BACKGROUND
mps <dig> , a serine/threonine kinase, plays multiple functions in the cell cycle  <cit> .
in mitosis, mps <dig> is indispensable for both the spindle assembly checkpoint  and chromosomal alignment.
mps <dig> activates sac by directing the assembly of the mitotic checkpoint complex .
the resulting complex binds and inactivates apc/c-mediated proteolysis of cyclin b and securin  <cit> .
mps <dig> can phosphorylate borealin to regulate chromosomal alignment independently of sac  <cit> .
mps <dig> also affects centrosome duplication by collaborating with substrates, including mortalin, centrin  <dig> and vdac <dig>  <cit> .
in interphase, mps <dig> interacts with p <dig> and chk <dig> and is involved in dna damage response  <cit> .
the subcellular distribution of mps <dig> correlates with the functions that it performs in the cell cycle.
during mitosis, mps <dig> appears on the kinetochore at prophase, stays consistently until metaphase initiation, and then comes off at metaphase upon mitotic entry  <cit> .
kinetochore association of mps <dig> is regulated by hec  <dig> and aurora b  <cit> .
the timely release of mps <dig> from kinetochore is required for mitotic progression  <cit> .
in interphase, mps <dig> resides in the cytoplasm, nuclear envelope and centrosomes  <cit> .
translocation of mps <dig> from cytoplasm to nucleus at the g2/m boundary requires two lxxll motifs at the n-terminal  <cit> .
the centrosome localization of mps <dig> is required for centrosome duplication  <cit>  and in the case of dna damage, mps <dig> is also recruited to dna damage foci, showing that mps <dig> plays a role in dna damage response  <cit> .
the regulatory mechanism retarding mps <dig> at cytoplasm is unclear so far.
in this paper, a putative nuclear export sequence  motif at the n-terminal of mps <dig> was identified and this motif shows a high sequence similarity to the classic nes, exporting egfp out of the nucleus in cases of fusion.
unexpectedly, replacing  <dig> isoleucine and  <dig> leucines in this pnes with alanines did not cause constitutive nuclear enrichment of mps <dig>  further studies suggest that the  <dig> amino acid mutation affects mps <dig> nuclear translocation at the g2/m boundary.
consistent with this finding, the mps <dig> mutant with  <dig> of the last  <dig> leucines is predominantly cytoplasmic while the other mutants behave as with the  <dig> amino acid mutation.
collectively, the observations showed that the pnes sequence at the n-terminal is a functional motif which is sufficient for driving mps <dig> nucleus export during interphase.
methods
dna constructs and stable cell lines generation
retroviral expression vector prex-yfp-mps1-ires-hygro in the prex background has been described previously  <cit> .
constructs prex-egfp-ires-hygro, prk5-myc-mps <dig>  pexl-fth-crm <dig>  pgst-4 t-mps <dig>  pexl-fth-crm <dig> were generated in a standard way.
to generate prex-pness-egfp-ires-hygro, the coding sequences for the putative ness were achieved by annealing pairs of complementary oligo-nucleotide primers and then they were sub-cloned into the n-terminal of egfp.
to introduce the pnes <dig> mutations in mps <dig>  construct prex-yfp-mps1-ires-hygro was subject to a standard quickchange mutagenesis procedure by pcr amplication with pairs of oligos sequentially.
primers used in this paper are showed in additional file 1: table s <dig>  all constructs were confirmed by dna sequencing.
the procedure for generation of sw <dig> stable cell lines expressing various mps <dig> mutants is described.
protein expression and purification
to prepare protein from e.coli, the construct pqe60-crm <dig> was transformed into bl <dig>  and then induced with  <dig>  mm iptg at 22°c overnight.
the cell pellet was washed and suspended in binding buffer , followed by a regular sonication procedure.
the cell debris was removed by centrifugation at  <dig>  g for 30 min.
the supernatant was then applied to ni-charged chelating-sepharose  and proteins were purified according to the protocol by the manufacturer.
to produce the purified proteins from mammalian cells, plasmids were transciently transfected into 293 t cells.
after 48 hrs, cells were washed by d-pbs twice and then subject to lysis buffer .then the supernatant was directly applied to ni-charged chelating-sepharose  or co-incubated with c-myc agarose affinity beads for immunoprecipitation  depending on the tag of proteins fused.
fusion proteins were quantified by bca assay  and the purity was confirmed by sds-page analysis.
cell synchronization
synchronization of cells at g1/s phase was accomplished by sequential double thymidine and ro <dig> treatment as described by zhang et al.
