plasmids and sirnas  plasmids encoding myc - cxcr4 and 
  glutathione s - transferase ( gst)-conjugated cxcr4 c terminus were 
  obtained from dr . gang pei ( shanghai institute of biochemistry and cell 
  biology , chinese academy of sciences ) . the enhanced green fluorescent protein 
  ( egfp)-conjugated cxcr4 was constructed by amplifying the cdnas of cxcr4 from 
  ha - cxcr4 in pcdna3 vector using pcr and inserting the cdna into the xhoi and 
  bamhi sites of the pegfp / n1 vector ( clontech ) . the epitope - tagged cxcr4 has 
  been tested by radioligand binding assay and cyclic amp assay and was 
  confirmed to function similarly as the nontagged receptor ( data not shown ) . 
  the mutant cxcr4 that can not be recognized for ubiquitination ( ha-3k / r cxcr4 ) 
  was obtained from dr . the usp14 
  plasmid was constructed by pcr amplification of usp14 cdna using lymphocyte 
  cdnas as template and inserted into the pcdna3 vector . pre - designed 
  usp14-specific sirna and pre - designed control ( scramble ) sirna were purchased 
  from ambion . massimo loda ( dana farber 
  cancer institute , harvard medical school , boston ) . roger everett ( institute of 
  virology , university of glasgow , scotland , uk ) . the pcdna3-usp7 was 
  constructed by amplifying the cdnas of usp7 in pci vector using pcr and 
  inserting the cdna into the xhoi and bamhi sites of the pcdna3 vector . cell culture and transfection  human embryonic kidney 
  ( hek293 ) and hela cells were grown in dulbecco 's modified eagle 's medium 
  containing 10% fetal bovine serum , 100 units / ml penicillin , and 100 g / ml 
  streptomycin in 5% co2 , 95% air at 37 c . transient transfection of recipient hek293 cells was performed using 
  lipofectamine 2000 ( invitrogen ) . cells stably expressing myc - cxcr4 or 
  egfp - cxcr4 were selected with 560 g / ml geneticin ( g418 ) . hek293 cells stably expressing myc - cxcr4 were transiently 
  transfected with ha - usp14 or usp14-specific sirna , treated with cxcl12 ( 10 
  nm ; pepro - tech , inc . ) for various time intervals , and lysed in 1 ml 
  of ripa buffer containing pbs ( ph.7.0 ) , 0.1% sodium deoxycholate , 0.01% sds , 
  and 1% nonidet p-40 to which additional sds was added to bring the final 
  concentration to 10% . the cell debris was removed by centrifugation 
  ( 13,000  g , 15 min ) . the supernatant was pre - cleared by 
  incubation with 40 l of protein a / g - agarose ( pierce ) for 1 h at 4 c 
  to reduce nonspecific binding . after removing the protein a / g - agarose by 
  centrifugation ( 13,000  g , 1 min ) , the cleared supernatant was 
  collected , and 10 l of mouse monoclonal anti - myc antibody ( santa cruz 
  biotechnology ) was added for an overnight incubation at 4 c . protein a / g 
  ( 40 l ) was then added to this mixture , and the incubation was continued 
  for 2 h at 4 c . the protein a / g - antibody - antigen complex was collected by 
  centrifugation and washing the pelleted resin three times with ice - cold 
  immunoprecipitation buffer . the final pellets were resuspended in 40 l of 
  sds sample buffer containing 5% -mercaptoethanol and heated to 50 c 
  for 10 min . the page - embedded proteins were transferred to nitrocellulose 
  membranes ( bio - rad ) by electrophoretic transfer at 60 v for 60 min at 4 c 
  in a buffer ( 25 mm tris , 192 mm glycine , 20% methanol ) . proteins were identified on the nitrocellulose filter by western blotting , 
  using the following antibodies . cxcr4 was detected using a mouse monoclonal 
  antibody directed against the myc epitope in the n - terminal domain of the 
  receptor . usp2a and usp7 were detected using a rabbit polyclonal antibody ( santa cruz 
  biotechnology , inc . ) usp4 was detected using a rabbit polyclonal antibody 
  ( santa cruz biotechnology ) directed against the his epitope engineered into 
  the n terminus of the protein . in vitro binding assay for protein association with the c terminus of 
  cxcr4for isolating proteins that interact with the cxcr4 c 
  terminus , we exploited a gst - cxcr4 c - terminal fusion protein , using gst alone 
  as a control . for the purification of the gst or gst - cxcr4 c - terminal fusion 
  proteins , dh5 bacteria transformed with plasmids encoding gst or 
  gst - cxcr4 c - terminal fusion proteins were cultured overnight at 37 c . isopropyl d - thiogalactopyranoside was added to the culture to 
  induce protein expression , and incubation was continued for another 3 h. the 
  bacteria were lysed in pbst buffer ( 10 mm sodium phosphate , 2 
  mm potassium phosphate ( ph 7.4 ) , 140 mm nacl , 3 
  mm kcl , 0.1% ( v / v ) tween 20 ) and then probe - sonicated on ice for 10 
  s. the supernatant of the bacterial lysate was incubated with 
  glutathione - sepharose ( pierce / thermo scientific , inc . ) for 30 min at 4 c 
  to isolate gst or the gst - cxcr4 c - terminal fusion protein . after washing three 
  times with pbst buffer , the purified gst- or gst - cxcr4 c - terminal fusion 
  protein - bound beads were resuspended in pbst buffer and stored on ice until 
  use that day . cell lysates that were to be incubated with sepharose - gst or 
  sepharose - gst - cxcr4 c terminus were prepared in the following way . hek293 
  cells not expressing cxcr4 ( and not stimulated with cxcl12 ) but transiently 
  expressing ha - usp14 , ha - usp2a , his - usp4 , or ha - usp7 ( with parental cells 
  serving as controls ) were lysed using pbst buffer ( 10 mm sodium 
  phosphate , 2 mm potassium phosphate ( ph 7.4 ) , 140 mm 
  nacl , 3 mm kcl , 0.1% ( v / v ) tween 20 ) 48 h after transfection . cell 
  lysates were pelleted to remove debris ( 13,000  g , 10 min at 4 
  c in a microcentrifuge ) , and the supernatants of this centrifugation were 
  used for the gst pulldown assay . for the gst pulldown assay , the supernatant of the cell lysates was 
  incubated with aliquots of the purified gst or gst - cxcr4 c - terminal fusion 
  proteins ( to bring the gst fusion protein concentration to 50 g / ml ) for 2 
  h at 4 c with rotation . to terminate the incubation , sepharose beads were 
  pelleted by centrifugation ( 13,000  g , 2 min ) and washed four 
  times with pbst buffer . proteins bound to the c - terminal cxcr4 receptor - gst 
  fusions protein ( or control gst ) were released by boiling in sds - page sample 
  buffer containing 5% -mercaptoethanol for 10 min , resolved by sds - page , 
  and analyzed by western blot , as outlined above . confocal microscopy  our initial studies revealed a 
  selectively greater association of cxcr4 with usp14 compared with other family 
  members ; thus , most of our studies focused on cxcr4 and its association with 
  usp14 . to examine the localization of usp14 compared with cxcr4 in cells 
  expressing both , hek293 cells stably expressing egfp - cxcr4 were transiently 
  transfected with ha - usp14 and grown on coverslips for 1 or 2 days . cells were 
