Download Protein kinases - Institut de recherches cliniques de Montréal

BIOLOGIE MOLÉCULAIRE ET CELLULAIRE
UdeM SMC6051/52 et BIM6026/27, et McGill 516-604D
Activation cellulaire. Tyrosine et Ser/Thr Kinase (MAP kinases)
Philippe P. Roux, Ph.D.
Chaire de recherche en signalisation et protéomique
Professeur agrégé, département de pathologie et biologie cellulaire
Institut de recherche en immunologie et en cancérologie (IRIC)
Université de Montréal
[email protected]
16 janvier 2014, 16h-18h
IRCM, salle André-Barbeau
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
The basics of protein phosphorylation

2-3% of the genome is dedicated to
phosphorylation and encodes protein
kinases
and phosphatases
What
percentage of the

is dedicated
~ 500 genome
kinase genes
and 150 to
phosphorylation?
phosphatase
genes in humans

~ 33 protein kinases (20 Tyr-K et 13
Ser/Thr-K) are mutated in human
How many protein kinases
diseases
γ β α
and protein phosphatases
 > 150 protein are
kinases associated to
there?
cancer (overexpression, amplification,
deletion, etc)
Protein phosphorylation: important dates
1906: Discovery of the first phosphorylated protein (Vitellin) by Phoebus A. Levene
1933: With Fritz Lipmann, Levene discovers a phosphoserine in the protein casein
1954: First description of a kinase activity on casein
1955: Fischer & Krebs and Sutherland demonstrate that conversion between phosphorylase a and
b results from one cycle of phosphorylation
1959: Identification of the first protein kinase, the phosphorylase kinase, by Fischer and Krebs
1968: Discovery of protein kinase A (PKA) by Krebs and characterization of the
first kinase cascade (PKA->phosphorylase kinase->phosphorylase)
1978: Demonstration of a kinase activity associated with the product
of the Src oncogene by Ray Erikson (Ser or Tyr kinase?)
1980: Discovery of tyrosine phosphorylation (by Src) by Tony Hunter
1981: Characterization of the protein phosphatase 2B (calcineurin)
1987: Discovery of the MAP kinases as the second kinase cascade
1991: Elucidation of the crystal structure of PKA by Susan Taylor
1992: Nobel prize in Physiology and Medicine to Krebs & Fischer
2001: FDA approval of Gleevec for the treatment of CML
2002: Description of the human kinome
Nobel prize 1992
How many phosphorylation sites are there?
If there are ~10,000 proteins per cell with an average length of 400 aa (~ 17% of
which are Ser, Thr or Tyr), then there are ~700,000 potential phosphorylation
sites for any given kinase (including hidden residues).
Although protein kinases have relatively similar
structures, even the most promiscuous can select
their many substrates from among the theoretical
700,000 potential phosphorylation sites:
For example,
ERK2
Akt
JAK
B-Raf
 MEK1 phosphorylates only two substrates: ERK1
and ERK2
 CaMK, CK2 and CDK phosphorylate hundreds of
substrates
Specificity is achieved using several mechanisms (we’ll get to that later!)
many phosphosites
are there?
and the totalThen…
number how
of phosphorylation
sites is probably
closer to 250,000
Available phosphorylation databases
1. Phospho.ELM 8.2: contains 4,687 experimentally verified phosphorylated proteins from different species with 2,217 tyrosine, 14,518
serine and 2,914 threonine sites collected from scientific literature (Diella, et al., 2004; Diella, et al., 2008).
2. PhosphoSitePlus: is a web-based database to collect protein modification sites, including protein phosphorylation sites from scientific
literature as well as high-throughput discovery programs. PhosphoSitePlus contains 73,017 phosphorylation sites (Hornbeck, et al., 2004).
3. PhosphoNET: PhosphoNET presently holds data on more than 26,000 phosphorylation sites in over 5350 human proteins that have
been collected from the scientific literature and other reputable websites.
4. HPRD: HPRD currently contains information for 16,972 PTMs which belong to various categories with phosphorylation (10,858),
dephosphorylation (3,118) and glycosylation (1,860) forming the majority of the annotated PTMs (Keshava Prasad, et al., 2009).
5. PHOSIDA: a phosphorylation site database, integrates thousands of high-confidence in vivo phosphosites identified by mass
spectrometry-based proteomics in various species (Gnad, et al., 2007).
