Discovery of MAPK

binding motifs in
Arabidopsis Thilana

Introduction
Protein phosphorylation
Ubiquitous
Most common mechanism of protein function regulation

Protein Kinases
In eukaryotes, phosphorylations are carried out by kinases

Abundance of Protein Kinases

~ 2 % of the proteins in eukaryotes are kinase

Mitogen-activated protein kinase (MAPK)

Here we focus on the interactions with substrates of MAPK

BIONDI et al. Biochemical Journal (2003)

MAPK (Mitogen Activated Protein Kinase)

MAPKs are involved in directing cellular responses to a diverse array of stimuli:

mitogens (chemical substance that encourages cell division)

osmotic stress
heat shock
Pro-inflammatory cytokines

MAPK regulate cell functions including

proliferation
gene expression
differentiation
mitosis
cell survival
apoptosis

Pearson et al. Endocrine Reviews (2001)

MAP Kinase (MAPK) Pathway

1. The growth factor ligand binds to its trans-membrane
receptor activates tyrosine kinases inherent in the
receptor molecule.
2. A series of protein-protein complexes form involving GRB2, and SOS activates Ras (core kinase).
3. Subsequent to this, GDP-Ras is converted to GTP-Ras
which activates MAP KKK MAP KK MAP K
4. Map kinases phosphorylate a number of transcription
factors including TCFs(Elk,Sap1a and Net) and other
proteins.
Major Players of MAPK Pathway
1. Signaling molecule: Growth factor
2. Receptor molecule: Growth receptor
3. Core Kinase : Ras
4. Messenger Kinase: MAPK
http://dna.brc.riken.jp/en/GENESETBANK/0200104Erk_cascade_2.html
http://www.biovision.com/signaling-pathways-1864/map-kinase-pathway-1912/

1
MAPK

in action

~350 residues
Protein kinase domain
MAPK mainly recognize their substrate with docking surface:
-ve charged CD (common docking) region
Hydrophobic docking groove

Bind to target proteins D(docking)-motif

Short motif from 7-18 residues
In disordered region
Far away from target phosphorylation sites

1. Zeke et al., Molecular Systems Biology (2015)

Motivation
MAPK are involved in broad spectrum of function
But the mechanism is not understood.

Discovery of MAPK network is not easy

They bind with low affinity and in less-characterized disordered region.

MAPK phosphorylates most of their substrates at Ser-Pro or Thr-Pro sequence motifs

But this is insufficient for specific target recognition
Additional binding motif ( e.g. D-motif ) provides the specificity

Organism:
Recently it is scanned and verified in proteome scale in Human
We are interested about plant (Arabidopsis Thaliana)

1. Zeke et al., Molecular Systems Biology (2015)

Variability in D-motif structural conformation

Two different spacing (1-2 residues) scheme

between hydrophobic residues (red pins)
Linker length between CD region and
hydrophobic groove varies with groove size

N terminal of D-motifs are variable in

shape:
Fully linear
Alpha-helical

1. Zeke et al., Molecular Systems Biology (2015)

Methods
Step-1: Basis Pattern Matching
Using different known D-motif types
We found 5 different class of D-motifs in human

Step-2: Checking disordered region

Despite their lack of stable structure, they are functionally
important
In some cases, adopt a fixed 3D structure after binding to other
macromolecules.

PDB: 1a5r
1. Zeke et al., Molecular Systems Biology (2015)

Methods (cont)
Step 3:MAPK accessibility
Motifs predicted in following regions are discarded
Extra cellular protein segment
Golgi
Endoplasmic Reticulum (ER)

Step 4: Structural Accessibility

Remove motifs which are in Pfam domains

Results
Step 1: ~5K motif
Step 2: ~3K motif
Step 3: In progress
Step 4: In progress

Questions

Disordered Region
An disordered region is a region
Lacks a fixed or ordered three-dimensional structure
fully unstructured to partially structured and include random
coils and large multi-domain proteins connected by flexible
linkers.

The discovery of this has challenged the traditional protein

structure paradigm, that protein function depends on a fixed
three-dimensional structure.
This dogma has been challenged over the last decades by
increasing evidence from various branches of structural biology,
suggesting that protein dynamics may be highly relevant for such
systems.
Despite their lack of stable structure, the are a very large and
functionally important class of proteins.
In some cases, they can adopt a fixed three-dimensional
structure after binding to other macromolecules.

PDB:1a5r

Dunker eg a. Journal of Mol. Grap. & Model. (2001)