  treated with cxcl12 ( 10 nm ) for various time intervals and fixed 
  with 100% methanol at room temperature before immediate transfer to 4 c 
  until further processing . fixed cells were washed with pbs and incubated with 
  a mouse monoclonal ha antibody ( santa cruz biotechnology ) for 30 min at room 
  temperature . cells were washed with pbs and incubated with a cy3-conjugated 
  anti - mouse antibody ( molecular probes , eugene , or ) for 30 min at room 
  temperature and then washed twice with pbs and then briefly with deionized 
  water to remove buffer salts . confocal microscopy was performed on an lsm-510 laser scanning microscope 
  ( carl zeiss , new york ) with a 63  1.3 numerical aperture oil immersion 
  lens using dual excitation ( 488 nm for egfp and 568 nm 
  for cy3 ) and emission ( 515 - 540 nm for egfp and 590 - 610 
  nm for cy3 ) filter sets . all digital images were captured at the 
  same settings to allow direct quantitative comparison of staining patterns . time course of cxcr4 ubiquitination and deubiquitination  to 
  assess whether ligand activation of cxcr4 altered the ubiquitination of this 
  receptor , hek293 cells stably expressing myc - cxcr4 were incubated with 10 
  nm cxcl12 for the time intervals indicated in the figures and 
  figure legends . the 
  cell debris was removed by centrifugation ( 13,000  g , 15 min ) . the supernatant was pre - cleared by incubation with 40 l of protein 
  a / g - agarose ( pierce ) for 1 h at 4 c to reduce nonspecific binding . after 
  removing the protein a / g - agarose by centrifugation ( 13,000  g , 
  1 min ) , the cleared supernatant was collected , and 10 l of mouse 
  monoclonal myc antibody ( santa cruz biotechnology ) was added for overnight 
  incubation at 4 c . protein a / g ( 40 l ) was then added , and the 
  incubation was continued for 2 h at 4 c . the protein a / g - antibody - antigen 
  complex was then collected by centrifugation , as above , and was washed three 
  times with ice - cold immunoprecipitation buffer . the final pellets were 
  resuspended in 40 l of sds sample buffer containing 5% 
  -mercaptoethanol and heated to 50 c for 10 min . forty microliters 
  of this preparation were separated by 10% sds - page , and the proteins 
  transferred to nitrocellulose membranes ( bio - rad ) , as described in detail 
  above for western blotting . the state of ubiquitination of the cxcr4 receptor 
  was detected by western blot analysis using a rabbit polyclonal anti - ubiquitin 
  antibody ( santa cruz biotechnology ) . two complementary strategies were used to determine the effect of usp14 on 
  cxcr4 modification , i.e. overexpression of usp14 in cxcr4-expressing 
  hek293 cells and rna silencing of endogenous usp14 in cells expressing cxcr4 . 
  for usp14 overexpression , cells were transiently transfected with vector 
  ( control ) or ha - usp14 ( see under  cell culture and transfection  ) 
  and incubated with ligand 48 h later . for knockdown of usp14 , cells were 
  transiently transfected with scrambled sirna ( control ) or usp14-sirna ( ambion , 
  inc . ) . in either case , cells were incubated with cxcl12 for 10 min to assess 
  the impact of ligand on the ubiquitination of cxcr4 . to assess the 
  reversibility of the ubiquitination , another set of cells was incubated for 10 
  min with cxcl12 followed by a 60-min  recovery from stimulation period ( after removal of ligand and replacement of fresh medium after the 10 
  min of stimulation ) . incubations were terminated by cell lysis in 1 ml of 
  ice - cold ripa buffer . receptor degradation assay  to assess the impact of usp14 and 
  reversal of ubiquitination on cxcr4 turnover and degradation , hek293 cells 
  expressing cxcr4 and transiently expressing ha - usp14 ( see figure legends ) were 
  pretreated with cycloheximide ( 5 g / ml ) for 15 min at 37 c to prevent 
  new protein synthesis during the course of our experiments . cells were 
  incubated with cxcl12 ( 10 nm ) for 8 h at 37 c to maximally 
  induce receptor ubiquitination and degradation . to terminate the incubation , cxcr4 was 
  detected by western blot analysis using an anti - cxcr4 antibody ( abcam ) . the 
  blots were stripped and reprobed with anti - tubulin antibody to confirm equal 
  loading ( santa cruz biotechnology ) . to assess the effect of usp14 modulation on endogenous cxcr4 turnover and 
  degradation , hela cells , which endogenously express cxcr4 , were transiently 
  transfected with ha - usp14 or usp14-specific sirna . cells were pretreated with 
  cycloheximide ( 5 g / ml ) for 15 min at 37 c , to prevent new protein 
  synthesis during the course of our experiments , and incubated with cxcl12 ( 10 
  nm ) for varying time points at 37 c ( see figure legends ) . to 
  terminate the incubation , cells were transferred to ice and then lysed in 
  ice - cold ripa buffer . cxcr4 was detected by western blot analysis using 
  an anti - cxcr4 antibody ( abcam ) . the blots were stripped and reprobed with 
  anti - tubulin antibody to confirm equal loading ( santa cruz biotechnology ) . densitometric analysis of western blot bands  the relative 
  amount of all western blot bands was measured using unscan - it gel version 6.1 
  ( silk scientific corp . ) . the relative density of the protein bands was 
  calculated in the area encompassing the immunoreactive protein band following 
  subtraction of the density of the background signal detected in an adjacent 
  area without protein signal in the same lane as the protein of interest . hek293 cells stably expressing myc - cxcr4 were exposed to cxcl12 
  ( 10 nm ) for the indicated time intervals ( a - c ) . myc - cxcr4 
  was immunoprecipitated ( ip ) from cell lysates using a mouse anti - myc 
  antibody ( see  experimental procedures  ) . the amount of 
  co - precipitated usp14 protein was detected by western blotting for the ha 
  epitope on usp14 . the membrane was stripped and reprobed using a rabbit 
  anti - myc antibody to evaluate myc - cxcr4 loading . the migration of molecular 
  weight markers is shown to the left of the gel . b , quantitation of the relative amount of usp14 co - precipitated with 
  cxcr4 was determined by densitometric scanning as outlined under 
   experimental procedures  ; n = 6 . experiments were performed as in 
  a and described in detail under  experimental 
  procedures . there was no detectable interaction of the cxcr4 with usp7 
  ( data not shown ) . n = 4 ) and 
  his - usp4 (  , n = 3 ) were detected using anti - ha and anti - his 
  antibodies ( see  experimental procedures  ) . d , usp14 
  interacts with the c terminus of cxcr4 ; gsh - sepharose - bound gst ( lane 
  2 , control ) or gst - cxcr4 c - terminal fusion protein ( lane 3 ) was 
  incubated with hek293 cell lysates prepared from control ( i.e. not 
  stimulated by cxcl12 ligand ) , as described under  experimental 
  procedures .  upper panel , ha - usp14 was detected by western 