6. PhosphoPep v2.0: contains MS-derived phosphorylation data from 4 different organisms, including fly (Drosophila melanogaster),
human (Homo sapiens), worm (Caenorhabditis elegans), and yeast (Saccharomyces cerevisiae) (Bodenmiller, et al., 2008).
7. Swiss-Prot knowledge base: for each protein annotation, the "Amino acid modifications" in the "Sequence annotation (Features)"
section collected the post-translational modification information of proteins (Farriol-Mathis, et al., 2004).
8. dbPTM 2.0: integrates experimentally verified PTMs from several databases, and to annotate the predicted PTMs on Swiss-Prot
proteins (Lee, et al., 2006).
9. SysPTM 1.1: provides a systematic and sophisticated platform for proteomic PTM research, equipped not only with a knowledge base
of manually curated multi-type modification data, but also with four fully developed, in-depth data mining tools. (Li, et al., 2009).
10. PhosphoPOINT: is a comprehensive human kinase interactome and phospho-protein database, containing 4195 phospho-proteins
with a total of 15,738 phosphorylation sites (Yang, et al., 2008).
11. NetworKIN 1.0: is a method for predicting in vivo kinase-substrate relationships, that augments consensus motifs with context for
kinases and phosphoproteins. It's a great resource of phospho-regulatory network (Linding, et al., 2007; Linding, et al., 2008).
http://www.uniprot.org/
http://www.phosphosite.org/
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
Protein kinases
S. cerevisiae: 118 (1.9%)
D. melanogaster: 236 (1.7%)
C. elegans: 435 (2.3%)
H. sapiens: 518 (1.7%)
A. thaliana: 1049 (4.1%)
The human kinomeThe
(based
on sequence homology only)
kinome
Protein kinases:
10 groups
134 families
201 sub-families
518 human genes
coding for protein
kinases
- 478 eucaryotic PKs
- 40 atypical PKs
TK, tyrosine kinase; TKL, tyrosine kinase-like; STE, homologs of Ste7,
Ste11, Ste20 kinases; CK1, casein kinase 1; AGC, containing PKA,
PKG, PKC families; CAMK, calcium/calmodulin-dependent protein
kinase; CMGC, containing CDK, MAPK, GSK3, CLK families; RGC,
receptor guanylate cyclase; Other; Atypical
Conservation of protein kinase sub-families during evolution
From the 201 sub-families found
in humans…

51 sub-families conserved in
the four kinomes = 25%

144 sub-families conserved in
metazoans (human, fly and
worm) = 72%

> 95% human kinases have an
orthologue in the mouse

13 sub-families of kinases
present only in humans (ex. Tie)
Protein Tyrosine Kinases
Tyrosine kinases
•
90 human genes
•
2 families of tyrosine kinases
1. receptor tyrosine kinases (58)
2. non-receptor tyrosine kinases (32)
Non-receptor
Receptor
ProteinSerine/Threonine
Serine/Threonine Kinases
Kinases
Protein
Serine/Threonine Kinases
•
352 human genes
•
2 families of Ser/Thr kinases
1. receptor Ser/Thr kinases (12)
2. cytoplasmic Ser/Thr kinases (340)
Ser/Thr and Tyr protein kinases have similar structures
5 β-sheets + 1 α-helix
Domains I-IV: Orient and
interact with the Mg-ATP
complex that gives γ-phosphate
N-term lobe
ATP
molecule
Domain V: Links both lobes
Domains VI-XI: Interact with
substrate and initiate the
transfer of phosphate
C-term lobe
Mostly α-helices
Human CDK2
Conserved structure of Ser/Thr and Tyr kinase domains
Typically 250-300 aa
Phosphorylation
site(s)
Interacts with
and orients ATP
GxGxxG
K
E
I
II
III
Activation loop
(T-loop)
Hinge
region
IV
V
HRDxxxxN
VIa
N-terminal lobe
VIb
Act as a base
acceptor (catalytic)
DFG APE
D
VII
IX
VIII
R
X
C-terminal lobe
The HRDxxxxN motif in subdomain VIb
varies between Ser/Thr and Tyr kinases:
The consensus signature of Ser/Thr
kinases: HRDLKxxN
The consensus signature of Tyr kinases:
HRDLAARN or HRDLRAAN
The nine amino acids
in red are invariable
XI
Domain structure of Ser/Thr and Tyr kinases
There are 9 invariable amino acids
(here in PKA)
Gly52 Lys72 Glu91
Asp166 Asn171 Asp184
Glu208 Asp220 Arg280
The highly conserved residues are
identified by an arrow. The region
underlined corresponds to the activation
loop, which often contains one or several
phosphorylation sites (underlined in black).
