MAPK  docking  sites

MAPK  cascades  regulate  a  variety  of  biological  functions,  such  as  cell  proliferation,   differentiation  and  stress  responses.  Moreover,  aberrant  MAPK  signalling  has  been   associated  with  numerous  diseases  such  as  cancer  (ERK1/2),  rheumatoid  arthritis  (p38   kinases)  and  Alzheimer’s  disease  (JNKs)  [119-­‐121].  Besides  the  need  for  tight  regulation,   these  cascades  require  high  efficiency  and  fidelity  in  signal  transduction,  which  is  

achieved  through  substrate  binding  motifs,  also  known  as  substrate  docking  sites.  

Conventional  MAPKs  and  their  substrate  binding  motifs  have  been  studied  extensively  in   search  for  putative  drug  inhibition  sites.  On  the  other  hand,  our  understanding  of  atypical   MAPKs  is  still  in  its  infancy  in  regards  to  regulation  and  biological  functions.  Thus,  no   docking  domains  have  yet  been  described  for  this  MAPK  subgroup.  

1.2.3.1 Common docking site

As  the  name  suggests,  this  docking  site  mediates  binding  of  numerous  proteins,  such  as   MEKs,  phosphatases  and  transcription  factors  [63].  It  was  originally  identified  in  an   attempt  to  abolish  MEK  binding  to  ERK2  through  mutations  and  termed  the  cytoplasmic   retention  sequence  (CRS)  [122].  Later,  however,  this  region  was  shown  to  facilitate   interaction  of  ERK  with  a  variety  of  proteins  and  therefore  termed  the  common  docking   (CD)  domain  [123].  Moreover,  this  binding  site,  which  is  located  C-­‐terminal  to  the  

catalytic  domain,  is  conserved  among  all  conventional  MAPKs  (Figure  1-­‐5)  and  comprises   negatively-­‐charged  aspartate  (D)  and  glutamate  (E)  residues  [123].  Crystal  structures  of   ERK2,  p38α  and  JNK3  have  shown  that  the  conserved  amino  acids  are  not  only  exposed   on  the  surface  of  these  enzymes,  but  also  reside  close  to  one  another,  thus  forming  a   negatively  charged  interaction  platform  opposite  to  the  catalytic  centre  [124-­‐126].  

Indeed,  this  region  was  shown  to  bind  to  a  conserved  sequence  of  basic  amino  acids  in   MAPK  substrates,  termed  the  D-­‐motif  [123,  127].  Notably,  ERK1/2  substrates  generally   possess  two  consecutive  basic  amino  acids  in  their  D-­‐motif,  whereas  substrates  for  JNK   and  p38  kinases  display  three  or  more  consecutive  lysines  (L)  and  arginines  (R)  (Table   1-­‐3).  Thus,  substrate  specificity  might  be  achieved  through  varying  numbers  of  positively   charged  amino  acids  on  the  D-­‐motif  [124,  128].  

MAPK substrate Proposed D-motif MAPK specificity

MAPKK MEK1 MPKKKPTPIQLNPNP

ERK1/2

MEK2 MLARRKPVLPALTINP

MKK3 KGKSKRKKDLRI

p38s

MKK6 SKGKKRNPGLKIP

MKK4 QGKRKALKLNF

JNKs

MKK7 EARRRIDLNLDISP

MEK5 LKKSSAELRKIL ERK5

MAPKAPK RSK1 SSILAQRRVRKLPSTTL

ERK1/2

RSK2 RSTLAQRRGIKKITSTAL

MAPKAPK2 NPLLLKRRKKARALEAAA

p38s

MAPKAPK3 NRLLNKRRKKQAGSSSAS

MKP MKP-3

(DUSP6)

PGIMLRRLQKGNLPVR

ERK1/2

MKP-5 (DUSP10)

CADKISRRRLQQGKITV

p38, JNK

Table 1-3 Overview of proposed D-motifs of various MAPK substrates

The D-motif is characterised by a cluster of positively charged amino acids (coloured in grey). The number of consecutive arginines or lysines determines the MAPK-binding specificity.

Figure 1-5 MAPK docking sites

A. Human amino acid sequences of the CD domains of various members of the MAPK family. Coloured characters represent negatively charged amino acids in the CD domain, which are supposed to be exposed at the surface and mediate substrate binding. Adapted from [124].

B. MAPKs comprise various docking domains, which mediate substrate binding. ED and CD domains mediate docking of D-motifs, whereas the FXFP-docking site allows binding of DEF-motifs.