<cit> .
briefly, cells were arrested at g1/s phase by treatment with 2 mm thymidine for 19 h and cells were washed  <dig> times with d-pbs before released into fresh dmem medium for another 16 h. these cells were washed with d-pbs another  <dig> times and then released into dmem medium with cdk <dig> inhibitor ro- <dig> for 12 h. the arrested cells were then fixed with 1% paraformaldehyde for 15 min and subjected to immunofluorescence staining.
pulldown assay and western blot analysis
to detect the direct interaction of proteins, equal amount purified proteins were co-incubated in buffer  <dig> ,  <dig>  m nacl,  <dig> % np <dig>  1 mm edta, 1 mm pmsf, 20 mm naf,  <dig>  mm na3vo <dig>  1×protease inhibitors) and rotated for 2 h. then the corresponding beads were added and rotated for another 1 h, the supernatant and beads were collected by centrifugation.
the proteins in beads were released by boiling for 10 min in buffer  <dig>  the proteins from supernatant and the boiled beads were resolved in 10% sds-page gel and transferred to nitrocellulose membrane.
primary and secondary antibodies were applied sequentially.
primary antibodies anti-α gapdh , anti-mps <dig> nt  and anti-6×his tag  were prepared at a 1: <dig> dilution.
the blots were developed in super signal westdura  according to the manufacturer’s instruction.
immunofluorescence
cells for immunofluorescence were grown on cover glasses.
prior to staining, cells were treated with chemicals as indicated both in duration and doses.
cells were washed  <dig> times with d-pbs and fixed for 10 min in d-pbs plus 1% paraformaldehyde.
anti-γ-tubulin  and anti-mps <dig> nt  was prepared at a 1: <dig> and 1: <dig> dilutions respectively.
the cells were stained with primary antibody for  <dig>  h at room temperature, followed by secondary antibodies conjugated with alexa fluor 488-conjugated goat anti-mice secondary antibodies .
to stain dna, 50 μg/ml propidium plus rnaase a or  <dig>  μg/ml dapi in d-pbs was used.
after staining, the coverglasses were mounted onto pre-cleaned microscope slides with d-pbs containing 50% glycerol and sealed with nail oil.
images were acquired on a zeiss lsm  <dig> equipped with a  <dig> × objective lens.
RESULTS
crm <dig> binds to and exports mps <dig> from the nucleus after mitosis
mps <dig> dominantly resides in the cytoplasm and relocates into the nucleus at the g2/m boundary  <cit> .
for the first time, it has been found that mps <dig> can be excluded from the nucleus gradually as the nuclear envelope reforms after mitosis .
cytoplasmic retardation of endogenous mps <dig> in the colon cancer cell line sw <dig> requires crm <dig>  as treating cells with crm <dig> inhibitor lmb can block mps <dig> nuclear transport .
a similar result was also achieved by using stable sw <dig> cells expressing an yfp fused mps <dig> .
these findings suggested that cytoplasmic retardation of mps <dig> is due to the crm1-mediated passive exclusion process.
to determine that crm <dig> directly regulates mps <dig> translocation, the association of mps <dig> with crm <dig> was examined.
293 t cells were co-transfected with pexl-fth-crm <dig> and prk5-myc-mps <dig> and collected for immuno-precipitation assay after 48 h. as shown in figure 1d, crm <dig> can bind to endogenous mps <dig>  to determine whether mps <dig> can bind to crm <dig> directly, an in vitro interaction of mps <dig> and crm <dig> was examined using purified proteins.
gst-tagged mps <dig> was purified from 293 t cells after a pfast-gst-mps <dig> baculovirus infection for 48 h. 6× his-tagged crm <dig> was expressed and purified from e. coli.
these  <dig> proteins were incubated with gst beads at room temperature for 2 h, followed by separating the supernatant and beads via centrifugation.
beads were washed 3× and the proteins on beads were subject to a western blot analysis.
as showed in figure 1e, a physical association of mps <dig> with crm <dig> was observed.
a reciprocal interaction was also performed.
as shown, his-crm <dig> can interact with a non-tag mps <dig> .
based on these findings, it was concluded that crm <dig> binds mps <dig> and may affect the nuclear export of mps <dig> directly.figure  <dig>
crm <dig> binds to and exports mps <dig> from the nucleus after mitosis.
export of mps <dig> from nucleus upon mitosis completion.
sw <dig> cells were arrested at prometaphase via 100 ng/ml nocodazole treatment and released into fresh medium for the duration indicated, before being fixed for immunofluorescence staining with anti-mps <dig> n <dig> antibody.
dna was counterstained with dapi.
mps <dig> relocated into the nucleus upon lmb treatment.
asynchronized sw <dig> cells were treated with 10 μm lmb and fixed for immunofluorescence staining at the indicated time points.
yfp-fused mps <dig> show same pattern in sw <dig> upon lmb treatment.
asynchronized sw <dig> cells expressing yfp-mps <dig> were treated with 10 μm lmb and fixed for immunofluorescence staining at the indicated time points.
interaction of mps <dig> and crm <dig>  293 t cells were transiently co-transfected with pexl-fth-crm <dig> and prk5-myc-mps <dig> and collected for the immuno-precipitation assay.
nonspecific binding product is indicated as symbol *.
reciprocal association of mps <dig> and crm1: the bead bound gst-mps <dig> was co-incubated with his-crm <dig> for 3 hrs in lysis buffer and then was collected by centrifuge.
the bead bound protein and the proteins in supernatant were examined by western-blot with antibodies indicated.
reciprocally, bead bound his-crm <dig> was incubated with a non-tagged mps <dig>  the interaction was also determined in the same standard protocol.