  blotting using an anti - ha antibody . data shown in b and c are mean  
  s.e . from the number of independent experiments outlined above . , p < .05 ; , p < .01 ; , 
  p < .001 , compared with cells not stimulated with cxcl12 
  ( control ) . hek293 cells stably expressing myc - cxcr4 were exposed to cxcl12 
  ( 10 nm ) for the indicated time intervals ( a - c ) . myc - cxcr4 
  was immunoprecipitated ( ip ) from cell lysates using a mouse anti - myc 
  antibody ( see  experimental procedures  ) . the amount of 
  co - precipitated usp14 protein was detected by western blotting for the ha 
  epitope on usp14 . the membrane was stripped and reprobed using a rabbit 
  anti - myc antibody to evaluate myc - cxcr4 loading . the migration of molecular 
  weight markers is shown to the left of the gel . b , quantitation of the relative amount of usp14 co - precipitated with 
  cxcr4 was determined by densitometric scanning as outlined under 
   experimental procedures  ; n = 6 . experiments were performed as in 
  a and described in detail under  experimental 
  procedures . there was no detectable interaction of the cxcr4 with usp7 
  ( data not shown ) . co - precipitated ha - usp2a (  , n = 4 ) and 
  his - usp4 (  , n = 3 ) were detected using anti - ha and anti - his 
  antibodies ( see  experimental procedures  ) . d , usp14 
  interacts with the c terminus of cxcr4 ; gsh - sepharose - bound gst ( lane 
  2 , control ) or gst - cxcr4 c - terminal fusion protein ( lane 3 ) was 
  incubated with hek293 cell lysates prepared from control ( i.e. not 
  stimulated by cxcl12 ligand ) , as described under  experimental 
  procedures .  upper panel , ha - usp14 was detected by western 
  blotting using an anti - ha antibody . data shown in b and c are mean  
  s.e . from the number of independent experiments outlined above . , 
  p < .05 ; , p < .01 ; , p < .001 , compared with cells not stimulated with cxcl12 
  ( control ) . statistical analysis student 's t tests were 
  performed to test statistical significance for the paired data comparisons . analysis of variance tests were used to test the significant differences for 
  the group data comparisons . chemotaxis assay  the migration of hek293 cells stably 
  expressing myc - cxcr4 and transiently expressing vector alone ( control ) , 
  ha - usp14 , usp14-specific sirna , or scrambled ( control ) sirna was evaluated 
  using a chemotaxis assay ( 36 ) . briefly , polycarbonate filters ( 10-m pore size ) coated with 20 g/l 
  human collagen type iv were placed between the upper and lower compartments of 
  the boyden chambers ( neuroprobe , gaithersburg , md ) . the lower compartment of 
  the chamber was loaded with 400-l aliquots of 1 mg / ml ovalbumin / dulbecco 's 
  modified eagle 's medium ( chemotaxis buffer ) or cxcl12 ( 0.01 - 100 nm ) 
  diluted in chemotaxis buffer . cells ( 5  10/100 l ) were 
  loaded into the upper compartment and incubated for 4 h at 37 c in a 5% 
  co2 atmosphere . cells that had migrated through the filter into the 
  bottom chamber of medium or medium containing cxcl12 were counted under the 
  microscope ( 20 objective ) after being stained with a diff - quik kit . the 
  migration index was calculated and was defined as number of cells crossing the 
  filter toward cxcl12 ( various concentrations)/number of cells migrating 
  toward medium alone ( control ) . each experiment was performed at least three 
  times in duplicate . to confirm that the modulation of chemotaxis was being mediated via the 
  receptor itself , and not some other usp14-interacting protein , and to confirm 
  the necessity of a ubiquitination / deubiquitination cycle , we assessed the 
  effect of cxcl12 stimulation on mutant cxcr4-mediated , ha-3k / r cxcr4 , cell 
  migration . as such , the chemotactic index of hek293 cells transiently 
  transfected with ha - wt cxcr4 or ha-3k / r cxcr4 was calculated as described 
  above . cxcl12-induced cxcr4-mediated erk activation  to exploit a 
  possible mechanism for the inhibition of cxcl12-induced cxcr4-mediated cell 
  chemotaxis , we explored possible changes in the activation of erk , a signaling 
  molecule located downstream of cxcr4 activation . hek293 cells transiently 
  transfected with ha - wt cxcr4 or ha-3k / r cxcr4 were stimulated with cxcl12 ( 10 
  nm ) for varying time points ( see figure legends ) at 37 c . stimulation was terminated by transferring cells to ice and then lysed with 
  ice - cold ripa buffer . lysates containing equal amounts of protein were 
  subjected to 10% sds - page , and activated erk was detected using an anti - p - erk 
  antibody ( santa cruz biotechnology ) . the blots were stripped and reprobed with 
  an antibody ( anti - erk2 antibody ; santa cruz biotechnology ) that recognizes erk 
  in both its phosphorylated and nonphosphorylated states to confirm equal 
  loading . an anti - ha antibody ( santa cruz biotechnology ) was also employed to 
  detect the level of expression of ha - cxcr4 . cxcl12 activation leads to a time - dependent association of usp14 with 
  cxcr4a previous series of experiments revealed the possibility 
  that usp14 was a cxcr4-interacting protein 
  ( 37 ) . this study verified that 
  cxcl12 incubation of hek293 cells stably expressing the receptor and 
  transiently expressing ha - usp14 leads to a time - dependent association of usp14 
  with cxcr4 ( fig . usp14 association with cxcr4 was not readily detected before ligand 
  stimulation , but exposure of cxcr4-expressing cells to cxcl12 led to detection 
  of usp14 association with the receptor at the earliest time point evaluated ( 2 
  min ) . association continued for 5 min , after which time detectable 
  receptor - dependent association declined 
  ( fig . it was not 
  necessary to overexpress usp14 to detect this interaction , because a similar 
  time course for cxcl12-induced association of usp14 with myc - cxcr4 was also 
  detected in hek cells expressing endogenous usp14 ( data not shown ) . of further 
  interest was our finding that usp14 interaction with cxcr4 is relatively 
  selective , compared with other members of the usp family , including usp2a , 
  usp4 , and usp7 , which were evaluated using the same experimental strategy 
  ( fig . cells stably expressing egfp - cxcr4 and transiently transfected with ha - usp14 
  were treated without ( control ) or with cxcl12 ( 10 nm ) for the 
  indicated time intervals . the cells were fixed and evaluated using 
  immunohistochemistry via confocal microscopy as detailed under 
   experimental procedures . representative laser - scanning confocal 
  micrographs demonstrating the distribution of egfp - cxcr4 ( green ) , 
  usp14 ( red ) , and overlay ( yellow ) are shown . cells stably expressing egfp - cxcr4 and transiently transfected with ha - usp14 
  were treated without ( control ) or with cxcl12 ( 10 nm ) for the 
  indicated time intervals . the cells were fixed and evaluated using 