3D localization of the 9 invariable amino acids in PKA
Where are these
residues located in
the folded protein?
Typical experimental mutations in protein kinases:
•
•
Mutation of Lys in subdomain II (K to R)
Mutations of Asp in subdomain IX (D to A)
The (un)targeted cancer kinome
1970s
Discovery of the first oncogene, vSrc, as an enzyme with Tyr kinase activity
1980s
First potent but non-selective tools for protein kinase inhibition (staurosporine)
2001
Approval of the first kinase inhibitor, imatinib (Gleevec), for the treatment of CML
by targeting BCR-Abl
Since 2001, only nine small-molecule kinase inhibitors have been
approved for cancer (Abl, PDGFR, cKit, EGFR and VEGFR family)
How much do we know about protein kinases?
What percentage of the kinome has been studied?
25-50% of the kinome remains unexplored!!!
Federov et al. (2010) Nature Chem Biol
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
Control of protein kinase activity
1. Regulated by ligands
- EGFR dimerization
2. Regulation by second
messengers
- PKA, PKC and Ca2+/CaM
kinases
3. Regulation by
phosphorylation
- Autophosphorylation
(most kinases)
- Upstream kinase (ERK,
RSK, etc)
4. Regulation by
regulatory subunits
- CDKs are dependent on
cyclins
- PI3K requires regulatory
subunit
5. Regulation by
interaction prot-prot
- Src and GSK3
intramolecular interactions
6. Regulation by
synthesis/degradation
- Mos, ERK3
Control of protein kinase activity
1. Regulated by ligands
- EGFR dimerization
2. Regulation by second
messengers
- PKA, PKC and Ca2+/CaM
kinases
3. Regulation by
phosphorylation
- Autophosphorylation
(most kinases)
- Upstream kinase (ERK,
RSK, etc)
4. Regulation by
regulatory subunits
- CDKs are dependent on
cyclins
- PI3K requires regulatory
subunit
5. Regulation by
interaction prot-prot
- Src and GSK3
intramolecular interactions
6. Regulation by
synthesis/degradation
- Mos, ERK3
Control of protein kinase activity
Activation loop sequences
1. Regulated by ligands
- EGFR dimerization
2. Regulation by second
messengers
- PKA, PKC and Ca2+/CaM
kinases
3. Regulation by
phosphorylation
- Autophosphorylation
(most kinases)
- Upstream kinase (ERK,
RSK, etc)
4. Regulation by
regulatory subunits
- CDKs are dependent on
cyclins
- PI3K requires regulatory
subunit
5. Regulation by
interaction prot-prot
- Src and GSK3
intramolecular interactions
6. Regulation by
synthesis/degradation
- Mos, ERK3
PKA
JNK
Control of protein kinase activity
1. Regulated by ligands
- EGFR dimerization
2. Regulation by second
messengers
- PKA, PKC and Ca2+/CaM
kinases
3. Regulation by
phosphorylation
- Autophosphorylation
(most kinases)
- Upstream kinase (ERK,
RSK, etc)
4. Regulation by
regulatory subunits
- CDKs are dependent on
cyclins
- PI3K requires regulatory
subunit
5. Regulation by
interaction prot-prot
- Src and GSK3
intramolecular interactions
6. Regulation by
synthesis/degradation
- Mos, ERK3
The Regulation of Cyclin-Dependent Kinase (CDK)
In the absence of cyclin, the C helix of CDK (also called the PSTAIRE helix) is
rotated so as to move a crucial catalytic glutamate out of the active site
(DeBondt et al., 1993). This is correlated with an inhibitory conformation of the
activation loop. Cyclin binding reorients the PSTAIRE helix so as to place the
glutamate within the active site (Jeffrey et al., 1995). The activation loop adopts
a near-active conformation upon cyclin binding, and its subsequent
phosphorylation further stabilizes the active form (Russo et al., 1996).