dapi, 4′,6-diamidino-2-phenylindole.
mps <dig> bears a putative nuclear export sequence
crm <dig> carries its cargo proteins through binding to a nuclear export sequence.
given the direct interaction of mps <dig> and crm <dig>  it was speculated that mps <dig> bears the nuclear export sequence.
to uncover the putative nes, the mps <dig> protein sequence was analyzed via  <dig> online software programs netnes  and elm .
as shown in figure 2a, only  <dig> putative nes was revealed in both elm and netnes.
to validate these prediction sequences, these putative nes sequences were fused to the n-terminal of enhanced-green fluorescence protein  and the distribution of the resulted fusion protein was examined after a transient transfection of 293 t cells.
only the pnes <dig> fused egfp resided in the cytoplasm while the other  <dig> pnes fusion proteins showed a similar distribution pattern as that of the egfp.
the cytoplasmic distribution of pnes1-egfp appears to rely on crm <dig>  as the treatment of cells with lmb caused the nuclear enrichment of pnes1-fused egfp .
the pnes <dig> seemed to be a classic nes sequence as the leucines enriched pattern is conserved when aligned to the classic nes sequence of cyclin b <dig>  hiv rev and mapk .
notably, replacement of these conserved leucines with alanine caused re-localization of egfp into the nucleus .
based on these findings, it is thought that pnes <dig> in mps <dig> is a functional nes.figure  <dig>
mps <dig> bears a putative nuclear export sequence.
prediction of nuclear export sequence in mps <dig> via  <dig> online procedures.
mps <dig> protein sequence was loaded into procedures according to the protocol by software developer.
the resulted nuclear sequences were showed as indicated.
functional validation of the putative nes.
the putative sequences were fused with egfp in frame and the resulted constructs were transfected into 293 t cells.
the subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated.
dna was counterstained with pi.
the ratio of fusion proteins in the nucleus and cytoplasm were measured via image j and the statistical result of these were generated by using graphpad software.
the amino acids alignment of nes motifs.
the pnes <dig> of mps <dig> is aligned with the classical nes from mapkk, cyclin b <dig>  hiv-rev.
the letters in red indicate identical or similar amino acids.
pi, propidium iodide.
mps <dig> with all leucines substituted in pnes <dig> reside in the cytoplasm
to further validate that pnes <dig> is a functional nes, the subcellular distribution of mps <dig> mutants, in which  <dig> isoleucine and  <dig> leucines were replaced with alanines  was determined.
the resulted fusion protein yfpmps1mutnes <dig> was forcibly expressed in 293 t cells and the subcellular distribution was examined under the indications as shown .
unexpectedly, the mps1mutnes <dig> did not reside in the nucleus, even in the presence of lmb , suggesting that pnes <dig> may be not sufficient for nuclear export, or that this mutation may affect both the nuclear import and export of mps <dig> in interphase.figure  <dig>
mps <dig> with all leucines substituted in pnes <dig> reside in the cytoplasm.
schematic of mps <dig> mutant with mutated pnes <dig> .
the lysines in red were mutated to alanines .
the sequencing result of the mutated pnes <dig> .
the subcellular distribution of yfp fused mps <dig> and mps1mutnes <dig> in presence of lmb and dmso.
the quantitative result of fusion distribution was generated as in figure 2c.
western blot of 293 t cells shows the expression of yfpmps <dig> and yfpmps1mutnes <dig> in sw <dig> cell lines.
mps <dig> with one leucine substituted in pnes <dig> relocate to the nucleus
mps <dig> moves from the cytosol to the nucleus at the g2/m boundary prior to the nuclear envelope breakdown  and this process depends on  <dig> lxxll motifs which contribute to the partial tetratricopeptide repeat domain  in the n-terminus of mps <dig> <cit> .
given that the pnes <dig> is adjacent to the tpr domain , it was proposed that the mutation of pnes <dig> may alter the conformation of these peptides adjacent to the lxxll motifs.
in this case, the mps1mutpnes <dig> would fail to get into the nucleus at the g2/m boundary.
to test this theory, it was observed that the distribution of yfp fused mps <dig> and mps1mutnes <dig> was expressed constantly in sw <dig> and was arrested at the g <dig> phase by sequentially treating cells with thymidine and cdk <dig> inhibitor ro- <dig>  consistent with the previous report  <cit> , the mps <dig> mutant loss of  <dig> lxxll motifs in the a domain  failed to get into the nucleus at late g <dig> phase as compared with mps <dig>  as a control, the nonphosphorylation mimics of mps <dig> at s <dig> , which is phosphorylated by map kinase, was translocated into the nucleus at the g2/m boundary  <cit> .
interestingly, mps1mutnes <dig> showed an impaired nuclear localization , showing that all leucine substitutions affect the nuclear localization of mps <dig>  in contrast, mps1mutnes <dig> localized to the centrosome as efficiently as wild type mps <dig> , suggesting the nuclear entry inefficiency is not due to the misfolding of the n-terminal.
