many studies have shown that lyric / aeg-1 has a pivotal role in the development and progression of tumourigenesis ( brown and ruoslahti , 2004 ; chen et  al . ; emdad et  al . , 2009 ; hu et  al . , 2009 ; jiang et  al . , 2012 ;
, 2011 ; song et  al . , 2009 ; wang et  al . , 2011 ; xia et  al . ; yoo et  al . , 2009 ; yu et  al . ,
differential lyric / aeg-1 expression and altered localisation has been demonstrated in a variety of cancers , with increased expression frequently linked to a poor prognosis ( chen et  al . ; jiang et  al .
, 2012 ; lee et  al . , 2009 ; li et  al . , 2008 ;
liao et  al . , 2011 ; song et  al . , 2009 ; thirkettle et  al .
, 2011 ; xia et  al . ; yoo et  al . , 2009 ; yu et  al . , 2009 ) .
previous studies have shown that lyric / aeg-1 increases chemo - resistance and cell survival in response to hypoxia and glucose starvation , possibly by initiating protective autophagy ( bhutia et  al . ;
lyric / aeg-1 is regulated by h - ras and pi3k and there is mounting evidence for its role in regulating ubiquitination ( ash et  al . , 2008 ; emdad et  al . , 2006 ;
ubiquitin modifications are known to result in a broad range of responses , dependent upon the number of ubiquitin molecules and their linkage ( denuc and marfany , 2010 ; ikeda and dikic , 2008 ) .
the addition of k48-linked ubiquitin moieties is associated with proteasomal dependent degradation of the substrate protein , whereas k63-linked ubiquitin moieties are associated with endocytosis and dna repair { aguilar and wendland , 2003 # 48}. signalling activation and protein trafficking are more closely associated with monoubiqutination .
ubiquitin monomers can also elicit a response analogous to phosphorylation ( ikeda and dikic , 2008 ) that regulates signalling or sub - cellular localisation of proteins such as pten , p53 and foxo3a ( salmena and pandolfi , 2007 ) .
topors is a unique e3 ligase which can ligate both ubiquitin and small ubiquitin - like modifier ( sumo ) to substrate proteins ( denuc and marfany , 2010 ) .
it contains an n - terminal ring - domain which ligates ubiquitin to its target proteins including dna topoisomerase i , p53 and nkx3.1 ( guan et  al . , 2008 ;
depending on cellular context , topors has been documented as a proto - oncogene or tumour suppressor ( guan et  al .
lyric / aeg-1 contains an usually large number of lysine residues ( > 12% ) with the majority clustered within extended nuclear localisation signal regions ( exnls ) .
these regions have previously been defined as exnls-1 ( aa78130 ) , exnls-2 ( aa415486 ) and exnls-3 ( aa546582 ) and shown to act as nuclear and nucleolar localisation signals ( thirkettle et  al . , 2009a ) .
however , only exnls-2 , which contains 18 lysine residues , was shown to be modified by mono - ubiquitin ( thirkettle et  al . ,
2009a ) causing almost all of lyric / aeg-1 to run at a higher mw of 105  kda .
here we identify the specific sites of lyric / aeg-1 ubiquitination by topors in the exnls-2 region .
mutagenesis of these residues leads to a highly significant reduction in lyric / aeg-1 ubiquitination which results in altered sub - cellular distribution and impairs the interaction between p65 , importin- and lyric / aeg-1 .
gfp - tagged lyric / aeg-1 constructs were created using pqbi25-fc3 plasmid ( q - biogene ) as described ( thirkettle et  al . , 2009a ) .
site directed mutagenesis was performed using quickchange ii site - directed mutagenesis kit ( stratagene ) following the manufacturer 's instructions to create gfp - k486r - lyric / aeg-1 alone , gfp - k491r - lyric / aeg-1 alone or a gfp - k486r / k491r - lyric / aeg-1 double mutant .
stefan weger ( the free university of berlin , germany ) ( weger et  al . , 2005 ) .