Control of protein kinase activity
1. Regulated by ligands
- EGFR dimerization
2. Regulation by second
messengers
- PKA, PKC and Ca2+/CaM
kinases
3. Regulation by
phosphorylation
- Autophosphorylation
(most kinases)
- Upstream kinase (ERK,
RSK, etc)
4. Regulation by
regulatory subunits
- CDKs are dependent on
cyclins
- PI3K requires regulatory
subunit
5. Regulation by
interaction prot-prot
- Src and GSK3
intramolecular interactions
6. Regulation by
synthesis/degradation
- Mos, ERK3
The Regulation of Src Tyrosine Kinase
In Src, intramolecular interactions between the phosphorylated tail and the SH2
domain, and between the SH2-kinase linker and the SH3 domain, stabilize
inhibitory conformations of both helix C and the activation loop (Schindler et al.,
1999; Xu et al., 1999). The conformation of C in the off state is quite similar to
that seen in CDK. Disengagement of the SH2 domain by dephosphorylation of the
tail (at Tyr527), combined with phosphorylation of the activation loop (at Tyr416),
allows the C helix to move into an active conformation.
Control of protein kinase activity
1. Regulated by ligands
- EGFR dimerization
2. Regulation by second
messengers
- PKA, PKC and Ca2+/CaM
kinases
3. Regulation by
phosphorylation
- Autophosphorylation
(most kinases)
- Upstream kinase (ERK,
RSK, etc)
4. Regulation by
regulatory subunits
- CDKs are dependent on
cyclins
- PI3K requires regulatory
subunit
5. Regulation by
interaction prot-prot
- Src and GSK3
intramolecular interactions
6. Regulation by
synthesis/degradation
- Mos, ERK3
Catalytically Inactive Protein Kinases
Inactive protein kinases
• ~ 50 kinase domains that are mising a catalytic residue and are predicted to be inactive
• non-catalytic role: modulation, scaffold, other?
• pathological roles (ex: Jak2, B-Raf, HER3)
Schematic of Janus Kinase Structure
Janus kinases comprise FERM, SH2, pseudokinase, and kinase
domains. The FERM domain mediates receptor interactions. Both
the FERM and pseudokinase domains regulate catalytic activity.
Fig. 2 Involvement of the cytokine receptor-tyrosine kinase axis in
MPN oncogenesis. The four main myeloid growth factor receptors
involved in MPN pathogenesis are represented with their schematic
principal downstream signaling involving the binding of JAK2, and the
phosphorylation of PI3K, AKT, STATs, and MAPK (red arrows and
brackets). The adaptor and E3 ubiquitinligase C-CBL down-regulates cKIT and JAK2 signaling. Red stars indicate the oncogenic mutations that
occur in MPN resulting in a constitutive or enhanced downstream signaling
(red) with modulation of transcription and protein levels for cell cycle,
proliferation, and apoptosis-related factors.
Protein Kinases as Therapeutic Targets
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
Determinants of substrate specificity
Factors that determines
substrate specificity
1.
Structure
of the
catalyticare
cleft
If protein
kinases
very similar…
2.
Phosphoacceptor sites in substrates
3.
Subcellular targeting
4.
ERK2
Akt
JAK
B-Raf
…what are the factors
that will determine
substrate
Docking
sites specificity?
1. Structure of the catalytic cleft – Tyr versus Ser/Thr
ATP
Hydroxyl
side chain
Complementary of
cleft with substrate
• Substrate is recognized
by subdomains VIB and VIII
• Depth of cleft provides
some specificity (Tyr vs
Ser/Thr)
• Complementarity of
residues in cleft in terms of
hydrophobicity and charge
Figure 1 | The catalytic clefts of Tyr kinases are deeper than those of Ser/Thr kinases and this determines their specificities for Tyr or
Ser/Thr. a | The structure of the Tyr kinase domain of the insulin receptor (IRK) bound to a Tyr substrate peptide (Protein Data Bank
(PDB) ID: 1IR3) and b | a modelled Ser substrate peptide. Unlike Ser, Tyr extends far enough into the catalytic cleft to be efficiently
phosphorylated. c | The structure of the Ser/Thr kinase cyclin-dependent kinase-2 (CDK2) bound to a Ser substrate peptide (PDB ID:
1QMZ) and d | a modelled Tyr substrate peptide. Tyr is too large to fit into the catalytic cleft. Structures and modelled substrates were
created using PyMol134. ATP is shown in red. Most of the peptide substrate is black, with hydroxyl sidechain oxygens shown in red.
Ser versus Thr residues, is there a difference?