to examine the amino acids essential for mps <dig> nuclear export, yfp fused mps <dig> mutants with only  <dig> mutated leucine were generated and evaluated for the efficiency of nuclear entry.
notably, these mutants show the diverse localization pattern: mutation  <dig> of the forward isoleucine  or leucine  predominantly resides in cytoplasm while the mutants with  <dig> of the last  <dig> leucines replaced  show an apparent nuclear localization .
similar result was obtained when sw <dig> cells expressing these mutants were arrested at the late g <dig> phase by using cdk <dig> inhibitor ro- <dig> .
these findings suggest the pnes <dig> is a functional nuclear export sequence and mutation of the pnes <dig> sequences affect the overall structure integrity of mps <dig> and thereby the distribution in interphase.figure  <dig>
mps <dig> with a single leucine substituted in pnes <dig> relocate to the nucleus.
pnes <dig> motif is adjacent to the lxxll motifs within tpr domain.
the subcellular distribution of wild type mps <dig>  mps <dig> s821a, mps1mutnes <dig>  mps1deltaa domain at g2/m boundary.
the centrosome localization of wild type mps <dig> and mps1mutnes.
the subcellular distribution of wild type mps <dig>  mps1mutnes1- <dig>  mps1mutnes1- <dig>  mps1mutnes1- <dig>  mps1mutnes1- <dig> in interphase.
discussion and
CONCLUSIONS
mps <dig> predominantly resides in cytoplasm during interphase and then translocates into the nucleus at the g2/m boundary  <cit> .
these observations reveal another mps <dig> translocation process in interphase.
upon completion of mitosis, mps <dig> was gradually excluded from the nucleus, mediated by mps1/crm <dig> interaction.
furthermore, a putative nes motif at the n-terminal of mps <dig> was identified.
fusion of this motif with egfp causes the latter to be restrained in the cytoplasm.
in addition, it was found that substitution of  <dig> leucine is sufficient to relocate the mps <dig> into the nucleus although the complete substitution of leucine actually hampers this effect.
this nes, however, seems to be not the only mediator for mps <dig> export after mitosis as not all mps <dig> accumulate in the nucleus, suggesting that another layer of regulation mechanisms is also involved in this process.
tp <dig> is an attractive candidate- this can bind mps <dig> in cases of dna damage response  <cit> .
nuclear export of tp <dig> is mediated by mdm <dig> which bears a classic nes  <cit> .
as a result, a straightforward hypothesis is that mps <dig> may be exported into cytoplasm by interacting with other proteins, such as tp <dig>
the n-terminal of mps <dig> is essential several versatile roles, including kinetochore localization, protein instability and nuclear translocation  <cit> .
recently, the crystal structure of the mps <dig> n-terminus was solved independently by  <dig> groups  <cit> .
the mps <dig> n-terminal is characterized with a tetratricopeptide repeat  domain, followed by the pnes <dig>  due to lxxll motifs being protein-protein interaction domains but not classic nuclear localization sequences  <cit> , it is speculated that the disruption of pnes <dig> may affect the nuclear import of mps <dig> in interphase.
consistent with this hypothesis, the nuclear translocation of mps1mutnes <dig> is impaired at the g2/m boundary, showing that the disruption of pnes <dig> integrity also affects mps <dig> nuclear translocation.
it is noted that the pnes <dig> sequence of mps <dig> is not orthologically conserved among human, mouse, frog and zebrafish .it is speculated that beside pnes <dig>  other unidentified sequence elements are involved in cytoplasmic restraint of mps <dig> in interphase.
the results reveal that mps <dig> is a novel target for crm <dig>  nevertheless, the function underlying the association of mps <dig> with crm <dig> has been unclear so far.
as well as mps <dig>  crm <dig> also regulates the subcellular distribution of several other mitotic regulators in interphase, including cyclin b, aurora b and aurora a  <cit> .
these substrates are crucial for several aspects of mitosis, including mitotic spindle formation, nuclear envelope breakdown, chromosomal alignment and sac generation.
crm <dig> is also an active mitotic regulator: including the recruitment of mad <dig> to the kinetochore, promotion of spindle assembly and regulation of mitotic progression and chromosome segregation  <cit> .
recently, it was found that crm <dig> may also regulate mitotic progression via mps <dig> as lmb treatment can promote kinetochore recruitment of mps <dig> through mitosis .
collectively, the results here add new regulatory roles of crm <dig> in mps <dig> translocation during interphase.
additional files
additional file 1: table s <dig>  the primers used in this paper.
additional file 2: figure s <dig>  plasmids bearing pnes1-egfp mutant as indicated were transfected to sw <dig> and imaged for the localization of fusion protein after 48 hours.
additional file 3: figure s <dig>  subcellular distribution of yfp-mps <dig> pnes <dig> mutants in sw <dig> cells arrested at the late g <dig> phase.
additional file 4: figure s <dig>  the alignment of mps <dig> orthologue from mouse, human, frog and zerafish.
the alignment process was conducted by dnassist software and presented by photoshop software.