cos-7 cells purchased from the cancer research uk cell bank were routinely cultured in dmem ( gibco ) supplemented with 10% fetal bovine serum ( labtech ) . for transfection cells were grown to 40% confluency .
dna ( 1  g/6 wells of a 24 well plate or 6  g / per 14  cm dish ) was transfected using fugene 6 ( roche ) following the manufacturer 's protocol .
cells were grown for a further 48  h prior to confocal microscopy or western analysis .
all western blotting procedures were performed as described ( whitaker et  al . , 2007 ) including the addition of 0.5% ( w / v ) sodium deoxycholate .
membranes were incubated with primary antibody ; anti - gfp ( 1:5000 ) , anti - actin ( 1:5000 ) , anti - p65 ( 1:1000 ) , anti - importin- ( 1:5000 ) ( all from abcam ) , anti - ubiquitin ( 1:1000 , santa cruz biotechnology ) and anti - topors ( 1:1000 , sigma ) .
protein bands were detected with ecl - plus ( ge healthcare ) and where they exceeded the dynamic range of film diaminobenzidine ( vector laboratories ) was used .
immunoprecipitation was performed at 4  c using the method previously described ( thirkettle et  al . , 2009a ) .
for densitometry all available western blots ( n  >  3 ) were scanned using a typhoon scanner ( ge healthcare ) and analysed using imagequant tl ( ge healthcare ) . to overcome any variation in transfection / translation efficiency
the amount of ubiquitination was normalised using gfp - lyric / aeg-1 values ( ubiquitination / gfp - lyric / aeg-1 ) .
proteins were separated using pre - cast 8% gels ( pierce ) and stained using biosafe coomassie ( molecular probes ) .
protein bands were excised and incubated overnight with destain ( 1:1 ratio of acetonitrile and 10  mm triethylammonium bicarbonate ) .
bands were washed in destain , dehydrated and reduced using 10  mm dtt , alkylated with 50  mm iodoacetamide then washed and dehydrated once more .
proteins were digested at 37  c overnight using porcine trypsin ( promega ) ( 10  ng/l ) .
digestion was stopped by flash freezing before the addition of 5% formic acid ( fluka , uk ) and sonication for 15  min , and repeated prior to sample dehydration .
digested samples were resuspended in 8  l 1% formic acid for 20  min prior to analysis using a nano - reverse phase lc- ms using a qtof 6510 mass spectrometer with chip cube source interface and 1200 series hplc running masshunter b.01.03 ( agilent , usa ) .
samples were loaded onto a chip containing integrated 40  nl enrichment and 150  mm analytical columns and electrospray needle using 3  l / min 0.1% formic acid .
nl / min using a linear gradient from 10 to 70% elution buffer ( 80% acetonitrile/20% 0.1% formic acid ( v / v ) ) for 10  min .
after 1  min the gradient was increased to 100% elution buffer and 100% buffer was continued for a further 6  min .
ms data was recorded in the 2902500 m / z range at 6 spectra per second .
ms / ms data was acquired in the 573000 m / z range at 4 spectra per second .
data was searched against uniprot ( kb15.5j ) and the known lyric / aeg-1 constructs using mascot daemon version 2.2.2 ( matrix science , uk ) using a peptide tolerance of 10  ppm and a fragment tolerance of 0.05  da in addition to statistical scoring , the validity of the data was confirmed by manual assessment of the data using scaffold software 2.1 ( proteome science , usa ) .
data were acquired using the agilent 's lc - chip cube- 6510 qtof data - dependent mode with the settings described above with the exception of collecting profile data .
data was acquired randomly as determined by list randomizer ( http://www.random.org/lists/ ) . using trapper version 4.2.0 ( natalie tasman , institute for systems biology , usa ) files were converted to mzxml format and imported into progenesis lc - ms version 2.5.3478.16299 ( nonlinear dynamics , uk ) for lc - ms run alignment and ms peak extraction for label - free quantitation after ion peak identification by mascot .