  immunohistochemistry via confocal microscopy as detailed under 
   experimental procedures . representative laser - scanning confocal 
  micrographs demonstrating the distribution of egfp - cxcr4 ( green ) , 
  usp14 ( red ) , and overlay ( yellow ) are shown . terminus of cxcr4 has been shown to interact with a variety 
  of intracellular proteins after ligand stimulation 
  ( 32 - 35 , 
  37 ) , we explored whether usp14 
  association with cxcr4 might occur via interactions with the c terminus of the 
  receptor . to test this hypothesis we created a cdna encoding a fusion of the 
  c - terminal domain of the receptor with gst . 1d , the gst - cxcr4 
  c - terminal domain fusion proteins interacted with usp14 when cell lysates 
  expressing usp14 were incubated with gsh - sepharose - gst - cxcr c - terminal fusion 
  protein , whereas no interaction of usp14 occurred with gst alone ( control ) . we 
  did not need to stimulate the hek cells with cxcl12 prior to harvesting the 
  cell lysates to see interaction of the c terminus with usp14 in those lysates , 
  presumably because in these in vitro assays , in contrast to the 
  intact cell assays shown in fig . 1 , 
  a and b , the concentration of the c terminus was 
  high enough to detect this interaction even in the absence of ligand . it is 
  probable that cxcl12 activation of the cxcr4 induces a receptor conformation 
  that preferentially interacts with usp14 via the receptor c terminus . in 
  nonstimulated cells , the c terminus of the membrane - bound receptor may be 
  limiting in concentration , perhaps by steric hindrance in an inactive receptor 
  conformation . however , in in vitro gst - pulldown experiments , the c 
  terminus is freely available and presumably in excess concentrations , such 
  that it is not necessary to have pre - stimulated the cells from which the 
  lysate is derived to see usp14 association with the c terminus of the cxcr4 . the finding , however , that the c terminus of cxcr4 can directly interact with 
  usp14 provides additional confirmation of a direct interaction between this 
  enzyme and cxcr4 . 1c , the cxcr4 c 
  terminus does not interact in a detectable fashion with usp2a , usp4 , or usp7 
  ( data not shown ) . cxcl12 stimulation induces usp14 apparent co - localization with 
  cxcr4to date , the subcellular distribution of usp14 has been 
  limited to proteasomes ( 38 ) 
  and synaptic vesicles ( 39 ) . we 
  were curious whether cxcl12 activation of cxcr4 might lead to redistribution 
  of usp14 to cxcr4-containing membrane compartments , consistent with the 
  ligand- and time - dependent association of usp14 with cxcr4 noted in 
  fig . 1 . cxcl12 treatment of 
  hek293 cells stably expressing egfp - cxcr4 and transiently expressing ha - usp14 
  led to a time - dependent co - localization of these proteins both on the cell 
  surface and in apparently internalized membrane compartments 
  ( fig . this co - localization 
  was more readily detected at 5 min when compared with a 60-min exposure to 
  cxcl12 , which is also consistent with the time course for cxcl12-induced 
  association of cxcr4 with usp14 described in 
  fig . 1 , a and 
  b. in studies not shown here , we observed that ligand 
  stimulation leads to detection of cxcr4 in a variety of rab gtpase - associated 
  compartments , including rab 7 ( late endosomal ) and rab 11a ( recycling 
  endosome ) compartments . reversal of cxcl12-induced cxcr4 ubiquitination is 
  usp14-dependent  although cxcr4 ubiquitination has been detected in 
  cells heterologously expressing ha - ubiquitin , we wanted to establish whether 
  detectable ubiquitination of cxcr4 occurs when only endogenous ubiquitin is 
  available , and what impact usp14 has on this covalent modification . cells 
  stably expressing myc - cxcr4 were treated with cxcl12 for varying times before 
  immunoisolation of myc - cxcr4 ; the extent of ubiquitination of the receptor at 
  these various time points was assessed using an anti - ubiquitin antibody . as 
  shown in fig . 3a , 
  cxcl12 increased the extent of detectable receptor - associated ubiquitin 
  ( upper gel panel ) at a migration in the sds - page that corresponds to 
  45,000 , as well as the laddering of cxcr4 ( bracketing the 54-kda 
  molecular mass marker ) , characteristic of ubiquitinated proteins , including 
  gpcrs ( 40 ) . when we blotted 
  for the myc - cxcr4 with an anti - myc antibody , these laddered proteins were not 
  detectable , suggesting that only a fraction of the expressed receptor was 
  being modified . a , antibody directed 
  against endogenous ubiquitin reveals the time - dependent ubiquitination of 
  myc - cxcr4 ( upper panel ) in response to cxcl12 ( 10 nm ) 
  treatment of hek293 cells ( see  experimental procedures  ) . the 
  lower panel , obtained by reprobing the western blot with an anti - myc 
  antibody directed against the myc - cxcr4 , readily detects the receptor protein 
  migrating at 45,000 , but not the higher molecular mass 
   ladders  at 54 kda and above . b , quantitation of the relative density of 
  bands representing cxcr4-endogenous ub complexes was determined by 
  densitometric scanning (  ; see  experimental procedures  ) . 
  superimposed on these data are the findings from the time course of usp14 
  association with the cxcr4 in cxcl12-exposed cells (  ) , reported in 
  fig . hek293 cells stably expressing myc - cxcr4 
  and transiently transfected with vector alone ( vector ) or ha - usp14 
  were incubated for 10 min with cxcl12 as in a. this incubation was 
  terminated immediately for some samples ( 10 min , no recovery ) but 
  allowed to continue after washing away the cxcl12 , for 60 min ( 10 
  min , + 60 min recovery ) . the gel data shown below the bar 
  graph , from one representative experiment , confirm that transfection of 
  the hek293 cells with the cdna encoding ha - usp14 indeed leads to 
  overexpression of this enzyme . d , rna interference knockdown of 
  endogenous usp14 eliminates deubiquitination of cxcl12-evoked ubiquitination 
  of myc - cxcr4 . hek293 cells transfected with scrambled ( control ) sirna 
  or usp14 sirnas were treated without cxcl12 ( none ) or with 10 
  nm cxcl12 for 10 min ( 10 min , no recovery ) or 10 min 
  followed by a 60-min recovery period ( 10 min , + 60 min 
  recovery ) as described under  experimental procedures . the 
  panel below the bar graph provides a representative gel that confirms 
  the ability of the sirna construct to successfully reduce the expression of 
  the usp14 protein in these cells under these conditions . myc - cxcr4 was 
  isolated by immunoprecipitation with an anti - myc antibody , and the cxcr4-ub 
  complexes were quantified by western blot using an antibody against endogenous 
  ubiquitin . blots were stripped and reprobed using a myc antibody to evaluate 
  loading of myc - cxcr4 . data in b - d represent the mean  s.e . 