abbreviations
mps1mono-polar spindles-1
pnesputative nuclear export sequence
sacspindle assembly checkpoint
nebnuclear envelope breakdown
tprtetratricopeptide repeat domain
apc/canaphase promoting complex/cyclosome
crm1chromosome region maintenance 1
vdac3voltage-dependent anion-selective channel protein 3
markmicrotubule affinity-regulating kinase
lmbleptomycin b
dapi <dig>  6-diamidino-2-phenylindole
egfpenhanced green fluorescent portein
yfpyellow fluorescent protein
hivhuman immunodeficiency virus
cdk1cyclin-dependent kinase 1
gstglutathione s transferase
gapdhglyceraldehyde-3-phosphate dehydrogenase
sds-pagesodium dodecyl sulfate-polyacrylamide gelelectrophoresis
haiwei jia, xiaojuan zhang and wenjun wang contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
hj, xj and ww carried out experiments and analyses.
xj and qx participated in drafting and revising the manuscript.
yl and yb carried out the immunoassays.
xw participated in the design of the study and performed the statistical analysis.
qx, cc, hz and rzm conceived of the study and participated in its design and coordination.
all authors read and approved the final manuscript.

 mps, a serine/threonine kinase, plays multiple functions in the cell cycle. in mitosis, mps is indispensable for both the spindle assembly checkpoint and chromosomal alignment. mps activates sac by directing the assembly of the mitotic checkpoint complex. the resulting complex binds and inactivates apc/c-mediated proteolysis of cyclin b and securin. mps can phosphorylate borealin to regulate chromosomal alignment independently of sac. mps also affects centrosome duplication by collaborating with substrates, including mortalin, centrin and vdac. in interphase, mps interacts with p and chk and is involved in dna damage response. the subcellular distribution of mps correlates with the functions that it performs in the cell cycle. during mitosis, mps appears on the kinetochore at prophase, stays consistently until metaphase initiation, and then comes off at metaphase upon mitotic entry. kinetochore association of mps is regulated by hec and aurora b. the timely release of mps from kinetochore is required for mitotic progression. in interphase, mps resides in the cytoplasm, nuclear envelope and centrosomes. translocation of mps from cytoplasm to nucleus at the g2/m boundary requires two lxxll motifs at the n-terminal. the centrosome localization of mps is required for centrosome duplication and in the case of dna damage, mps is also recruited to dna damage foci, showing that mps plays a role in dna damage response. the regulatory mechanism retarding mps at cytoplasm is unclear so far. in this paper, a putative nuclear export sequence motif at the n-terminal of mps was identified and this motif shows a high sequence similarity to the classic nes, exporting egfp out of the nucleus in cases of fusion. unexpectedly, replacing isoleucine and leucines in this pnes with alanines did not cause constitutive nuclear enrichment of mps further studies suggest that the amino acid mutation affects mps nuclear translocation at the g2/m boundary. consistent with this finding, the mps mutant with of the last leucines is predominantly cytoplasmic while the other mutants behave as with the amino acid mutation. collectively, the observations showed that the pnes sequence at the n-terminal is a functional motif which is sufficient for driving mps nucleus export during interphase. methods dna constructs and stable cell lines generation retroviral expression vector prex-yfp-mps1-ires-hygro in the prex background has been described previously. constructs prex-egfp-ires-hygro, prk5-myc-mps pexl-fth-crm pgst-4 t-mps pexl-fth-crm were generated in a standard way. to generate prex-pness-egfp-ires-hygro, the coding sequences for the putative ness were achieved by annealing pairs of complementary oligo-nucleotide primers and then they were sub-cloned into the n-terminal of egfp. to introduce the pnes mutations in mps construct prex-yfp-mps1-ires-hygro was subject to a standard quickchange mutagenesis procedure by pcr amplication with pairs of oligos sequentially. primers used in this paper are showed in additional file 1: table s all constructs were confirmed by dna sequencing. the procedure for generation of sw stable cell lines expressing various mps mutants is described. protein expression and purification to prepare protein from e.coli, the construct pqe60-crm was transformed into bl and then induced with  mm iptg at 22°c overnight. the cell pellet was washed and suspended in binding buffer, followed by a regular sonication procedure. the cell debris was removed by centrifugation at  g for 30 min. the supernatant was then applied to ni-charged chelating-sepharose and proteins were purified according to the protocol by the manufacturer. to produce the purified proteins from mammalian cells, plasmids were transciently transfected into 293 t cells. after 48 hrs, cells were washed by d-pbs twice and then subject to lysis buffer.then the supernatant was directly applied to ni-charged chelating-sepharose or co-incubated with c-myc agarose affinity beads for immunoprecipitation depending on the tag of proteins fused. fusion proteins were quantified by bca assay and the purity was confirmed by sds-page analysis. cell synchronization synchronization of cells at g1/s phase was accomplished by sequential double thymidine and ro treatment as described by zhang et al.. briefly, cells were arrested at g1/s phase by treatment with 2 mm thymidine for 19 h and cells were washed times with d-pbs before released into fresh dmem medium for another 16 h. these cells were washed with d-pbs another times and then released into dmem medium with cdk inhibitor ro- for 12 h. the arrested cells were then fixed with 1% paraformaldehyde for 15 min