Supuestos y metodología 54

1.2.2   Substrate  specificity  and  modular  structure  of  PTPs    

Studies  have  shown  that  cytosolic  PTPs  have  a  high  degree  of  sequence  identity   but  distinct  specificity  (Andersen  et  al.,  2001).  The  substrate  specificity  of  PTPs   is   controlled   by   the   intrinsic   sequence   specificity   of   their   catalytic   domains   (Myers   et   al.,   2001;   Ren   et   al.,   2011;   Salmeen   et   al.,   2000).   In   particular,   interactions   between   residues   flanking   the   pY   in   the   primary   sequence   of   the   substrate   and   the   residues   surrounding   the   PTP   active   site   contribute   to   their   affinity   for   substrates.   For   example,   the   ER   localized   PTP1B   showed   a   70-­‐fold   higher   affinity   for   tandem   pY   containing   peptides   compared   to   mono   pY   substrates  (Salmeen  et  al.,  2000).  Such  a  finely  tuned  regulation  allows  the  PTP   activity   to   be   adjusted   according   to   the   given   amount   of   phosphotyrosine   residues.   More   recent   work   shows   that   each   PTP   has   a   different   degree   of   sequence  specificity  and  unique  substrate  specificity  profiles  that  can  range  from   stringent   sequence   dependency   to   a   more   broad   specificity   (Ren   et   al.,   2011).   Furthermore,   additional   binding   domains   or   sequences   that   flank   the   PTP  

Figure   1.2   Classification  

and   substrate   specificity   of  PTPs.  The  PTP  families   are   color   coded:   class   I   Cys-­‐based   PTPs   (green),   class   II   Cys-­‐based   PTPs   (pale   yellow),   class   III   Cys-­‐based   PTPs   (pale   blue),   and   Asp-­‐based   PTPs  (red).  The  substrate   specificity   of   each   group   or   class   of   PTPs   is   listed   (Alonso  et  al.,  2004).    

domain   can   also   bind   to   potential   substrates   or   mediate   the   recruitment   to   distinct  cellular  regions  to  bring  the  PTP  domain  in  close  proximity  to  its  targets   (Tonks  and  Neel,  2001).    

In  summary,  the  substrate  specificity  of  PTPs  is  dependent  on  the  primary   sequence   specificity   of   the   catalytic   domain,   but   is   also   dictated   by   secondary   interactions  with  substrates.  Secondary  substrate  interactions  or  distinct  cellular   localizations  of  PTPs  are  determined  by  the  modular  domain  structure  of  PTPs.   Most   PTPs   consists   of   at   least   one   additional   motif   or   non-­‐catalytic   domain   beside   their   catalytic   phosphatase   domain   (Figure   1.3).   In   a   classical   example   and  as  described  for  SH2/PTB  adaptor  proteins,  the  presence  of  a  SH2  domain   facilitates  direct  binding  of  a  PTP  to  phosphorylated  tyrosine  signaling  proteins,   including   active   RTKs   (Neel   et   al.,   2003;   Pao   et   al.,   2007).   PTPs   also   contain   several   phospholipid-­‐binding   motifs   that   target   PTPs   to   different   cellular   membranes   including   endosomes   or   the   PM   where   they   can   contribute   to   the   assembly  of  RTK-­‐activated  downstream  effectors.  Some  PTPs  contain  a  nuclear   localization  sequence  (NLS)  and  shuttle  between  the  nucleus  and  the  cytoplasm.   Moreover,   growth   factor   stimuli   can   induce   a   shift   in   the   nuclear   and   cytosolic   fractions   of   a   PTP,   which   could   have   important   consequences   for   their   accessibility   to   substrates   (He   et   al.,   2005;   Tiganis   et   al.,   1998).   In   summary,   binding   domains   or   sequence   motifs   function   as   a   “zip-­‐code”   to   direct   PTPs   to   their  defined  cellular  address  (Mauro  and  Dixon,  1994)  (Figure  1.4).  Beside  the   broad  group  of  cytosolic  PTPs,  RPTPs  contain  a  membrane  spanning  α-­‐helix  and   are  located  predominantly  at  the  PM,  where  they  are  exposed  to  the  extracellular   milieu  in  a  receptor-­‐like  fashion.  Most  RPTPs  contain  a  tandem  of  PTP-­‐domains   that  is  uncommon  for  cytosolic  PTPs  (Figure  1.3).    

Figure   1.3   Domain   structure   of   all   PTPs.   Schematic   view   of   the   domain   composition   of   all  

members  of  the  four  PTP  families.  Abbreviations:  BRO,  baculovirus  BRO  homology;  C1,  protein   kinase  C  conserved  region  1;  C2,  protein  kinase  C  conserved  region  2;  CA,  carbonic  anhydrase-­‐ like;   CAAX   box,   farnesylation   signal;   CH2,   cdc25   homology   region   2;   CRAL/TRIO,   cellular   retinaldehyde   binding   protein/trio   homology   (Sec14p   homology);   FERM,   band   4.1/ezrin/radixin/moesin   homology;   FN,   fibronectin-­‐like;   FYVE,   Fab1/Yotb/Vac1p/early   endosomal   antigen-­‐1   homology;   Ig,   immunoglobulin-­‐like;   KIM,   kinase   interaction   motif;   KIND,   kinase   N   lobe-­‐like   domain;   MAM,   meprin,   A2,   RPTPμ   homology;   PBM,   PDZ   binding   motif;   PDZ,   postsynaptic   density-­‐95/discs   large/ZO1   homology;   PH,   pleckstrin   homology   (including   GRAM   domains);   PTB,   phosphotyrosine-­‐binding   domain;   SH2,   src   homology   2;   SH3B,   src   homology   3   domain   binding   motif;   SH4,   src   homology   4   (myristylation   signal);   coil,   coiled-­‐coil   domain;   GB,   glycogen   binding;   mRC,   mRNA   capping;   PBM,   PDZ   binding   motif;   pepN,   N-­‐terminal   peptidase-­‐ like;  PH-­‐G,  pleckstrin  homology-­‐“GRAM”  domain;  Pro-­‐rich,  proline-­‐rich;  Sec14,  Sec14p  homology   (or  CRAL/TRIO).  In  addition,  a  small  black  box  signifies  transmembrane  stretch  and  a  red  cross   over  a  PTP  domain  signifies  catalytically  inactive  domain.  (Alonso  et  al.,  2004).  

