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Regulation  of  spindle  checkpoint  signaling  by  UbcH10/UbcX

  UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

 

39   In  the  following  section,  the  implications  of  these  novel  findings  on  our  view  of   the  spindle  checkpoint  arrest  as  well  as  on  CSF  arrest  will  be  discussed,  models   for  the  physiological  function  of  APC/C  dependent  XErp1  ubiquitylation  will  be   suggested   and   open   questions   as   well   as   possible   future   directions   will   be   proposed.    

3.1. Regulation  of  spindle  checkpoint  signaling  by  UbcH10/UbcX  

3.1.1. The  spindle  assembly  checkpoint  can  be  inactivated  by  UbcX  in  Xenopus   egg  extract  

In   mitosis,   the   presence   of   improperly   attached   kinetochores   leads   to   the   inactivation  of  Cdc20  by  the  checkpoint  proteins  Mad2,  BubR1  and  Bub3  by  the   formation   of   the   inhibitory   MCC–Cdc20   complex,   therefore   the   APC/C   is   inactive   and   anaphase   is   delayed   (Musacchio   and   Salmon,   2007).   The   crystal   structure   of   Mad2   bound   to   a   Cdc20   mimicking   peptide   argues   for   a   tight   interaction   between   the   two   proteins   (Sironi   et   al.,   2002)   and   it   implies   that   the   Mad2-­‐Cdc20   complex   has   to   be   actively   dissociated.   As   described   previously,   contradictory   mechanism   have   been   proposed   in   human   cells   to   inactivate  the  mitotic  checkpoint  complex  (MCC)  and  to  provide  free  Cdc20  for   APC/C   activation,   including   APC/C   dependent   proteolysis   of   Cdc20   (“Cdc20   inactivation   model”,(Nilsson   et   al.,   2008)   or   the   polyubiquitylation,   but   not   degradation,   of   Cdc20   to   dissociate   the   MCC-­‐Cdc20-­‐APC/C   complex   (“Cdc20-­‐

activation  model”;(Reddy  et  al.,  2007).    

In   support   of   the   Cdc20-­‐activation   model,   UbcH10   has   been   shown   to   be   responsible   for   the   dissociation   of   the   MCC-­‐Cdc20   complex   by   ubiquitylating   Cdc20   and   thereby   releasing   the   cell   from   the   mitotic   arrest   (Reddy   et   al.,   2007).  However,  a  major  criticism  on  this  Cdc20-­‐activation  model  concerns  the   ability   of   UbcH10   to   override   SAC   arrest   under   physiological   conditions.  

Indeed,   the   overexpression   of   UbcH10   has   been   linked   to   cancer,   as   it   was   shown  that  UbcH10  levels  are  increased  in  several  cancer  cell  lines  and  tumors  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

  (Berlingieri  et  al.,  2007;  Okamoto  et  al.,  2003;  Pallante  et  al.,  2005;  Wagner  et   al.,   2004)   and   therefore   UbcH10   overexpression   might   inactivate   the   spindle   checkpoint   in   these   cells.   The   physiological   significance   of   UbcH10   mediated   spindle  checkpoint  inactivation  has  been  put  into  question,  since  the  amounts   used  in  the  original  studies  by  Reddy  et  al.  were  suggested  to  be  hundred  times   the  endogenous  levels  and  thus  do  not  represent  physiological  conditions  that   could   be   observed   in   cancer   cells   (Walker   et   al.,   2008).   Accordingly,   overexpression   of   UbcH10   to   three   times   the   endogenous   levels   did   not   significantly  induce  APC/C  activation  and  exit  from  mitosis  in  cells  arrested  by   the  spindle  checkpoint  (Walker  et  al.,  2008).    

In   both   studies,   overexpression   of   UbcH10   was   induced   by   transient   transfection   of   cells   with   a   plasmid   encoding   UbcH10   (Reddy   et   al.,   2007;  

Walker   et   al.,   2008).   Therefore,   the   transfection   efficiency   and   thus   the   amount   of   overexpression   might   vary   from   cell   to   cell   and   the   amount   of   overexpression   relative   to   the   endogenous   protein   can   only   be   estimated.  

Instead,   cell   free  Xenopus  egg   extracts   allow   the   addition   of   recombinant   protein,  therefore  the  increase  in  protein  levels  is  exactly  measureable  and  the   same   in   every   condition.   To   estimate   more   accurately   the   levels   of   UbcX   required  for  SAC  inactivation,  we  determined  the  amount  of  endogenous  UbcX   in  extracts  by  quantitative  westernblotting  (Figure  2.5.).  When  eight  times  the   endogenous  levels  of  UbcX  were  added  to  SAC  arrested  extracts,  we  observed   APC/C   activation   and   SAC   override   (Figure   2.1.).   Since   this   increase   in   UbcX   levels  is  only  modest  and  can  be  observed  in  cancer  cells  (van  Ree  et  al.,  2010),   we   conclude   that   increased   UbcX   or   UbcH10   levels   can   compromise   the   spindle  checkpoint  under  physiological  conditions.  