peaks of interest were analysed and validated using the statistical software r ( version 2.10.1 ) ( team , 2010 ) . to normalize for variability in protein input
the data was analysed using a mixed effects model , where the two fixed effects were gfp - lyric / aeg-1 constructs and ubiquitin . to reflect the pairing of measurements within these fixed effects ( induced by the fact that each replicate provides two measurements ) we also had a single mixed effects term indicating which replicate ion abundance mass spectrometric measurement it came from ( analogous to the paired t - test that would have been carried out for a single fixed effect ) .
analysed data was the log  2 median of the unmodified ubiquitin normalized abundances obtained from progenesis lc - ms label - free quantitation software .
cells were fixed stained and mounted as previously described ( thirkettle et  al . , 2009a ) .
a yeast two - hybrid assay was performed by dualsystems biotech ag , zurich , switzerland using plexa - dir - lyric / aeg-1 aa73 - 582 as bait and a human placental cdna library as described ( thirkettle et  al . , 2009b ) .
previously , we have demonstrated that cytoplasmic lyric / aeg-1 is modified within the exnls-2 region by mono - ubiquitin , leading to a 105  kda mw band and no increase in lyric / aeg-1 degradation ( thirkettle et  al . , 2009a ) .
using immunoprecipitation of gfp - tagged wtlyric / aeg-1 and exnls constructs ( shown in figure  1a ) we have previously demonstrated that deletion of the entire exnls-2 region ( aa415486 ) , and not exnls-1 ( aa78130 ) or exnls-3 ( aa546582 ) , leads to an almost complete loss of lyric / aeg-1 mono - ubiquitination ( thirkettle et  al . , 2009a ) .
we identified two potential specific mono - ubiquitination sites by mass spectrometry at lysine residues k486 and k491 .
k486 lies within the exnls-2 region while k491 lies 5 residues upstream of exnls-2 ( figure  1b , top panel ) . to determine if k486 and k491 are the primary sites of ubiquitin modification single and double point mutations were made to ablate mono - ubiquitination without disrupting the protein charge or the potential to act as an nls ( k486r , k491r and k486/491r ) .
gfp - tagged wild - type and mutant constructs and ubiquitin were over - expressed in mammalian cells and immunoprecipitated using the gfp - tag .
mutation of either k486 or k491 resulted in a significant reduction in lyric / aeg-1 mono - ubiquitination . when both k486 and k491 were mutated mono - ubiquitination was almost entirely ablated ( figure  1b ) . to validate these findings we employed a mass spectrometry quantification method .
ubiquitination was normalised to the amount of gfp - tagged construct using the number of defined gfp peptides per construct as a control for gfp - lyric / aeg-1 input ( figure  1c ) . due to the lysine - rich nature of lyric / aeg-1
the standard mass spectrometry methods were unsuitable and ubiquitination of each construct was quantified using a label - free mass spectrometry and a mixed effects model . using this method the most consistent and robust ubiquitin and gfp peptides that could be detected
were defined and measured for each construct ( shown in supplementary figures s2 and s3 ) . using mass spectrometry
we confirmed that deletion of exnls-2 resulted in a significant loss of ubiquitination compared to wtlyric / aeg-1 ( p  =  0.0012 ) ( figures 1a and 1c ) . when k486 and k491 were modified a highly significant loss of ubiquitination
was seen ; k486r mutant ( p  =  0.0028 ) , k491r mutant ( p  =  0.0004 ) and the k486/491r double point mutant ( p  =  0.0009 ) ( figures 1b and 1c ) .
there was also a small , but not significant , loss of ubiquitination when exnls1/3 was deleted ( p  =  0.43 ) .
we have previously demonstrated that deletion of the entire exnls-2 region had no effect on lyric / aeg-1 function or stability and that treatment with mg132 failed to promote lyric / aeg-1 poly - ubiquitination , both of which are consistent with mono - ubiquitination ( thirkettle et  al . , 2009a ) .
to establish if ubiquitination of lyric / aeg-1 had any effect on its function we examined the interaction between lyric / aeg-1 and p65 , a known interacting protein proposed to shuttle into the nucleus with lyric / aeg-1 ( emdad et  al .