  from three independent experiments . , p < 0.05 ; 
  , p < 0.01 ; , p < 0.001 , 
  compared with controls cells ( no cxcl12 treatment ) . a , antibody directed 
  against endogenous ubiquitin reveals the time - dependent ubiquitination of 
  myc - cxcr4 ( upper panel ) in response to cxcl12 ( 10 nm ) 
  treatment of hek293 cells ( see  experimental procedures  ) . the 
  lower panel , obtained by reprobing the western blot with an anti - myc 
  antibody directed against the myc - cxcr4 , readily detects the receptor protein 
  migrating at 45,000 , but not the higher molecular mass 
   ladders  at 54 kda and above . b , quantitation of the relative density of 
  bands representing cxcr4-endogenous ub complexes was determined by 
  densitometric scanning (  ; see  experimental procedures  ) . 
  superimposed on these data are the findings from the time course of usp14 
  association with the cxcr4 in cxcl12-exposed cells (  ) , reported in 
  fig . hek293 cells stably expressing myc - cxcr4 
  and transiently transfected with vector alone ( vector ) or ha - usp14 
  were incubated for 10 min with cxcl12 as in a. this incubation was 
  terminated immediately for some samples ( 10 min , no recovery ) but 
  allowed to continue after washing away the cxcl12 , for 60 min ( 10 
  min , + 60 min recovery ) . the gel data shown below the bar 
  graph , from one representative experiment , confirm that transfection of 
  the hek293 cells with the cdna encoding ha - usp14 indeed leads to 
  overexpression of this enzyme . d , rna interference knockdown of 
  endogenous usp14 eliminates deubiquitination of cxcl12-evoked ubiquitination 
  of myc - cxcr4 . hek293 cells transfected with scrambled ( control ) sirna 
  or usp14 sirnas were treated without cxcl12 ( none ) or with 10 
  nm cxcl12 for 10 min ( 10 min , no recovery ) or 10 min 
  followed by a 60-min recovery period ( 10 min , + 60 min 
  recovery ) as described under  experimental procedures . the 
  panel below the bar graph provides a representative gel that confirms 
  the ability of the sirna construct to successfully reduce the expression of 
  the usp14 protein in these cells under these conditions . cxcr4 was 
  isolated by immunoprecipitation with an anti - myc antibody , and the cxcr4-ub 
  complexes were quantified by western blot using an antibody against endogenous 
  ubiquitin . blots were stripped and reprobed using a myc antibody to evaluate 
  loading of myc - cxcr4 . data in b - d represent the mean  s.e . 
  from three independent experiments . , p < 0.05 ; 
  , p < 0.01 ; , p < 0.001 , 
  compared with controls cells ( no cxcl12 treatment ) . ubiquitination of the cxcr4 is time - dependent , as summarized for multiple 
  experiments in fig . 3b 
  also superimposes the time course for cxcr4 ubiquitination with the time 
  course for cxcr4 association with usp14 (  ) , as already reported above in 
  fig . this 
  comparison indicates that when the receptor association with usp14 begins to 
  decline , receptor ubiquitination continues to increase , as might be expected 
  in a continuing ubiquitination / deubiquitination cycle on the receptor 
  molecule . the data in fig . 3c 
  provide additional evidence for the reversibility of cxcr4 ubiquitination . 
  when stimulation of the receptor with cxcl12 for 10 min was followed by ligand 
  removal , cell washing , and an additional 60-min incubation , we observed 
  decreased ubiquitination of cxcr4 , suggesting an ongoing and parallel process 
  of cxcr4 ubiquitination and deubiquitination 
  ( fig . when usp14 was overexpressed in these 
  cells , no accumulated ubiquitinated cxcr4 was detected , even at the end of the 
  first 10-min incubation with cxcl12 . these data are consistent with the 
  interpretation that not only does usp14 associate with cxcr4 in a 
  ligand - modulated fashion ( as in fig . 
  1 ) , but also that usp14 recognizes this receptor as a substrate 
  for deubiquitination . we used a complementary experimental strategy to test whether endogenous 
  usp14 catalyzes the deubiquitination of cxcr4 . for this purpose , we exploited 
  silencing rna to reduce the expression of endogenous usp14 in our hek293 cells 
  stably expressing the cxcr4 receptor ( fig . cells expressing usp14-specific sirna manifest a 
  greater basal ubiquitination of cxcr4 , as well as a greater ligand - induced 
  ubiquitination of the receptor following a 10-min stimulation with cxcl12 . these data suggest that the usp14 gene product indeed serves as a catalyst to 
  deubiquitinate the cxcr4 , because when its expression is reduced there is a 
  reciprocal increase in cxcr4 ubiquitination . reduced expression of endogenous 
  usp14 ( following sirna exposure ) eliminated the reduction of ligand - evoked 
  ubiquitination of the receptor during the 60-min under  recovery 3 , c and 
  d , support the conclusion that the usp14 gene product 
  endogenously contributes to a ligand - enhanced cxcr4-ub cycle . receptor degradation is regulated by usp14 expression  as for 
  other proteins , ubiquitin has been implicated as a  tag  
  identifying cxcr4 for proteasome- or lysosome - mediated degradation 
  ( 41 ) . deubiquitination of 
  cxcr4 by usp14 would thus be expected to reduce the rate of ligand - accelerated 
  receptor degradation and result in an increased steady state level of 
  receptors . to test this hypothesis , hek293 cells stably expressing egfp - cxcr4 
  and overexpressing usp14 were stimulated with cxcl12 ( 10 nm ) for 0 
  or 8 h ; we chose this 8-h time point because previous studies have shown that 
  prolonged incubation of the cxcr4 with cxcl12 permits more facile detection of 
  ligand - accelerated receptor degradation 
  ( 28 ) . 4a , sustained 
  incubation with cxcl12 led to an 35% decrease in the steady state level 
  of cxcr4 . ( preliminary studies using cycloheximide to block new receptor 
  synthesis resulted in quantitatively indistinguishable findings . ) overexpression of usp14 , however , led to two readily detectable consequences 
  in cxcr4-expressing cells . figure 4.usp14 prevents cxcl12-mediated egfp - cxcr4 degradation and increases the 
  steady state level of the receptor . a , hek293 cells stably 
  expressing egfp - cxcr4 were treated with cxcl12 for 8 h ( + ) ; this prolonged 
  incubation allowed detection of cxcl12-evoked receptor down - regulation . egfp - cxcr4 levels were detected by western blot using an anti - egfp antibody . b , quantitation of the relative amount of cxcr4 was determined by 
  densitometric scanning as outlined under  experimental 
  procedures . data are mean  s.e . from three independent 
  experiments . , p < 0.05 ; compared with control cells 
  ( no cxcl12 treatment ( - ) ) . usp14 prevents cxcl12-mediated egfp - cxcr4 degradation and increases the 