1.2.3.2 ERK docking site

The  ERK  docking  (ED)  site  is  located  close  to  the  CD  domain  in  the  crystal  structure  and   consists  of  hydrophobic  residues  from  helices  αD,  αE  and  a  reverse  turn  of  β7-­‐β8  [129].  

This  docking  site  is  significantly  different  in  ERK1/2,  p38  kinases  and  JNKs  and  therefore   provides  a  means  for  substrate  specificity  within  the  MAPK  family  [130].  Moreover,  the   CD  and  ED  domains,  which  are  close  to  one  another  in  the  folded  protein,  form  a  docking   groove  on  the  surface  of  the  kinases  (Figure  1-­‐5).  Thus,  structural  differences  brought   about  by  different  hydrophobic  residues  can  confer  substrate  specificity  and  alter   substrate  affinity  [124].  Indeed,  exchange  of  only  two  amino  acids  in  the  ED  domain  of   p38α  and  ERK2  (Glu160  and  Asp161  in  p38  with  Thr157  and  Thr158  in  ERK2)  is  enough  to   alter  substrate  specificity  [124].  It  has  to  be  noted,  however,  that  amino  acids  close  to  the   docking  groove  are  likely  to  also  be  involved  in  docking  interactions.  Moreover,  substrates   might  differentially  recognise  the  ED  and  CD  domain,  thus  providing  another  means  of   variability.  

1.2.3.3 FXFP binding site

In  addition  to  the  hydrophobic  groove,  another  interaction  motif  has  been  described  for   ERK1/2  and  p38α,  which  is  called  the  FXFP  binding  site.  This  site  was  identified  using   hydrogen  exchange  mass  spectrometry,  and  is  marked  by  a  cluster  of  hydrophobic  amino   acids  distinct  from  the  ED  domain,  which  specifically  interacts  with  a  Phe-­‐X-­‐Phe  (FXF)   motif  [131,  132].  The  FXFP  binding  site  is  situated  close  to  the  active  centre  and  is   occluded  in  the  inactive  enzyme  through  intramolecular  interactions  (Figure  1-­‐5).  Many   ERK1/2  substrates,  including  SAP-­‐1  and  Elk-­‐1,  have  been  shown  to  bind  to  the  FXFP   binding  site  with  their  corresponding  DEF  domain  (docking  site  for  ERK  and  FXFP)  [132].  

Notably,  the  hydrophobic  residues  important  in  ERK-­‐DEF  binding  are  conserved  among   various  MAPK  family  members,  yet  DEF  motif  interactions  have  only  been  observed  in   ERK1/2  and  p38α,  but  not  p38β/γ/δ  and  JNK2  [131].  This  suggests  differences  in  the   tertiary  structure  of  these  kinases  which  lead  to  the  exposure  of  specific  substrate   recognition  patterns.  

1.2.3.4 Other MAPK-binding domains

Although  many  MAPK  substrates  and  regulators  contain  one  or  more  MAPK  dockings  sites   described  above,  some  interacting  proteins  lack  these  conserved  domains,  but  still  bind    

to  the  enzymes  efficiently.  One  such  MAPK  target  is  the  well-­‐known  transcription  factor   Ets-­‐1,  which  has  been  shown  to  bind  to  ERK2  via  a  unique  pointed  domain  [133].  MITF   (microphtalmia-­‐associated  transcription  factor)  also  falls  into  this  category,  as  its  C-­‐

terminal  sequence  required  for  ERK2  binding  does  not  resemble  a  D-­‐  or  DEF-­‐motif  [134].  

1.2.3.5 Kinase inhibitor binding sites

Due  to  the  involvement  of  MAPKs  in  numerous  diseases,  significant  effort  has  been  made   by  the  pharmaceutical  industry  to  develop  inhibitors  that  block  specific  MAPK  pathways.  

This  has  led  to  the  identification  of  two  novel  inhibitor  binding  sites,  i.e.  backside  binding   pocket  and  the  “DFG-­‐out”-­‐site,  where  inhibitor  interactions  do  not  compete  with  ATP   binding  [135].  The  backside  binding  pocket  is  a  region  in  the  vicinity  of  the  CD  domain  in   p38α  and  binds  inhibitors  such  as  PD98056  [136].  In  contrast,  the  “DFG-­‐out”-­‐site  is  a   docking  domain  adjacent  to  the  active  site.  Inhibitor  binding  to  the  conserved  DFG   sequence  induces  a  conformational  change  in  the  activation  loop  of  the  enzyme  and   thereby  blocks  its  activity  [135,  137].  “DFG-­‐out”-­‐sites,  however,  are  not  unique  for  MAPK   as  they  have  also  been  described  for  MEK1/2  [138]  and  c-­‐Abl  [139].