The  Xenopus  extract  system  is  well  established  in  SAC  research  and  has  been  a   valuable  tool  for  the  identification  and  characterization  of  proteins  important   for  spindle  checkpoint  signaling  in  vertebrate  cells  (Chen  et  al.,  1998;  Chen  et   al.,   1996;   Minshull   et   al.,   1994),   hence   results   obtained   using  Xenopus   egg  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

 

41   these  studies,  we  contribute  to  the  knowledge  of  spindle  checkpoint  signaling,   specifically,  in  its  inactivation,  by  confirming  that  elevated  UbcX  activity  causes   SAC   inactivation.   We   could   show   that   this   mechanism   is   conserved   between   human   and  Xenopus.   However,   we   did   not   test   in   detail   whether   Cdc20   destabilization  contributes  to  spindle  checkpoint  arrest  in  Xenopus  egg  extract.  

Since  we  only  confirmed  one  of  the  two  models,  the  Cdc20-­‐activation  model,   we   cannot   exclude   that   the   Cdc20-­‐inactivation   pathway   could   operate   in   parallel  to  regulate  SAC  mediated  APC/C  inhibition.    

3.1.2. Is  an  APC/C  inhibitor  targeted  for  ubiquitylation  during  SAC  signaling?  

The  similarities  between  UbcX  induced  SAC-­‐  and  CSF-­‐override  presented  here   suggest  that  ubiquitylation  of  APC/C  inhibitors  could  be  a  general  mechanism   to  regulate  the  activity  of  APC/C  inhibitory  complexes.  We  could  show  that  an   increase  in  UbcX  levels  induces  the  ubiquitylation  of  XErp1  rather  than  Cdc20.    

Maybe,  also  during  spindle  checkpoint  signaling,  the  APC/C  inhibitor  needs  to   be  ubiquitylated,  and  not  Cdc20,  to  dissociate  the  MCC-­‐Cdc20  complex.  Could   this  be  an  explanation  for  the  controversial  findings  on  Cdc20  regulation  during   SAC  signaling?  

Under  SAC  arrest  conditions,  it  has  been  suggested  that  APC/CCdc20  bound  to   the   MCC   proteins   is   inactive   (Musacchio   and   Salmon,   2007).   Electron   microscopy   studies   of   the   APC/CCdc20-­‐MCC   complex   favor   this   idea,   as   they   revealed  that  substrate  engagement  to  the  APC/C  is  hindered  when  the  MCC   proteins  are  bound  (Herzog  et  al.,  2009).  Additionally,  the  non-­‐APC/C-­‐bound,   free   pool   of   Cdc20   is   either   in   an   inhibitory   complex   bound   to   the   MCC   proteins   (Musacchio   and   Salmon,   2007)   or   degraded   upon   ubiquitylation   by   the  APC/C  (Nilsson  et  al.,  2008).  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                            Discussion  

 

 

Figure  3.1.  Model  of  the  dynamic  spindle  checkpoint  mediated  APC/C  inhibition  regulated  by   ubiquitylation.   During   SAC   arrest,   the   MCC   proteins   bind   to   Cdc20   to   inhibit   the   APC/C.  

Ubiquitylation   of   the   MCC   proteins   and   Cdc20   by   the   APC/C   could   result   in   MCC-­‐Cdc20   complex  dissociation  and  free  ubiquitylated  of  Cdc20  is  targeted  for  degradation.  During  SAC   arrest,  the  dissociation  could  be  counteracted  by  the  deubiquitylating  activity  of  USP44.  Once   the   SAC   is   satisfied,   the   existing   MCC-­‐Cdc20   complexes   are   rapidly   dissociated   by   ubiquitylation,  free  Cdc20  activates  the  APC/C  and  cells  progress  to  anaphase.  

A  possible  model  for  the  dynamic  regulation  of  APC/C  by  the  SAC  (Figure  3.1.)   could  be  that  Cdc20  once  synthesized,  is  incorporated  in  the  MCC  complex  and   recruited   to   the   APC/C.   The   APC/C   ubiquitylates   Cdc20   and   targets   it   for   degradation,   perhaps   to   keep   Cdc20   at   a   constant   level   despite   continuous   synthesis.   Additionally,   the   APC/C   ubiquitylates   another   component   of   the   inhibitory  MCC  to  induce  the  dissociation  of  the  complex.  It  has  been  shown   previously   that   BubR1   is   ubiquitylated   by   the   APC/C  in   vitro  (Garnett   et   al.,   2009),  which  was  suggested  to  be  important  for  progression  through  mitosis   (Choi  et  al.,  2009).  Therefore  it  is  tempting  to  speculate  that  BubR1  could  be   the   target   of   APC/C   mediated   ubiquitylation.   The   stability   of   the   Cdc20-­‐MCC   complex  could  be  additionally  promoted  by  USP44  to  counteract  dissociation   of  ubiquitylated  Cdc20  to  a  certain  degree.  In  this  scenario,  Cdc20  bound  to  the   MCC   complex   is   stable   but   inactive,   whereas   free,   ubiquitylated   Cdc20   is   targeted  for  degradation.  As  a  result  there  is  no  free  Cdc20  available  for  APC/C   activation,   and   at   the   same   time,   due   to   e.g.   BubR1   ubiquitylation   and   deubiquitylation,  the  Cdc20-­‐MCC  complex  remains  dynamic  and  responsive  to   changes  i.e.  fully  attached  kinetochores.  Together,  these  mechanisms-­‐  Cdc20  

    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