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UbcX  mediated  ubiquitylation  of  XErp1  regulates  its  APC/C  inhibitory   activity

  UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

 

43   a   robust   SAC   arrest   in   the   presence   of   unattached   kinetochores,   but   at   the   same   time   a   fast,   switch-­‐like   inactivation   of   the   SAC   once   it   is   satisfied,   thus   ensuring   the   faithful   segregation   of   the   genetic   material   into   the   two   descending  daughter  cells.    

In   future,   it   will   be   interesting   to   test   if   also   the   Cdc20   degradation   can   be   verified  in  SAC  arrest  conditions  in  Xenopus,  or  if  the  two  models  are  mutually   exclusive  and  only  the  Cdc20  activation  model  applies.  Additionally,  it  could  be   tested   whether   BubR1,   Mad2   and/or   Bub3   are   regulated   by   ubiquitylation   similar  to  XErp1.  If  this  is  the  case,  lysine  less  mutants  of  the  relevant  proteins   could   be   generated   which   cannot   be   inactivated   by   ubiquitylation   anymore.  

These   mutants   should   be   inducing   a   spindle   checkpoint   arrest   when   overexpressed  in  somatic  cells  or  a  delay  in  anaphase  onset  despite  a  satisfied   spindle   checkpoint,   since   the   APC/C   cannot   inactivate   inhibitory   MCCs   anymore.  

3.2. UbcX  mediated  ubiquitylation  of  XErp1  regulates  its  APC/C  inhibitory   activity  

In  both,  the  Cdc20-­‐acitvation  and  Cdc20-­‐inactivation  model,  the  primary  target   of  the  spindle  assembly  checkpoint  is  APC/CCdc20.  Similarly,  the  primary  target   of   CSF   and   hence   XErp1   is   the   APC/C  Cdc20,   however   it   is   unclear   how   XErp1   regulates   the   APC/C.   Therefore,   we   tested   the   two   contradictory   models   of   APC/C  regulation  during  SAC  arrest  in  the  CSF  mediated  metaphase  arrest.    

3.2.1. Cdc20  is  not  destabilized  in  CSF  arrested  egg  extract  

It  has  been  proposed  that  the  APC/C  inhibitory  MCC  proteins  bind  to  the  APC/C   coactivator   Cdc20   and   turn   it   into   an   APC/C   substrate   (Nilsson   et   al.,   2008),   thereby   promoting   its   degradation   and   APC/C   inactivation.   We   hypothesized   that   during   CSF   arrest,   Cdc20   could   be   recruited  via   XErp1   to   the   APC/C,   resulting   in   Cdc20   ubiquitylation   and   degradation   and   the   APC/C   is   kept  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

  inactive.   Our   studies   however   demonstrate   that   destruction   of   the   APC/C   activator   Cdc20   does   not   occur   and,   hence,   does   not   contribute   to   the   CSF   arrest.   This   important   finding   is   based   on   the   observation   that   inhibition   of   neither  protein  translation  nor  protein  degradation  affected  protein  levels  of   Cdc20   in   CSF-­‐extract.   Given   that   the   second   APC/C   activator   Cdh1   is   not   expressed   in  Xenopus   egg   extract   (Lorca   et   al.,   1998),   these   experiments   revealed  that  the  amounts  of  Cdc20  present  in  translation  inhibited  extracts  is   sufficient  to  promote  the  destruction  of  APC/C  substrates.  This  implicates  that   CSF  arrest  is  not  mediated  by  the  destabilization  of  Cdc20.  

3.2.2. UbcX  mediated  ubiquitylation  of  XErp1  regulates  its  APC/C  inhibitory   activity  

Using  UbcH10  dependent  APC/C  activation  during  SAC  signaling  (Reddy  et  al.)   as   a   model,   we   tested   whether   the  Xenopus   ortholog   of   UbcH10,   UbcX   can   induce   APC/C   activation   and   CSF   override   in  Xenopus.   The   incubation   of   CSF   arrested   egg   extract   with   UbcX   two   times   the   endogenous   levels   induced   APC/C   activation   and   the   degradation   of   APC/C   substrates.   Eight   times   the   endogenous  amount  of  UbcX  was  sufficient  to  cause  CSF  override  and  mitotic   exit,   as   indicated   by   APC/C   substrate   degradation,   dephosphorylation   of   mitotic   phosphoproteins   like   Cdc27   and   Cdc20   (data   not   shown)   and   sperm   nuclei  decondensation.  UbcX  addition  was  accompanied  by  XErp1  release  from   the   APC/C,   which   was   analyzed   by   glycerol   gradient   centrifugation   and   co-­‐

immunoprecipitation  assays.  XErp1  and  UbcX  bind  to  the  APC/C,  and  if  UbcX   and  XErp1  compete  for  the  same  binding  site,  wild  type  and  catalytic  inactive   UbcX   would   lead   to   the   dissociation   of   XErp1.   Since   only   the   catalytic   active   version   was   able   to   decrease   XErp1   binding   to   the   APC/C,   we   conclude   that   UbcX  mediated  ubiquitylation  is  required  for  this  effect.    