and subjected to immunofluorescence staining. pulldown assay and western blot analysis to detect the direct interaction of proteins, equal amount purified proteins were co-incubated in buffer,  m nacl, % np 1 mm edta, 1 mm pmsf, 20 mm naf,  mm na3vo 1×protease inhibitors) and rotated for 2 h. then the corresponding beads were added and rotated for another 1 h, the supernatant and beads were collected by centrifugation. the proteins in beads were released by boiling for 10 min in buffer the proteins from supernatant and the boiled beads were resolved in 10% sds-page gel and transferred to nitrocellulose membrane. primary and secondary antibodies were applied sequentially. primary antibodies anti-α gapdh, anti-mps nt and anti-6×his tag were prepared at a 1: dilution. the blots were developed in super signal westdura according to the manufacturer’s instruction. immunofluorescence cells for immunofluorescence were grown on cover glasses. prior to staining, cells were treated with chemicals as indicated both in duration and doses. cells were washed times with d-pbs and fixed for 10 min in d-pbs plus 1% paraformaldehyde. anti-γ-tubulin and anti-mps nt was prepared at a 1: and 1: dilutions respectively. the cells were stained with primary antibody for  h at room temperature, followed by secondary antibodies conjugated with alexa fluor 488-conjugated goat anti-mice secondary antibodies. to stain dna, 50 μg/ml propidium plus rnaase a or  μg/ml dapi in d-pbs was used. after staining, the coverglasses were mounted onto pre-cleaned microscope slides with d-pbs containing 50% glycerol and sealed with nail oil. images were acquired on a zeiss lsm equipped with a × objective lens.  crm binds to and exports mps from the nucleus after mitosis mps dominantly resides in the cytoplasm and relocates into the nucleus at the g2/m boundary. for the first time, it has been found that mps can be excluded from the nucleus gradually as the nuclear envelope reforms after mitosis. cytoplasmic retardation of endogenous mps in the colon cancer cell line sw requires crm as treating cells with crm inhibitor lmb can block mps nuclear transport. a similar result was also achieved by using stable sw cells expressing an yfp fused mps. these findings suggested that cytoplasmic retardation of mps is due to the crm1-mediated passive exclusion process. to determine that crm directly regulates mps translocation, the association of mps with crm was examined. 293 t cells were co-transfected with pexl-fth-crm and prk5-myc-mps and collected for immuno-precipitation assay after 48 h. as shown in figure 1d, crm can bind to endogenous mps to determine whether mps can bind to crm directly, an in vitro interaction of mps and crm was examined using purified proteins. gst-tagged mps was purified from 293 t cells after a pfast-gst-mps baculovirus infection for 48 h. 6× his-tagged crm was expressed and purified from e. coli. these proteins were incubated with gst beads at room temperature for 2 h, followed by separating the supernatant and beads via centrifugation. beads were washed 3× and the proteins on beads were subject to a western blot analysis. as showed in figure 1e, a physical association of mps with crm was observed. a reciprocal interaction was also performed. as shown, his-crm can interact with a non-tag mps. based on these findings, it was concluded that crm binds mps and may affect the nuclear export of mps directly.figure crm binds to and exports mps from the nucleus after mitosis. export of mps from nucleus upon mitosis completion. sw cells were arrested at prometaphase via 100 ng/ml nocodazole treatment and released into fresh medium for the duration indicated, before being fixed for immunofluorescence staining with anti-mps n antibody. dna was counterstained with dapi. mps relocated into the nucleus upon lmb treatment. asynchronized sw cells were treated with 10 μm lmb and fixed for immunofluorescence staining at the indicated time points. yfp-fused mps show same pattern in sw upon lmb treatment. asynchronized sw cells expressing yfp-mps were treated with 10 μm lmb and fixed for immunofluorescence staining at the indicated time points. interaction of mps and crm 293 t cells were transiently co-transfected with pexl-fth-crm and prk5-myc-mps and collected for the immuno-precipitation assay. nonspecific binding product is indicated as symbol *. reciprocal association of mps and crm1: the bead bound gst-mps was co-incubated with his-crm for 3 hrs in lysis buffer and then was collected by centrifuge. the bead bound protein and the proteins in supernatant were examined by western-blot with antibodies indicated. reciprocally, bead bound his-crm was incubated with a non-tagged mps the interaction was also determined in the same standard protocol. dapi, 4′,6-diamidino-2-phenylindole. mps bears a putative nuclear export sequence crm carries its cargo proteins through binding to a nuclear export sequence. given the direct interaction of mps and crm it was speculated that mps bears the nuclear export sequence. to uncover the putative nes, the mps protein sequence was analyzed via online software programs netnes and elm. as shown in figure 2a, only putative nes was revealed in both elm and netnes. to validate these prediction sequences, these putative nes sequences were fused to the n-terminal of enhanced-green fluorescence protein and the distribution of the resulted fusion protein was examined after a transient transfection of 293 t cells. only the pnes fused egfp resided in the cytoplasm while the other pnes fusion proteins showed a similar distribution pattern as that of the egfp. the cytoplasmic distribution of pnes1-egfp appears to rely on crm as the treatment of cells with lmb caused the nuclear enrichment of pnes1-fused egfp. the pnes seemed to be a classic nes sequence as