In  contrast  to  the  discussed  PTPs  that  are  targeted  to  several  intracellular   locations   PM   localization   is   an   important   feature   of   the   RPTPs   because   they   share  the  same  compartment  where  RTKs  become  activated  upon  growth  factor   binding.   In   summary,   research   from   the   last   few   years   has   demonstrated   that   PTPs   are   a   very   diverse   family   with   much   higher   substrate   specificity   than   assumed  in  the  past.  The  distinct  localization  and  specific  substrate  recognition   of   PTPs   suggests   that   there   is   a   spatial   dependency   that   tightly   controls   RTK   phosphorylation.  In  the  next  subsection  we  will  describe  different  mechanisms   that   ensure   that   PTPs   are   regulated   enzymes   which   are   integrated   in   RTK   signaling.                                        

Figure   1.4   Subcellular   localization   of   PTPs.   Cytoplasmic   PTPs   are   recruited   to   activated   cell-­‐

surface   receptors   by   SH2,   proline-­‐rich   FERM   (band  4.1,   ezrin,   radixin,   moesin   homology)   and   PDZ   (postsynaptic   density   protein  95,   discs   large,   Zonula   occludens)   domains.   RPTPs   are   also   engaged  in  these  complexes.  Nuclear  localization  signals  (NLS)  and  ER  targeting  domains  direct   PTPs   to   these   compartments.   A   Sec14-­‐homology   domain   (Sec14h)   mediates   functional   association   with   secretory   vesicles.   Cytoplasmic   PTPs   are   recruited   into   lipid   rafts   by   different   domains.   The   kinase-­‐interacting   motif   (KIM)   in   PTPs   mediates   binding   to   MAPK.   Proteolysis   releases   the   catalytic   domain   of   (R)PTPs   into   the   cytoplasm   and   possibly   also   into   the   nucleus   (den  Hertog  et  al.,  2008).  

1.2.3   Regulation  of  PTP  activity    

Multiple  mechanisms  regulate  the  activity  of  PTPs.  For  example,  PTP  activity  can   be  dependent  on  alternative  splicing  or  proteolysis.  On  the  other  hand,  PTPs  can   be   activated   by   direct   recruitment   to   RTKs   or   inhibited   by   growth   factor-­‐ mediated   production   of   reactive   oxygen   species   (ROS).   The   latter   two   mechanisms   highlight   that   the   activity   of   many   PTPs   is   directly   coupled   to   the   activity  of  RTKs.    

1.2.3.1    Regulation  of  PTPs  by  splicing  and  proteolysis  

To   explain   the   regulation   by   splicing   or   protein   proteolysis   we   will   start   with   example   based   on   PTPN1   (PTP1B)   and   PTPN2   (TCPTP,   TC48).   Both   PTPs   are   targeted   to   the   cytoplasmic   site   of   the   endoplasmatic   reticulum   (ER)   via   a   C-­‐ terminal   hydrophobic   sequence   (Cool   et   al.,   1989;   Frangioni   et   al.,   1992).   Alternative   splicing   of   TCPTP   generates   two   additional   isoforms,   a   45   kDa   (TC45)  and  a  41  kDA  (termed  TC41  in  this  work),  which  differ  in  their  C-­‐termini.   In  contrast  to  the  full  length  48  kDA  form  (TC48)  that  is  targeted  to  the  ER,  TC45   lacks   the   hydrophobic   segment   exposing   a   N-­‐terminally   located   NLS   targeting   TC45   to   the   nucleus   (Lam   et   al.,   2001).   TC41   lacks   the   NLS   and   is   therefore   present  in  both  the  nucleus  and  the  cytosol.  Similarly  to  the  regulation  of  TC45   by   splicing,   PTP1B   contains   a   site   for   proteolytic   cleavage   by   calpain,   which   generates   a   truncated,   soluble   PTP1B   with   enhanced   activity   (Frangioni   et   al.,   1993).  This  demonstrates  the  importance  of  targeting  motifs  in  PTP  regulation.   The  examples  of  PTP1B  and  TCPTP  illustrate  that  the  subcellular  localization  is   directly   coupled   to   PTP   activity.   In   the   following   part   we   discuss   a   general   regulatory  mechanism  based  on  RTK-­‐mediated  activation  of  PTPs.    

1.2.3.2    PTP  activation  by  RTKs    

PTPs   can   be   activated   following   recruitment   to   phosphorylated   RTKs.   For   example,  crystal  structures  of  the  SH2  tandem  containing  PTP,  PTPN11  (SHP2)   have  shown  that  its  catalytic  site  is  occluded  by  an  interaction  with  residues  of