To   identify   the   target   of   UbcX   dependent   ubiquitylation,   we   treated   CSF   arrested   extracts   with   wild   type   ubiquitin   or   as   a   negative   control  

  observe  Cdc20  ubiquitylation  either.  Interestingly,  when  we  concentrated  for   APC/C  bound  Cdc20  and  performed  an  in  vitro  ubiquitylation  assay,  we  could   to  multiubiquitylated  XErp1  specifically  in  extracts  treated  with  UbcX  and  wild   type  ubiquitin  (see  figure  2.9.).  Since  the  purification  of  ubiquitylated  proteins  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

  3.2.3. Are  ubiquitin  hydrolases  counteracting  the  activity  of  UbcX  during  CSF  

arrest?  

We   could   show   that   the   APC/C   can   free   itself   from   the   inhibitory   activity   by   ubiquitylating   XErp1  via   its   E2   enzyme   UbcX.   The   APC/C   and   UbcX   are   both   present   during   CSF   arrest,   raising   the   question   of   why   is   XErp1   is   not   ubiquitylated  and  inactivated  under  this  condition.  In  cells  arrested  by  the  SAC,   Cdc20   is   ubiquitylated   by   APC/C-­‐UbcH10   and   this   ubiquitylation   of   Cdc20   needs  to  be  removed  by  the  activity  of  the  deubiquitylating  enzyme  USP44  to   maintain   SAC   arrest   (Stegmeier   et   al.,   2007).   If   the   balance   between   ubiquitylation  and  deubiquitylation  is  disturbed,  for  example  by  increasing  the   activity  of  UbcH10  or  depleting  the  antagonizing  USP44,  cells  cannot  maintain   the  SAC  and  exit  mitosis  prematurely.    

During  CSF  arrest,  shifting  the  balance  towards  ubiquitylation  by  increasing  the   amounts  of  UbcX  caused  APC/C  activation.  To  test  whether  a  deubiquitylating   activity   is   required   to   maintain   CSF   arrest   by   counteracting   APC/C-­‐   UbcX   dependent   ubiquitylation   and   inactivation   of   XErp1,   we   first   tested   the   most   obvious  candidate  for  mediating  this  activity-­‐  the  Xenopus  homolog  of  USP44.  

To   test   the   requirement   of   USP44   during   CSF   arrest,   we   wanted   to   immunodeplete  USP44  from  CSF  egg  extracts.  This  approach  is  dependent  on   antibodies   that   can   recognize   the   protein   in   vivo   and   that   are   able   to   specifically  deplete  it.  To  obtain  such  antibodies,  the  native  protein  is  normally   used  to  immunize  rabbits.  However,  we  were  unable  to  obtain  native  protein   due   to   expression   and   solubility   problems   in   bacteria   and   insect   cells.  

Therefore  we  used  two  USP44  peptides  to  generate  antibodies  against  USP44   in   two   different   rabbits.   Both   of   them   recognized   denatured   protein   by   westernblotting,   but   were   unable   to   deplete   USP44   from   egg   extracts,   probably   because   the   epitopes   of   these   antibodies   are   masked   in   the   native   protein.  

  the  proteasome  could  efficiently  degrade  ubiquitylated  proteins  like  cyclin  B.  

The   proteasome   needs   for   its   activity   ubiquitin   hydrolases   that   remove   the   ubiquitin   before   the   substrates   enter   the   proteasome   tunnel   (Hershko   and   Ciechanover,   1998).   This   process   is   inhibited   when   ubiquitin-­‐aldehyde   is   incorporated   into   the   chains,   and   substrates   can   no   longer   be   degraded   and   are   thus   stabilized.   In   our   hands,   extracts   treated   with   ubiquitin-­‐aldehyde   efficiently   degraded   ubiquitylated   substrates,   suggesting   that   ubiquitin-­‐

aldehyde  was  not  efficiently  incorporated  in  the  ubiquitin  chains,  possibly  due   efficient  incorporation  of  ubiquitin-­‐aldehyde  into  the  ubiquitin  chain  of  XErp1.  

Next,   endogenous   XErp1   would   be   replaced   by   ubiquitin-­‐aldehyde   modified  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

  ubiquitylated  and  to  make  mutant  XErp1  proteins  that  lack  these  critical  lysine   residues.   We   would   expect   that   such   a   mutant   of   XErp1   cannot   be   ubiquitylated  by  UbcX  anymore  and  its  ability  to  regulate  the  APC/C  during  CSF   arrest  and  oocyte  maturation  is  compromised  when  compared  to  the  wild  type   protein.   Nevertheless,   the   discovery   of   apparent   regulation   of   XErp1   by   ubiquitylation  is  novel  and  adds  a  new  type  of  complexity  to  the  mechanism  of   CSF  arrest.  

3.3. Is   the   regulation   of   UbcX   activity   important   during   the   meiotic   cell