wtlyric / aeg-1 interacts with p65 but this interaction was lost with the non - ubiquitinated k486r / k491r mutant ( figure  2a ) .
mass spectrometry data identified a number of peptides corresponding to importin- , a nuclear import chaperone , suggesting that it may interact with lyric / aeg-1 ( supplementary figure  s4 ) ( harel and forbes , 2004 ) .
this interaction was confirmed by immunoprecipitating gfp - lyric / aeg-1 or the gfp - lyric / aeg-1 k486/491r mutant from cos-7 cells overexpressing the constructs .
importin- was shown to interact with the wtlyric / aeg-1 but only at very low levels in the k486/491r mutant suggesting the ubiquitination of lyric / aeg-1 might regulate its interaction with importin-. using confocal microscopy we examined the localisation of the gfp - tagged lyric / aeg-1 constructs .
previously we have reported that wtlyric / aeg-1 localises throughout the cytoplasm , nucleolus and to a lesser extent the nucleoplasm , as shown by co - localisation with fibrillarin ( figure  3 ) ( thirkettle et  al . , 2009a ) .
all of the lyric / aeg-1 ubiquitin mutants are expressed at lower levels than wtlyric / aeg-1 despite being transfected with identical amounts of dna .
all of these mutants exhibit loss of the wild - type diffuse cytoplasmic and nucleoplasmic localisation . k491r and
all nuclear lyric / aeg-1 in the k486r , k491r and k486r / k491r double mutants resides within the nucleolus and nuclear bodies and is lost from the nucleoplasm .
to identify potential e3 ligases for the ubiquitination of lyric / aeg-1 we used a yeast two - hybrid assay with c - terminal lyric / aeg-1 , ( aa 73582 ) , as bait ( thirkettle et  al .
the screen identified only one e3 ligase , the n - terminal fragment of topors ( aa 45250 ) , containing the ring finger domain , as an lyric / aeg-1 interacting partner ( figure  4a ) ( rajendra et  al . , 2004 ) .
the interaction between wtlyric / aeg-1 was supported using immunoprecipitation of gfp - tagged wtlyric / aeg-1 from cos-7 cells co - transfected with wtgfp - lyric / aeg-1 and topors ( figure  4b ) . to determine if lyric / aeg-1 is a potential substrate for the topors ubiquitin e3 ligase activity we immunoprecipitated wtlyric / aeg-1 from cos-7 cells transfected with an increasing concentration of topors ( figure  4c ) .
western blot analysis showed that the ubiquitination of lyric / aeg-1 increased with increasing topors expression .
we have shown in previous studies using mg132 that ubiquitination of lyric / aeg-1 does not regulate its degradation by the proteasome ( thirkettle et  al . , 2009a ) .
mono - biquitination is known to regulate the localisation and function of a number of proteins including pten , p53 and foxo3a ( denuc and marfany , 2010 ; salmena and pandolfi , 2007 ) .
the large number of functions attributed to lyric / aeg-1 suggests it plays a pivotal role in tumourigenesis ( ash et  al . , 2008 ;
bhutia et  al . ; brown and ruoslahti , 2004 ; emdad et  al . , 2009 ; hu et  al .
, 2009 ; li et  al . , 2012 ; yoo et  al . ) .
previously we have shown that the exnls-2 region of lyric / aeg-1 is modified by mono - ubiquitin , rather than poly - ubiquitin and that this modification does not alter lyric protein stability ( thirkettle et  al . , 2009a ) .
we also saw modification by sumo-1 , but at much lower levels than mono - ubiquitin suggesting that mono - ubiquitin may play a more important role in post - translational modification of lyric / aeg-1 .
deletion of exnls-2 , but not exnls-1 or exnls-3 , results in an abrogation of lyric / aeg-1 modification by mono - ubiquitin ( figure  1a ) .
we have previously reported that lyric / aeg-1 localisation and expression is up - regulated in prostate tumourigenesis supported by reports of overexpression multiple other cancers ( brown and ruoslahti , 2004 ; chen et  al . ;
, 2011 , 2009 , 2008 ; liao et  al . , 2011 ; song et  al . ;
; yoo et  al . , 2009 ; yu et  al . , 2009 ) .