  steady state level of the receptor . a , hek293 cells stably 
  expressing egfp - cxcr4 were treated with cxcl12 for 8 h ( + ) ; this prolonged 
  incubation allowed detection of cxcl12-evoked receptor down - regulation . egfp - cxcr4 levels were detected by western blot using an anti - egfp antibody . b , quantitation of the relative amount of cxcr4 was determined by 
  densitometric scanning as outlined under  experimental 
  procedures . data are mean  s.e . from three independent 
  experiments . , p < 0.05 ; compared with control cells 
  ( no cxcl12 treatment ( - ) ) . degradation of endogenous cxcr4 receptor also is regulated by usp14 
  expression  to expand our findings and assess the effects of usp14 
  modulation on the turnover and degradation of endogenous cxcr4 , we extended 
  our studies to hela cells , which endogenously express the cxcr4 receptor . hela 
  cells were transiently transfected with ha - usp14 or usp14-specific sirna and 
  stimulated with cxcl12 ( 10 nm ) at 37 c for varying time points 
  ( see figure legend ) . as shown in fig . 5a , overexpression of ha - usp14 ( confirmed in the 
  accompanying sds - page ) increased steady state levels of cxcr4 and also blocked 
  cxcl12-evoked down - regulation of cxcr4 levels characteristic of control cells 
  ( vector alone ) . we also explored the impact of reducing usp14 expression in 
  hela cells by transfection with usp14-specific sirna ( confirmed in the 
  accompanying sds - page ) . unexpectedly , reduction of usp14 expression 
  also led to slightly increased steady state levels of cxcr4 and elimination of 
  the ability of cxcl12 to down - regulate the receptor , even after 8 h of 
  exposure to cxcl12 . as mentioned above , these findings of sustained or 
  slightly increased levels of cxcr4 following suppression of usp 14 expression 
  were unexpected , as we had expected a decline in receptor levels under 
  conditions where the level of usp14 , a deubiquitinating enzyme for this 
  receptor , was reduced . one interpretation of these data is an ongoing cycle of 
  ubiquitination / deubiquitination is critical for cxcl12-modulated changes in 
  receptor density . cxcr4 ubiquitination / deubiquitination cycle appears to be critical for 
  cxcl12-induced cell chemotaxis  we were curious whether 
  ubiquitination of cxcr4 would have functional consequences beyond altering 
  cxcl12-induced receptor down - regulation . consequently , we examined 
  cxcl12-mediated chemotaxis in hek293 cells stably expressing myc - cxcr4 . as 
  shown in fig . 6a , 
  cxcl12 led to a concentration - dependent movement of cells across the filter of 
  the boyden chamber , with a maximal chemotactic index being achieved between 1 
  and 10 nm cxcl12 , and declining at higher concentrations , 
  presumably because of a desensitization response 
  ( 36 ) . interestingly , sirna knockdown of endogenous usp14 also diminished 
  cxcl12-induced chemotaxis ( fig . these findings are extremely interesting , because 
  they are consistent with the hypothesis that it is not the ubiquitinated 
  versus nonubiquitinated receptor that is critical for 
  receptor - mediated signaling , but rather the ability of the receptor to undergo 
  an accelerated ub cycle as part of the chemotactic process . this requirement 
  for a cxcr4-ub cycle for chemotaxis is reminiscent of the requirement for a 
  gtpase cycle for cellular migration / chemotaxis . one of the limitations of directing 
  all of our experimental efforts to manipulating the levels of expression of 
  usp14 is that the ubiquitination of a plethora of proteins could be altered in 
  parallel with the cxcr4 as a consequence of these experimental alterations in 
  usp14 levels . to assess whether cxcl12-induced chemotaxis requires a 
  ubiquitination cycle that involves the receptor protein itself , we compared 
  the ability of cxcl12 to evoke chemotaxis of hek293 cells expressing either a 
  wt receptor or the 3k / r mutant cxcr4 that can not be ubiquitinated , as 
  described previously ( 28 ) . as 
  shown in fig . 6c , 
  elimination of the ability of the cxcr4 to undergo ubiquitination also 
  eliminates the ability of cxcl12 to evoke chemotaxis of the cells expressing 
  this 3k / r mutant receptor . thus , taken together , these findings provide the 
  first evidence that cxcr4 mediates chemotaxis in a fashion that requires a 
  ubiquitination / deubiquitination cycle on the receptor protein itself . figure 5.degradation of endogenous cxcr4 in hela cells is disrupted by modulation 
  of usp14 expression . a , hela cells transiently transfected with 
  vector or ha - usp14 were treated with cxcl12 for 0 , 3 , 5 , or 8 h as indicated . experimental procedures .  data 
  obtained at time 0 and 8 h data are mean  s.e . from three independent 
  experiments . , p < 0.05 ; , p 
  < 0.01 ; , p < 0.001 compared with control cells 
  ( no cxcl12 treatment , i.e. time 0 ) ; the data obtained at 3 and 5 h 
  after cxcl12 incubation are from only two independent experiments , and thus 
  statistical analyses were not performed for these time points . b , 
  hela cells transiently transfected with scrambled sirna ( control ) or 
  usp14-specific sirna were treated with cxcl12 for 0 , 3 , 5 , or 8 h , as 
  indicated . degradation of endogenous cxcr4 in hela cells is disrupted by modulation 
  of usp14 expression . a , hela cells transiently transfected with 
  vector or ha - usp14 were treated with cxcl12 for 0 , 3 , 5 , or 8 h as indicated . quantitation of relative amount of cxcr4 was determined by densitometric 
  scanning as outlined under experimental procedures .  data 
  obtained at time 0 and 8 h data are mean  s.e . from three independent 
  experiments . , p < 0.05 ; , p 
  < 0.01 ; , p < 0.001 compared with control cells 
  ( no cxcl12 treatment , i.e. time 0 ) ; the data obtained at 3 and 5 h 
  after cxcl12 incubation are from only two independent experiments , and thus 
  statistical analyses were not performed for these time points . b , 
  hela cells transiently transfected with scrambled sirna ( control ) or 
  usp14-specific sirna were treated with cxcl12 for 0 , 3 , 5 , or 8 h , as 
  indicated . data are mean  s.e . from three independent 
  experiments . , p < 0.05 ; , p 
  < 0.01 . a , chemotaxis was evaluated in hek293 cells stably expressing 
  myc - cxcr4 ( and endogenous levels of usp14 , i.e.  vector 
  alone  ) or in cells overexpressing ha - usp14 as described in detail under 
   experimental procedures . overexpression of usp14 , which 
  dramatically reduces cxcl12-evoked cxcr4 ubiquitination ( cf . b , knockdown of 
  endogenous usp14 expression with usp14-directed sirna , which leads to enhanced 
  cxcl12-induced cxcr4 ubiquitination ( cf . 