The assumption is that there is little preference for one or another,
but in fact, this assumption is incorrect…
In proteins from humans, worm and
yeast, the expected Ser:Thr ratio is:
1.5 : 1
- 8,5% of residues are Ser
- 5,7% of residues are Thr
- 3.0% of residues are Tyr
However…
• Partial acid hydrolysis and phosphoamino-acid analysis of 32P labelled
cells gives a ratio of 9:1 pSer:pThr.
• Recent global mass spectrometry
studies
of phosphorylation have
What happens
in reality?
found the distribution of pSer, pThr and ptyr sites to be around 79.3%,
16.9% and 3.8%, respectively, or a 5:1 ratio of pSer:pThr.
The reality is that most Ser/Thr kinases have a preference for phosphorylating Ser residues
and
Most Ser/Thr phosphatases show a striking bias towards dephosphorylating Thr residues.
2. Phosphoacceptor sites in substrates
• Consensus phosphorylation sequences : sequences situated just before and after the
phospho-acceptor residue (Ser, Thr, Tyr)
• In most cases, four residues before (minus) and after (plus) the phosphorylation site
determine specificity for the catalytic cleft
• Complementarity of residues in cleft and substrate in terms of hydrophobicity and charge
A method to determine the consensus phosphorylation sequence
-5 -4 -3 -2 -1
0
+1 +2 +3 +4
Y-A-X-X-X-X-X-S/T-X-X-X-X-A-G-K-K(biotin)
Array peptide library on streptavidin membrane
Incubate with kinase and [γ-32P]-ATP
Quench and wash membrane
Expose to phosphor screen
Scanning the human proteome for probable substrates
Translate into probabilities
for each position
P
G
A
C
S
T
V
I
L
M
F
Y
W
H
K
R
Q
N
D
E
pT
pY
-5
0.63
1.55
0.62
0.82
0.72
0.56
0.47
0.57
0.35
0.58
0.62
0.55
0.63
0.91
1.70
7.18
0.61
0.53
0.18
0.22
0.38
0.71
-4
-3
1.15 0.80
1.19 0.20
0.96 0.26
0.94 0.36
1.33 0.37
0.88 0.39
0.92 0.32
0.60 0.38
0.77 0.24
1.22 0.42
0.82 0.38
0.78 0.35
0.97 0.28
0.96 0.37
1.75 0.67
2.17 13.58
1.06 0.33
0.79 0.15
0.31 0.07
0.42 0.09
0.45 0.04
0.66 0.03
-2
0.58
1.28
0.30
1.03
4.45
0.70
0.16
0.23
2.33
0.29
0.07
0.61
0.03
2.30
2.95
1.53
0.61
0.41
0.06
0.09
0.02
0.13
-1
2.68
1.33
1.18
0.19
1.31
0.71
0.63
0.41
0.65
0.85
0.81
1.11
0.77
2.03
1.22
0.93
0.83
1.28
0.69
0.39
0.17
0.46
+1
0.26
0.41
1.05
0.26
1.90
1.83
1.75
1.48
0.86
1.29
1.38
1.41
0.73
1.37
0.52
0.54
0.80
0.76
0.52
0.90
0.77
1.13
+2
1.18
1.48
1.26
0.15
1.59
0.95
0.21
0.35
0.80
0.78
0.72
1.95
0.12
2.01
0.93
1.31
0.99
1.55
1.22
0.44
1.34
0.84
+3
1.23
1.05
1.16
0.47
1.97
0.66
0.89
0.77
0.62
0.99
1.12
1.03
0.94
1.12
0.53
1.08
1.13
0.81
1.22
1.20
1.60
1.42
+4
1.69
1.14
0.67
0.43
2.23
1.12
0.85
0.63
0.61
0.95
0.85
0.60
0.68
1.74
0.92
0.89
1.60
0.77
1.18
0.76
1.02
1.00
Prediction of kinase-specific phosphorylation sites
1. ScanProsite: consists of documentation entries describing protein domains, families and functional sites as well as associated patterns
and profiles to identify them (de Castro, et al., 2006; Hulo, et al., 2008).
2. ELM: is a resource for predicting functional sites in eukaryotic proteins (Puntervoll, et al., 2003).
3. PhosphoMotif Finder: contains known kinase/phosphatase substrate as well as binding motifs that are curated from the published
literature. It reports the PRESENCE of any literature-derived motif in the query sequence (Amanchy, et al., 2007).