the leucines enriched pattern is conserved when aligned to the classic nes sequence of cyclin b hiv rev and mapk. notably, replacement of these conserved leucines with alanine caused re-localization of egfp into the nucleus. based on these findings, it is thought that pnes in mps is a functional nes.figure mps bears a putative nuclear export sequence. prediction of nuclear export sequence in mps via online procedures. mps protein sequence was loaded into procedures according to the protocol by software developer. the resulted nuclear sequences were showed as indicated. functional validation of the putative nes. the putative sequences were fused with egfp in frame and the resulted constructs were transfected into 293 t cells. the subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated. dna was counterstained with pi. the ratio of fusion proteins in the nucleus and cytoplasm were measured via image j and the statistical result of these were generated by using graphpad software. the amino acids alignment of nes motifs. the pnes of mps is aligned with the classical nes from mapkk, cyclin b hiv-rev. the letters in red indicate identical or similar amino acids. pi, propidium iodide. mps with all leucines substituted in pnes reside in the cytoplasm to further validate that pnes is a functional nes, the subcellular distribution of mps mutants, in which isoleucine and leucines were replaced with alanines was determined. the resulted fusion protein yfpmps1mutnes was forcibly expressed in 293 t cells and the subcellular distribution was examined under the indications as shown. unexpectedly, the mps1mutnes did not reside in the nucleus, even in the presence of lmb, suggesting that pnes may be not sufficient for nuclear export, or that this mutation may affect both the nuclear import and export of mps in interphase.figure mps with all leucines substituted in pnes reside in the cytoplasm. schematic of mps mutant with mutated pnes. the lysines in red were mutated to alanines. the sequencing result of the mutated pnes. the subcellular distribution of yfp fused mps and mps1mutnes in presence of lmb and dmso. the quantitative result of fusion distribution was generated as in figure 2c. western blot of 293 t cells shows the expression of yfpmps and yfpmps1mutnes in sw cell lines. mps with one leucine substituted in pnes relocate to the nucleus mps moves from the cytosol to the nucleus at the g2/m boundary prior to the nuclear envelope breakdown and this process depends on lxxll motifs which contribute to the partial tetratricopeptide repeat domain in the n-terminus of mps. given that the pnes is adjacent to the tpr domain, it was proposed that the mutation of pnes may alter the conformation of these peptides adjacent to the lxxll motifs. in this case, the mps1mutpnes would fail to get into the nucleus at the g2/m boundary. to test this theory, it was observed that the distribution of yfp fused mps and mps1mutnes was expressed constantly in sw and was arrested at the g phase by sequentially treating cells with thymidine and cdk inhibitor ro- consistent with the previous report, the mps mutant loss of lxxll motifs in the a domain failed to get into the nucleus at late g phase as compared with mps as a control, the nonphosphorylation mimics of mps at s, which is phosphorylated by map kinase, was translocated into the nucleus at the g2/m boundary. interestingly, mps1mutnes showed an impaired nuclear localization, showing that all leucine substitutions affect the nuclear localization of mps in contrast, mps1mutnes localized to the centrosome as efficiently as wild type mps, suggesting the nuclear entry inefficiency is not due to the misfolding of the n-terminal. to examine the amino acids essential for mps nuclear export, yfp fused mps mutants with only mutated leucine were generated and evaluated for the efficiency of nuclear entry. notably, these mutants show the diverse localization pattern: mutation of the forward isoleucine or leucine predominantly resides in cytoplasm while the mutants with of the last leucines replaced show an apparent nuclear localization. similar result was obtained when sw cells expressing these mutants were arrested at the late g phase by using cdk inhibitor ro-. these findings suggest the pnes is a functional nuclear export sequence and mutation of the pnes sequences affect the overall structure integrity of mps and thereby the distribution in interphase.figure mps with a single leucine substituted in pnes relocate to the nucleus. pnes motif is adjacent to the lxxll motifs within tpr domain. the subcellular distribution of wild type mps mps s821a, mps1mutnes mps1deltaa domain at g2/m boundary. the centrosome localization of wild type mps and mps1mutnes. the subcellular distribution of wild type mps mps1mutnes1- mps1mutnes1- mps1mutnes1- mps1mutnes1- in interphase. discussion and  mps predominantly resides in cytoplasm during interphase and then translocates into the nucleus at the g2/m boundary. these observations reveal another mps translocation process in interphase. upon completion of mitosis, mps was gradually excluded from the nucleus, mediated by mps1/crm interaction. furthermore, a putative nes motif at the n-terminal of mps was identified. fusion of this motif with egfp causes the latter to be restrained in the cytoplasm. in addition, it was found that substitution of leucine is sufficient to relocate the mps into the nucleus although the complete substitution of leucine actually hampers this effect. this nes, however, seems to be not the only mediator for mps export after mitosis as not all mps accumulate in the nucleus, suggesting that another layer of regulation mechanisms is also involved in this process. tp is an attractive candidate- this can bind mps in cases of dna damage response. nuclear export of tp