 fig . c , chemotaxis in 
  response to cxcl12 was evaluated in cells expressing an ha - wt cxcr4 and in 
  cells expressing a 3k / r mutant receptor , as described under 
   experimental procedures . taken together , a and 
  b suggest that the cxcr4-ub cycle , and not a particular ubiquitinated 
  state of cxcr4 , is essential for cxcl12-mediated chemotaxis , and the data in 
  c confirm that it is the cxcr4 molecule itself that must undergo a 
  ubiquitination / deubiquitination cycle for chemotaxis to occur . values 
  represent the mean  s.e . from three independent experiments performed 
  in duplicate . all chemotaxis data are expressed as the chemotactic index , 
  which is calculated as the ratio of the number of cells that migrate across 
  the boyden chamber in the presence of cxcl12 at a given concentration compared 
  with the number of cells migrating in the absence of cxcl12 ; the value of 1 
  means that there was no migration greater than that observed in control , 
  nonstimulated cells . , p < 0.05 ; , p < 0.01 ; 
  , p < 0.001 , and compared with control cells , 
  e.g. cells expressing endogenous usp14 ( a ) , scrambled sirna a , chemotaxis was evaluated in hek293 cells stably expressing 
  myc - cxcr4 ( and endogenous levels of usp14 , i.e.  vector 
  alone  ) or in cells overexpressing ha - usp14 as described in detail under 
   experimental procedures . overexpression of usp14 , which 
  dramatically reduces cxcl12-evoked cxcr4 ubiquitination ( cf . b , knockdown of 
  endogenous usp14 expression with usp14-directed sirna , which leads to enhanced 
  cxcl12-induced cxcr4 ubiquitination ( cf . 
 fig . c , chemotaxis in 
  response to cxcl12 was evaluated in cells expressing an ha - wt cxcr4 and in 
  cells expressing a 3k / r mutant receptor , as described under 
   experimental procedures . taken together , a and 
  b suggest that the cxcr4-ub cycle , and not a particular ubiquitinated 
  state of cxcr4 , is essential for cxcl12-mediated chemotaxis , and the data in 
  c confirm that it is the cxcr4 molecule itself that must undergo a 
  ubiquitination / deubiquitination cycle for chemotaxis to occur . values 
  represent the mean  s.e . from three independent experiments performed 
  in duplicate . all chemotaxis data are expressed as the chemotactic index , 
  which is calculated as the ratio of the number of cells that migrate across 
  the boyden chamber in the presence of cxcl12 at a given concentration compared 
  with the number of cells migrating in the absence of cxcl12 ; the value of 1 
  means that there was no migration greater than that observed in control , 
  nonstimulated cells . , p < 0.05 ; , p < 0.01 ; 
  , p < 0.001 , and compared with control cells , 
  e.g. cells expressing endogenous usp14 ( a ) , scrambled sirna mutation of the cxcr4 to eliminate receptor ubiquitination does not 
  alter the time course or extent of cxcl12-induced erk activation a 
  commonly monitored consequence of cxcr4 activation by cxcl12 is the increase 
  in tyrosine phosphorylation on erk . as shown in 
  fig . 7 , the time course of this 
  stimulation by cxc12 is indistinguishable in hek293 cells expressing wt 
  versus 3k / r cxcr4 . as an increase in threonine phosphorylation on erk 
  also can be initiated by cxcl12-induced cxcr4 activation , it is possible that 
  changes in thr(p ) phosphorylation of erk occurred in response to cxcl12 
  activation of the 3k / r cxcr4 , which we would not have detected using only an 
  anti - tyr antibody . thus , we assessed erk phosphorylation in response to cxcl12 
  activation of wt and 3k / r cxcr4 using a p - erk p42/p44 thr / tyr antibody ( cell 
  signaling technologies ) . interestingly , even with this antibody , we observed 
  no difference in the time course or the extent of activation of erk upon 
  cxcl12 exposure to cells expressing wt versus 3k / r cxcr4 . with this 
  p - erk p42/p44 thr / tyr antibody , we detected 3-fold increase in erk 
  activation at 5 min following cxcl12 ( the time of peak stimulation detected by 
  this antibody , as was detected with the anti - tyr(p ) antibody ) in wt and 3k / r 
  cxcr4-expressing cells . after continued stimulation , erk activation 
  diminished , returning to basal levels at 60 min of stimulation ( data not 
  shown ) . the finding that equivalent erk activation occurs in hek cells 
  expressing the wt versus 3k / r cxcr4 provides additional functional 
  evidence that a comparable level of cxcr4 expression is likely occurring in 
  both circumstances , and thus the loss of chemotaxis observed for the 3k / r 
  cxcr4 in fig . 6c finally , these 
  data provide evidence that although erk is indeed a downstream target of 
  cxcl12 activation of cxcr4 , this pathway is not necessarily essential for 
  cxcr4-evoked chemotaxis . figure 7.erk activation by cxcr4 occurs independently of the ability of the cxcr4 
  to be ubiquitinated . a , erk activation was evaluated in hek293 
  cells transiently transfected with ha - wt cxcr4 or ha-3k / r cxcr4 and stimulated 
  with cxcl12 ( 10 nm ) for 0 , 5 , 15 , 30 , or 60 min , and erk activity 
  in cell lysates assessed was as described under b , quantitation of erk activation was based on the 
  amount of erk detected using an anti - p - erk antibody ; total erk , assessed using 
  the erk2 antibody , was indistinguishable in all conditions , and thus the data 
  were not normalized to total erk . erk activation by cxcr4 occurs independently of the ability of the cxcr4 
  to be ubiquitinated . a , erk activation was evaluated in hek293 
  cells transiently transfected with ha - wt cxcr4 or ha-3k / r cxcr4 and stimulated 
  with cxcl12 ( 10 nm ) for 0 , 5 , 15 , 30 , or 60 min , and erk activity 
  in cell lysates assessed was as described under  experimental 
  procedures . b , quantitation of erk activation was based on the 
  amount of erk detected using an anti - p - erk antibody ; total erk , assessed using 
  the erk2 antibody , was indistinguishable in all conditions , and thus the data 
  were not normalized to total erk . although ubiquitination mechanisms and the resulting degradative fates of 
  proteins , including cell surface receptors , have been exhaustively studied , 
  the mechanisms and functional consequences of protein deubiquitination are 
  less understood . our present studies reveal that cxcl12 activation of cxcr4 , a 
  member of the g protein - coupled receptor superfamily , results in reversible 
  ubiquitination of the receptor ( fig . 
  3 ) . receptor deubiquitination is paralleled by cxcl12-dependent 
  association of usp14 with cxcr4 , an interaction that occurs , at least in part , 
  via the c terminus of the receptor . usp14 interaction with cxcr4 is not 
  mimicked by other deubiquitinating enzymes of the usp family evaluated , 
  including usp2a , usp4 , and usp7 ( fig . 1 , 
  a - c ) . cxcl12 activation of cells also leads to 
  the redistribution of usp14 to cellular compartments shared by cxcr4 
  ( fig . similar 
  redistribution of gpcr - interacting proteins has been observed previously , 
  including the redistribution of arrestin 
  ( 44 ) and of spinophilin 
  ( 44 , 
  45 ) . our findings also are consistent with the interpretation that cxcr4 
  undergoes a ligand - modulated ubiquitination - deubiquitination cycle that 
  regulates the steady state level of cxcr4 . based on pre - existing data in the 
  literature , it would have been expected that cxcl12 incubation of 
  receptor - expressing cells would lead to receptor ubiquitination , receptor 
  internalization , and lysosomal degradation , which over time would lead to 
  cxcl12-evoked down - regulation of cxcr4 density . consequently , we expected that 
  overexpression of usp14 , a deubiquitinating enzyme , would lead to an increase 
  in the steady state level of the receptor and blockade of cxcl12-induced 
  down - regulation of receptor density . although this is what we observed , we 
  also observed that suppression of usp14 expression using sirna , which should 
  favor accelerated receptor degradation , does not . instead , we observed a loss 
  of the ability of cxcl12 to evoke down - regulation of cxcr4 following either 
  overexpression or suppression of usp14 expression . these findings indicate 
  that when the ubiquitination cycle is perturbed , the ability of cxcl12 
  stimulation of the cxcr4 to lead to receptor down - regulation also is 
  perturbed , and suggest that ubiquitination of the receptor is perhaps involved 
  in more than simply marking the receptor for degradation . a second initially unexpected finding was that eliminating the ability of 
  the cxcr4 to be ubiquitinated , i.e. by expressing the 3k / r mutant of 
  the cxcr4 , as well as by overexpressing or blocking usp14 expression ( which 
  modifies the ability of this enzyme to modify all of its targets in the cell ) , 
  leads to inhibition of receptor - activated chemotaxis . this may mean , in 
  addition to the known role of ubiquitination in targeting receptors for 
  lysosomal degradation , that the covalent modification of the receptor by 
  ubiquitin is also important for receptor signaling . a number of possible 
  molecular events might explain this role for a ubiquitination cycle on the 
  cxcr4 . for example , the ubiquitinated state of the receptor could dictate a 
  particular conformation of the receptor , unique protein - protein interactions 
  of the cxcr4 , or particular receptor localization on the surface that favors 
  cxcr4 involvement in chemotaxis . because chemotaxis requires a sustained sensing  of a concentration gradient , often involving cycling of 
  receptors between activated and un - activated states , it is possible that the 
  ubiquitination - deubiquitination cycle on the cxcr4 also is causally involved 
  in this gradient sensing role . our findings that a 
  ubiquitination / deubiquitination cycle involving the cxcr4 receptor appears to 
  be essential for receptor - mediated chemotaxis elevate the role of receptor 
  ubiquitination beyond a mechanism simply to target the receptor for a 
  particular degradative fate . the selective role of usp14 for interaction with cxcr4 is also of interest 
  ( fig . 1 ) . other members of the 
  deubiquitinating enzyme family have been shown to interact with a variety of 
  receptor families , including the association of usp2a with the androgen 
  receptor ( 42 ) and usp4 with 
  the adenosine receptor ( 43 ) . 
  however , we are the first to show a preferential interaction of usp14 with 
  cxcr4 . our finding that the c terminus of cxcr4 , on its own , can interact with 
  usp14 may be related to the existence of a purported  degradation 
  motif  in this region of cxcr4 necessary for ubiquitin - mediated receptor 
  degradation ( 28 ) . it is also 
  important to note that the c terminus of the cxcr4 receptor , like the 
  holoreceptor , can distinguish among several usp family members studied , and 
  selectively recognize and bind usp14 ( data not shown ) . there is one aspect of our findings that differs from previous reports 
  regarding the ubiquitination of cxcr4 . our studies show a laddering of the 
  cxcr4 receptor in response to cxcl12 that can be detected with an antibody to 
  endogenous ubiquitin . however , our data also suggest that this laddering 
  involves only a small fraction of the receptor population , because an antibody 
  against the myc - cxcr4 used to assess protein loading on gels also did not 
  reliably detect higher molecular weight  ladders  of the cxcr4 . laddering  of the cxcr4 
  receptor in response to cxcl12 stimulation , but those studies were examining 
  the incorporation of ha - tagged ubiquitin following transient transfection of a 
  cdna encoding this epitope - tagged ubiquitin molecule 
  ( 28 ) . we suspect that the 
  difference in appearance / detection of the ubiquitinated cxcr4 receptor 
  following cxcl12 stimulation is likely because of our differences in 
  experimental strategies . another interesting finding of our study was the observation that the time 
  course of erk activation by cxcl12-induced activation of cxcr4 was 
  indistinguishable for the wt and 3k / r receptor structures , providing evidence 
  that a signaling pathway independent of erk activation links cxcl12 activation 
  of the receptor to chemotaxis . in summary , our findings demonstrate that cxcl12 activation of the cxcr4 
  leads to a dynamic ubiquitination / deubiquitination cycle and that usp14 
  preferentially interacts with and deubiquitinates cxcr4 . the functional 
  consequences of this cxcr4-ub cycle not only modulate ub - targeted receptor 
  degradation but also cxcl12-evoked chemotaxis . thus , our findings suggest the 
  exciting possibility that regulation of the cxcr4-ub cycle will have a 
  plethora of signaling as well as turnover consequences that may be a 
  generalized property of a variety of other gpcrs .
the chemokine receptor cxcr4 plays important roles in the immune and 
  nervous systems . 
 abnormal expression of cxcr4 contributes to cancer and 
  inflammatory and neurodegenerative disorders . 
 although ligand - dependent cxcr4 
  ubiquitination is known to accelerate cxcr4 degradation , little is known about 
  counter mechanisms for receptor deubiquitination . 
 cxcl12 , a cxcr4 agonist , 
  induces a time - dependent association of usp14 with cxcr4 , or its c terminus , 
  that is not mimicked by usp2a , usp4 , or usp7 , other members of the 
  deubiquitination catalytic family . 
 co - localization of cxcr4 and usp14 also is 
  time - dependent following cxcl12 stimulation . 
 the physical interaction of cxcr4 
  and usp14 is paralleled by usp14-catalyzed deubiquitination of the receptor ; 
  knockdown of endogenous usp14 by rna interference ( rnai ) blocks cxcr4 
  deubiquitination , whereas overexpression of usp14 promotes cxcr4 
  deubiquitination . 
 we also observed that ubiquitination of cxcr4 facilitated 
  receptor degradation , whereas overexpression of usp14 or rnai - induced 
  knockdown of usp14 blocked cxcl12-mediated cxcr4 degradation . 
 most 
  interestingly , cxcr4-mediated chemotactic cell migration was blocked by either 
  overexpression or rnai - mediated knockdown of usp14 , implying that a 
  cxcr4-ubiquitin cycle on the receptor , rather than a particular ubiquitinated 
  state of the receptor , is critical for the ligand gradient sensing and 
  directed motility required for chemokine - mediated chemotaxis . 
 our observation 
  that a mutant of cxcr4 , ha-3k / r cxcr4 , which can not be ubiquitinated and does 
  not mediate a chemotactic response to cxcl12 , indicates the importance of this 
  covalent modification not only in marking receptors for degradation but also 
  for permitting cxcr4-mediated signaling . 
 finally , the indistinguishable 
  activation of erk by wild typeor 3k / r - cxcr4 suggests that chemotaxis in 
  response to cxcl12 may be independent of the erk cascade .