4. PREDIKIN 1.0: produces a prediction of substrates for serine/threonine protein kinases based on the primary sequence of a protein
kinase catalytic domain (Brinkworth, et al., 2003).
5. ScanSite 2.0: searches for motifs within proteins that are likely to be phosphorylated by specific protein kinases or bind to domains
such as SH2 domains, 14-3-3 domains or PDZ domains (Obenauer,et al., 2003).
6. NetPhosK 1.0: produces neural network predictions of kinase specific eukaryotic protein phosphoylation sites. Currently NetPhosK
covers 17 kinases (Blom, et al., 2004).
7. GPS 2.1:The current version of GPS system. We renamed the tool as the Group-basedPrediction System. GPS 2.1 software was
implemented in JAVA and could predict kinase-specific phosphorylation sites for 408 human Protein Kinases in hierarchy (Xue, et al.,
2008).
8. KinasePhos 2.0: New version of kinase-specific phosphorylation site prediction tool that is based the sequenece-based amino acid
coupling-pattern analysis and solvent accessibility as new features of SVM (support vector machine) (Wong, et al., 2007).
9. NetPhorest: is a non-redundant collection of 125 sequence-based classifiers for linear motifs in phosphorylation-dependent signaling.
The collection contains both family-based and gene-specific classifiers (Miller, et al., 2008).
http://scansite.mit.edu/
3. Subcellular targeting
Roles of scaffolding proteins
1. Specificity of signaling modules
2. Amplification of signal
3. Spatial restriction to certain substrates
4. Prevents unwanted phosphorylation
AKAP mediated subcellular targeting
Scaffolds within the MAPK pathways
4. Docking sites (the MAPK example)
• Protein kinases often (but
not always) form stable
interactions with their
substrates and regulators
• Docking sites may simply
function by increasing the
local concentration of
substrate around the kinase
• Docking sites may also
precisely align the substrate
with the kinase catalytic
domain
• Also serves to activate or
inhibit the kinase activity
Conditional (phospho-dependent) docking sites
• PLK1
Contains a polo-box binding domain
(PBD) that binds to phosphorylated
substrates that have the consensus
S-pS/T-P/X. PLK1 may target
substrates that have been
previously phosphorylated by CDK1,
a proline-directed kinase.
• GSK3
Often requires a phosphorylated Ser
residue at position +4 for efficient
phosphorylation (priming). GSK3 is
itself inactivated by a
phosphorylation event in its Nterminus that loops back and inhibits
the kinase by binding to a docking
groove.
Many docking sites have been included in scansite for general searches…
http://scansite.mit.edu/
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
1. Change of conformation: the MAP kinase ERK2
Phosphorylation-mediated conformational changes usually results from the creation of new
hydrogen bonds between the phosphate groups and neighboring amino acid residues.
Structural examination of phosphorylated proteins revealed two types
of hydrogen bonds:
- with the positively-charged guanidinium side chain of arginine residues
- with the main-chain nitrogens of α-helices
The hydrogen bondings induced by phosphorylation consequently alter the conformations of the target
proteins, thereby modulating their functional properties.
Dual phosphorylation of the
activation loop segment residues (TEY)
changes the conformation by three ways:
1. Orients the C helix
2. Promotes lobe closure (phospho-TEY
associates with Arg residues in N-term lobe)
3. Organizes the C-term extension
The Regulation of MAP Kinase by Phosphorylation of the Activation Segment. Phosphorylation of the activation
segment creates a network of interactions that properly orient the C helix, promote lobe closure, and organize the Cterminal extension (shown in yellow) into a functionally important homodimerization interface (Zhang et al., 1994;
Canagarajah et al., 1997; Khokhlatchev et al., 1998).
2. Steric hindrance: Isocitrate dehydrogenase
Isocitrate dehydrogenase (IDH) is an enzyme which participates in the citric acid cycle. It catalyzes
the third step of the cycle: the oxidative decarboxylation of isocitrate, producing alphaketoglutarate (α-ketoglutarate) and CO2 while converting NAD+ to NADH. This is a two-step process,
which involves oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by
the decarboxylation of the carboxyl group beta to the ketone, forming alpha-ketoglutarate.
isocitrate
Phosphorylation blocks substrate binding to isocitrate dehydrogenase. A,
Surface representation with isocitrate (blue) bound to the active site. B,
Phosphorylation of serine 113 (yellow) blocks isocitrate binding.