we and others have previously shown that lyric / aeg-1 can move between the cytoplasm and nucleus ( figure  3 ) ( emdad et  al . , 2006 ;
sarkar et  al . , 2008 ; thirkettle et  al . , 2009a ) .
we have demonstrated that shuttling into the nucleus and interaction with proteins known to shuttle into the nucleus is attenuated by mono - ubiquitination of two key lysine regions at k486 and k491 ( figure  3 ) .
mutating k486 and k491 of lyric / aeg-1 resulted in a dramatic effect on its localisation to peri- and sub - nuclear compartments and complete loss of cytoplasmic and nucleoplasmic lyric / aeg-1 ( figure  3 ) .
this suggests the retention of non - ubiquitinated lyric / aeg-1 within inactive sub - cellular compartments where it is unable to bind with known interacting protein p65 and the nuclear import protein importin- ( figure  2 and supplementary figure  s4 ) ( emdad et  al . , 2006 ;
we have shown that mutation of k486 and k491 results in a dramatic loss of lyric / aeg-1 mono - ubiquitination ( figure  1b and c ) .
this suggests that k486 and k491 are the major sites of mono - ubiquitination and that multiple other neighbouring lysine residues do not act co - operatively to compensate for the loss of a single lysine at either k486 or k491 .
although k486 lies with the reported exnls-2 region , k491 lies just outside the exnls-2 region previously identified as the mono - ubiquitinated region of lyric - aeg-1 .
this is consistent either with co - operativity between the k486 and k491 mono - ubiquitination sites or binding of the e3 ligase needed to modify k491 to a site within exnls-2 .
lyric / aeg-1 has previously been demonstrated to interact with brca2 and cdkn1a - interacting protein and indirectly alter its stability ( ash et  al . ,
we identified topors , an oncogene and e3 ligase known to ubiquitinate proteins including p53 , topoisomerase i and ib kinase epsilon ( renner et  al . ) , as an e3 ligase that potentially interacts with lyric / aeg-1 .
topors was shown to interact with lyric / aeg-1 by yeast two - hybrid assay and co - immunoprecipitation figure  4a and b. in addition , increasing amounts of topors alters lyric / aeg-1 ubiquitination in a dose - dependent manner ( figure  4c ) .
lyric / aeg-1 has been implicated in tumour development , progression and treatment resistance ( bhutia et  al . ;
chen et  al . ; hu et  al . , 2009 ; jiang et  al .
, 2012 ; lee et  al . , 2009 ; li et  al . , 2012 , 2008 ;
thirkettle et  al . , 2009a ; wang et  al . , 2011 ; xia et  al .
understanding the mechanism of lyric / aeg-1 modification by ubiquitin and its effects on lyric / aeg-1 sub - cellular trafficking provides new information on the how lyric / aeg-1 function is regulated in cells and offers potential new avenues for therapeutic intervention .
 the pivotal role of lyric / aeg-1 in malignant transformation , tumourigenesis and chemo - resistance has previously been demonstrated in different cell types and sub - cellular compartments . 
 the localisation of lyric / aeg-1 appears crucial to its function and is regulated by three lysine - rich nuclear localisation signal regions , one of which was previously demonstrated to be modified by ubiquitin . here 
 we show that mutation of lyric / aeg-1 at k486 and k491 results in a loss of ubiquitination . 
 a k486/491r double mutant that is incapable of ubiquitination shows reduced binding to the nfb subunit p65 or importin- resulting in a distinctive peri - nuclear localisation of lyric / aeg-1 . 
 we also provide evidence to suggest that topors , an e3 ligase that also regulates p53 modification may be responsible for lyric / aeg-1 ubiquitin modification . 
 overall we demonstrate that specific sites of lyric / aeg-1 ubiquitination are essential for regulating lyric / aeg-1 localisation and functionally interacting proteins . 
Introduction
Materials and methods
Results
Discussion