is mediated by mdm which bears a classic nes. as a result, a straightforward hypothesis is that mps may be exported into cytoplasm by interacting with other proteins, such as tp the n-terminal of mps is essential several versatile roles, including kinetochore localization, protein instability and nuclear translocation. recently, the crystal structure of the mps n-terminus was solved independently by groups. the mps n-terminal is characterized with a tetratricopeptide repeat domain, followed by the pnes due to lxxll motifs being protein-protein interaction domains but not classic nuclear localization sequences, it is speculated that the disruption of pnes may affect the nuclear import of mps in interphase. consistent with this hypothesis, the nuclear translocation of mps1mutnes is impaired at the g2/m boundary, showing that the disruption of pnes integrity also affects mps nuclear translocation. it is noted that the pnes sequence of mps is not orthologically conserved among human, mouse, frog and zebrafish.it is speculated that beside pnes other unidentified sequence elements are involved in cytoplasmic restraint of mps in interphase. the results reveal that mps is a novel target for crm nevertheless, the function underlying the association of mps with crm has been unclear so far. as well as mps crm also regulates the subcellular distribution of several other mitotic regulators in interphase, including cyclin b, aurora b and aurora a. these substrates are crucial for several aspects of mitosis, including mitotic spindle formation, nuclear envelope breakdown, chromosomal alignment and sac generation. crm is also an active mitotic regulator: including the recruitment of mad to the kinetochore, promotion of spindle assembly and regulation of mitotic progression and chromosome segregation. recently, it was found that crm may also regulate mitotic progression via mps as lmb treatment can promote kinetochore recruitment of mps through mitosis. collectively, the results here add new regulatory roles of crm in mps translocation during interphase. additional files additional file 1: table s the primers used in this paper. additional file 2: figure s plasmids bearing pnes1-egfp mutant as indicated were transfected to sw and imaged for the localization of fusion protein after 48 hours. additional file 3: figure s subcellular distribution of yfp-mps pnes mutants in sw cells arrested at the late g phase. additional file 4: figure s the alignment of mps orthologue from mouse, human, frog and zerafish. the alignment process was conducted by dnassist software and presented by photoshop software. abbreviations mps1mono-polar spindles-1 pnesputative nuclear export sequence sacspindle assembly checkpoint nebnuclear envelope breakdown tprtetratricopeptide repeat domain apc/canaphase promoting complex/cyclosome crm1chromosome region maintenance 1 vdac3voltage-dependent anion-selective channel protein 3 markmicrotubule affinity-regulating kinase lmbleptomycin b dapi 6-diamidino-2-phenylindole egfpenhanced green fluorescent portein yfpyellow fluorescent protein hivhuman immunodeficiency virus cdk1cyclin-dependent kinase 1 gstglutathione s transferase gapdhglyceraldehyde-3-phosphate dehydrogenase sds-pagesodium dodecyl sulfate-polyacrylamide gelelectrophoresis haiwei jia, xiaojuan zhang and wenjun wang contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions hj, xj and ww carried out experiments and analyses. xj and qx participated in drafting and revising the manuscript. yl and yb carried out the immunoassays. xw participated in the design of the study and performed the statistical analysis. qx, cc, hz and rzm conceived of the study and participated in its design and coordination. all authors read and approved the final manuscript. 
additionally, mps mutant loss of pnes integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm. mps kinasesubcellular distributioncrm1nuclear export sequenceissue-copyright-statement© the author 2015 furthermore, a putative nuclear export sequence motif at the n-terminal of mps was identified by analyzing the motif of mps this motif shows a high sequence similarity to the classic nes, a fusion of this motif with egfp results in dramatic exclusion of the fusion protein from the nucleus. taken these findings together, it was concluded that the pnes sequence is sufficient for the mps export from nucleus during interphase. mps an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in dna damage response, cytokinesis and centrosome duplication.
 mps an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in dna damage response, cytokinesis and centrosome duplication. mps predominantly resides in the cytoplasm and relocates into the nucleus at the late g phase. so far, the mechanism underlying the mps translocation between the cytoplasm and nucleus has been unclear.  in this work, a dynamic export process of mps from the nucleus to cytoplasm in interphase was revealed- a process blocked by the crm inhibitor, leptomycin b, suggesting that export of mps is crm dependent. consistent with this speculation, a direct association between mps and crm was found. furthermore, a putative nuclear export sequence motif at the n-terminal of mps was identified by analyzing the motif of mps this motif shows a high sequence similarity to the classic nes, a fusion of this motif with egfp results in dramatic exclusion of the fusion protein from the nucleus. additionally, mps mutant loss of pnes integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm.  taken these findings together, it was concluded that the pnes sequence is sufficient for the mps export from nucleus during interphase. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mps kinasesubcellular distributioncrm1nuclear export sequenceissue-copyright-statement© the author 2015 