3. Modification of protein interaction
Table 2 | Phosphotyrosine-binding domains
SH2
PTB
pY
N-P-X-pY
Diverse
RTK Signaling
SH2 Domain
SH2 domains contain a central anti-parallel b-sheet surrounded by two ahelices. The phosphopeptide generally binds as an extended b-strand that lies
at right angles to the SH2 b-sheet. Conserved residues contribute to the
hydrophobic core or are involved in pY recognition while more variable
residues contribute to specific recognition of C-terminal residues. An
invariant Arg residue in the SH2 domain coordinates the phosphate
oxygens of pY and is essential for high affinity phosphopeptide binding. The
figure shows the SH2 domain of v-src bound to a pYEEI peptide ligand.
• non-catalytic module of ~ 100 amino acids
homologous to c-Src
• binding affinity for Tyr-P >> Tyr (Kd ~50-500
nM)
• SH2 binds to specific phosphopeptide sequences
(C-terminal)
• autophosphorylation of RTKs acts as molecular
‘switch’ for signal transduction
Topics for today’s lecture
1. PROTEIN PHOSPHORYLATION AND DATABASES
2. PROTEIN KINASES
3. CONTROL OF PROTEIN KINASE ACTIVITY
BREAK
4. DETERMINANTS OF SUBSTRATE SPECIFICITY
5. IMPACT OF PHOSPHORYLATION ON PROTEIN FUNCTION
6. MAPK SIGNALLING PATHWAYS
Protein Kinase Cascades
MAP Kinase Signalling Pathways
Extracellular stimuli
Effectors
The MAP Kinase Family
ERK1
ERK2
ERK5
p38α
p38β
p38γ
p38δ
JNK1
JNK2
JNK3
ERK7
NLK
ERK3
ERK4
GFLTEYVATRWYR
IML217
180
GFLTEYVATRWYRAPEIML198
216
YFMTEYVATRWYRAPELML234
177
DEMTGYVATRWYRAPEIML195
177
EEMTGYVATRWYRAPEIML195
180
SEMTGYVVTRWYRAPEVIL198
177
AEMTGYVVTRWYRAPEVIL195
180
FMMTPYVVTRYYRAPEVIL198
180
FMMTPYVVTRYYRAPEVIL198
218
FMMTPYVVTRYYRAPEVIL236
172
QAVTEYVATRWYRAPEVLL190
283
RHMTQEVVTQYYRAPEILM301
186
GHLSEGLVTKWYRSPRLLL204
183
GYLSEGLVTKWYRSPRLLL201
199
Pleiotropic Functions of MAP Kinases
Wnt signaling,
HSC stroma
embryo growth,
lung function
proliferation,
angiogenesis
proliferation,
survival,
senescence
inflammation,
development,
cell cycle
neural
apoptosis,
obesity,
T-cell function
DDR,
autophagy
Multiple Roles of ERK1/2 in Cell Proliferation
Activation of the Ras-ERK1/2 Pathway in Cancer
Regulation of ERK1/2 MAP Kinases by Mitogens
Mitogens
A-Raf
B-Raf
C-Raf
MEK1
MEK2
ERK1
ERK2
?
> 160 cellular targets
growth
proliferation
survival
Activation of MAP Kinases by Phosphorylation
Dual phosphorylation of the
activation loop segment residues (TEY)
changes the conformation by three ways:
1. Orients the C helix
2. Promotes lobe closure (phospho-TEY
associates with Arg residues in N-term lobe)
3. Organizes the C-term extension
Dephosphorylation and Inactivation of MAP Kinases
Regulation of ERK1/2 Pathway by Feedback Loops
Targeting the ERK1/2 Pathway in Cancer
vemurafenib
selumetinib
Lessons from Raf Inhibition in Melanoma
A 38-year-old man with BRAF-mutant melanoma and miliary,
subcutaneous metastatic deposits. Photographs were taken (A) before
initiation of PLX4032, (B) after 15 weeks of therapy with PLX4032, and
(C) after relapse, after 23 weeks of therapy.
Questions?
Some general references...
• Ubersax and Ferrell (2007) Nature Rev Mol Cell Biol 8:530-41
• Manning and Cantley (2007) Cell 129:1261-74
• Frame and Cohen (2001) Biochem J 359:1-16
• Manning et al. (2002) Science 539:1912-34
• Manning et al. (2002) Trends Biochem Sci 27:514-20
• Adams (2001) Chem Rev 101:2271-90