphorylation by the serine - threonine protein kinase akt at ser-102 in the csd of yb-1 [ 3941 ] .
this phosphorylation also induces translation of several cellular mrnas . since both the csd and the carboxy terminal acidic - basic domains are missing in our fusion proteins , many of the functions carried out by the full length endogenous yb-1 molecule are disrupted . as a consequence , the cellular protein(s ) that interact with the n - terminal yb-1 sequence affect downstream signaling leading to cellular damage , which keeps the cells in g2 , giving an opportunity for cells to recover or undergo apoptosis probably in a p53-dependent pathway [ 24 , 37 ] .
the data provided here suggests that the interaction between yb-1 and cyclin d1 play a critical role in regulating cyclin d1 function at the g2/m phase of the cell cycle .           
stabilization and nuclear accumulation of cyclin d1 depends on pi3-k , akt pathway , which can negatively regulate gsk-3 , a serine / threonine protein kinase , regulates cyclin d1 expression by regulating mrna transcription and protein degradation ( reviewed in ) .
phosphorylation of cyclin d1 as well as yb-1 depends on the pi3-k and akt pathway .
yb-1 , upon phosphorylation is translocated into nucleus where it directly induces the p110 catalytic subunit of pi3-kinase [ 39 , 44 ] .
similarly , cyclin d1 is also regulated by pi3-kinase , akt and gsk-3 ( reviewed in ) .
cyclin d1 in the nucleus is important for cell cycle progression and export from cytoplasm to nucleus is critical for g2/m esit .
normally cyclin d1 is exported to the cytoplasm where it is degraded by the ubiquitin pathway .         
the fact that wortmannin , an inhibitor of pi3-kinase - akt pathway , stimulates the nuclear localization of cyclin d1 in nocodazole - treated cells , suggests that the interaction between cyclin d1 and yb-1 and resulting cytoplasmic localization of cyclin d1 depends on the phosphorylation of yb-1 or cyclin d1 or both .
yb-1 is known to be phosphorylated on its ser 102 in csd by both akt and the p90 ribosomal s6 kinase .
this phosphorylation allows yb-1 to shuttle into nucleus where it induces transcriptioin of pi3-k 110 catalytic subunit .
however , in the apyb77gfp and apyb26gfp , this phosphorylation site is not present and as a result this n - terminal domain sequesters the cyclin d1 and prevents its movement to the nucleus .
yb-1 is known to interact with several cellular proteins including those involved in dna synthesis , dna repair , transcription , and translation .
depending on the type of cell and the cell cycle stage these interactions may be required for nuclear translocation and/or for cytoplasmic retention of various cellular factors .
it is generally known that cells halted in g2/m are unstable , and during this time the cell survival depends on several proteins including p53 , p21 and cyclins , which determine the fate of the cells , that is , whether to go for another round of cell cycle or apoptosis .
when full length yb-1 binds to appropriate targets and elicit proper response , the cell cycle progresses normally ; however , when the n - terminal sequence binds to proteins such as p53   or cyclin d1 and keeps them in a different compartment of the cell , then it can not elicit the same response and hence the defects .        
it has been shown that cells blocked in the g2/m phase of cell cycle appear to be unstable and undergo apoptosis [ 33 , 34 , 46 ] .
in addition , it is known that repair of any damaged dna takes place during g2 and yb-1 has been implicated in this process .   in our experiments we found that the n - termainal region of yb-1 stimulated cells to arrest in the g2/m phase of the cell cycle .
this dominant effect of the n - terminus of yb-1 may be due to the ability of yb-1 to regulate centrosome function by binding to actin   and centrosomes during mitosis .
inhibition of centrosome function can lead to chromosomal instability and aneuploidy [ 46 , 49 ] , which may lead to a block in cell cycle and apoptosis .  
our data , which are consistent with these previous observations , suggest a critical role for the yb-1 n - terminal domain in regulating cell proliferation and apoptosis .           in summary ,
in this report we demonstrated co - localization of yb-1 and cyclin d1 in the cytoplasm of g2/m phase blocked cells .
furthermore , we present evidence for the interaction of cyclin d1 with fusion proteins apyb26gfp and apyb77gfp in g2/m phase blocked cells .
this causes the cells to arrest in g2/m phase of the cell cycle and protects cyclin d1 from degradation , which is important for g2/m exit . as a consequence of this abnormal arrest in g2/m arrest
since cyclin d1 immunoprecipitates apyb77gfp and apyb26gfp , but not apyb36gfp and correlates with increased apoptosis , it is likely that the interaction with cyclin d1 occurs through the alanine and proline - rich sequence at the n - terminus of yb-1 .
further studies are necessary to elucidate the physiological importance of the yb-1/cyclin d1 interaction and to precisely identify the domains involved in this interaction .
 y - box binding protein yb-1 is a multifunctional protein involved in cell proliferation , regulation of transcription and translation . 
 our previous study indicated that disruption of one allele of chk - yb-1b gene in dt-40 cells resulted in major defects in the cell cycle . 
 the abnormalities seen in heterozygous mutants could be attributed to a dominant negative effect exerted by the disrupted yb-1 allele product . to test this hypothesis the n - terminal sequence of the yb-1 
 was fused with the third helix of antennapedia and the green fluorescent protein . 
 these purified fusion proteins were introduced into rat hepatoma cells and their effect on cell proliferation was studied .   
 results indicate that the n - terminal 77 amino acid domain of the yb-1 protein induced the cells to arrest in g2/m phase of the cell cycle and undergo apoptosis .   
 additional deletion analysis indicated that as few as 26 amino acids of the n - terminus of yb-1 can cause these phenotypic changes . 
 we further demonstrated that this n - terminal 77 amino acid domain of yb-1 sequesters cyclin d1 in the cytoplasm of cells at g2/m phase of cell cycle . 
 we conclude that the n - terminal domain of yb-1 plays a major role in cell cycle progression through g2/m phase of cell cycle . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion