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Molecular physiology of protein kinases
(and phosphatases): Molecules,
mechanisms, medicines…
Figure 3.
From: Eck & Yun. Biochem.
Biophys. Acta 1804(3): 559-566,
2010.
Freely accessible at
http://www.ncbi.nlm.nih.gov/pm
c/articles/PMC2859716/
Michael Eck
Dana-Farber Cancer Institute
Harvard Medical School
~518 protein
kinases in the
human genome…
Kinase signal transduction touches
essentially every aspect of human
physiology.
Kinases of central importance in
regulating cell growth and proliferation,
metabolism, motility, differentiation,
gene expression, intercellular
communication….
Kinases have become important drug
targets in diverse therapeutic
areas, including:
Cancer Immunology & inflammation
Metabolism
Infectious disease
Human Kinome poster available at
kinase.com/human/kinome/
Outline:
Overview – the reaction, structure
Mechanism of regulation by activation loop
phosphorylation – Insulin receptor as an example
Modular signaling domains, SH2, SH3, PH, PTB
Epidermal growth factor receptor (EGFR) signaling –
a representative example of a receptor tyrosine
kinase signaling network
EGFR in Cancer
Non-receptor tyrosine kinases: Src, Abl
SHP2 – a tyrosine phosphatase
Kinases catalyze transfer of the g-phosphate of ATP
to hydroxyl acceptor on substrate protein:
Ser-OH or Thr-OH for serine/threonine kinases
Tyr-OH for Tyrosine kinases
Tyrosine kinases are (almost) unique to higher
eukaryotes:
evolved to facilitate communication/coordination
between cells/tissues. They account for ~90 kinases
in human genome. Ser/thr kinases and signaling
pathways tend to be conserved in all eukaryotes.
Tyrosine kinase signaling pathways interconnect
extensively with ser/thr kinase cascades. For
example, growth factor receptors activate ras/map
kinase pathway.
Protein kinases interconnect with virtually every
regulatory mechanism in the cell: Heterotrimeric Gproteins, small G proteins, small-molecule second
messengers, Ca++, lipid kinases, phospholipases…
The effects of phosphorylation are remarkably diverse. A
few examples:
1) enzyme activation or inhibition [including kinases,
phosphatases and many other enzymes; activation of
glycogen phosphorylase by protein kinase A (PKA) and
phosphorylase kinase is a classic example]
2) Induce protein-protein interactions
3) Induce protein dimerization - for example to activate
transcription factors
4) Alter subcellular localization; nuclear translocation
5) Promote or prevent protein degradation: control
recognition by ubiquitin ligases
Overview of protein kinase structure
Active Site
Kinases:
1. Usually regulated by phosphorylation of activation loop
2. Autoinhibition typical, often by non-phosphorylated act. loop
3. Diverse mechanisms of inactivation/regulation, both within kinase
domain and by associated domains and sequences
a. inactivation deconstructs or blocks active site (many variations)
b. active kinases look very similar in active site
4. Recognize substrate peptides in extended form, b-sheet interactions
in insulin receptor tyrosine kinase (IRTK; only Tyr kinase/substrate
complex available).
5. Sequence specificity varies, determined both at level of kinase domain
and by associated protein-interaction domains.
“Generic” Growth factor receptor: Ligand-induced dimerization
results in activation, autophosphorylation of kinase. Phosphorylated
tyrosines recruit downstream signaling proteins.
Some Tyrosine kinase receptors recruit “adapter” proteins for docking sites…
Specific, regulated interactions
Give rise to a signaling pathway
Figure 4.
From: Hubbard. EMBO J. 16: 5572 – 5581, 1997
Freely accessible at
www.nature.com/emboj/journal/v16/n18/fig_ta
b/7590532a_F4.html#figure-title
Conformational changes in the kinase domain upon activation
Modular domains are key elements of kinase signaling pathways.
They recognize specific sequences in partner proteins, often
phosphorylation-dependent. Many have evolved multiple classes
of specificity…
Figure 2.
From: Pawson and Scott. Science 278(5346): 2075-2080, 1997
Freely accessible at www.sciencemag.org/content/278/5346/2075.full
Assembly of cell regulatory systems through protein interaction domains
Figure 1.
From Pawson and Nash. Science 300(5618):445-52, 2003.
Freely accessible at
www.sciencemag.org/content/300/5618/445.full
Figure 1A.
From: Poy et al. Mol. Cell 4: 555-561,
1999
Freely accessible at
http://www.sciencedirect.com/science/
article/pii/S1097276500802063
Two binding pockets revealed
by solvent-accessible surface
of Lck SH2 domain
SH2 domain architecture:
Central sheet flanked by helices
Conserved pTyr binding pocket
on one side of sheet. Specificity
pocket on the other side binds
3-5 residues C-terminal to pTyr.
Figure 4A.
From: Eck et al. Nature 362:
87-91, 1993
Available at
http://www.nature.com/nature
/journal/v362/n6415/abs/3620
87a0.html
A two-pronged plug: pYEEI-motif peptide inserts into pockets
Figure 2.
From: Boggon & Eck. Oncogene 23: 7918–7927, 2004
Freely accessible at
www.nature.com/onc/journal/v23/n48/full/1208081a.html
Two PH/PTB mechanisms for joint phospholipid/protein recognition
IRS-1: One domain for each job
Figure 5B.
From: Dhe-Paganon et al. Proc.
Natl. Acad. Sci. 96: 8378-8383,
1999
Freely accessible at
http://www.pnas.org/content/96/1
5/8378.full.pdf
Figure 2A, top.
From: Dhe-Paganon et al. Proc.
Natl. Acad. Sci. 96: 8378-8383,
1999
Freely accessible at
http://www.pnas.org/content/96/1
5/8378.full.pdf
Dab1: One domain does it all…
The ErbB signaling network
A simplified diagram showing the complicated ErbB signaling
network. EGFR as the most important ErbB member, has the
broadest target spectrum and so plays a very important role in a
lot of cell processes, including cell growth, differentiation,
adhesion, migration and apoptosis. Most of these processes are
more or less connected with carcinogenesis.
From: Yarden and Sliwkowski. Nat. Rev. Mol. Cell Biol. 2: 127-137, 2001
Available at www.nature.com/nrm/journal/v2/n2/full/nrm0201_127a.html
Structure of EGFR extracellular domain in the absence of EGF
reveals autoinhibition…
Figure 1A.
From: Ferguson et al. Molec. Cell 11: 507-517, 2003
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S10972
76503000479
Comparison of active and inactive conformations and
a model for activation…
Figure 4.
From: Ferguson et al. Molec. Cell 11: 507-517, 2003
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S10972
76503000479
How dimerization of the extracellular portion
of the EGFR induces activation of the kinase domain:
formation of an asymmetric dimer
Figure 7B.
From: Zhang et al. Cell 125: 1137-1149, 2006
Freely accessible at
www.cell.com/fulltext/S0092-8674%2806%2900584-8
.
An allosteric mechanism for activation of the kinase
domain of epidermal growth factor receptor
Figure 4A.
From: Zhang et al. Cell 125: 1137-1149, 2006
Freely accessible at
www.cell.com/fulltext/S0092-8674%2806%2900584-8
Somatic mutations in EGFR kinase domain in lung cancer
EGFR mutations drive ~15% of all
non-small cell lung cancer
mutations highlighted red
(30% or more in some populations)
C-Helix
Figure 1b.
From: Shigematsu and Gazdar. Int. J.
Cancer 118: 257-262, 2006
Freely accessible at
http://onlinelibrary.wiley.com/doi/10.10
02/ijc.21496/full
Mutations activate the kinase
by disrupting the inactive conformation
WT/Lapatinib
L858R/Iressa
(Wood et al. Cancer Res 64: 6652-6659, 2004)
2007)
(Yun et al. Cancer Cell 11: 217-227,
Figure 2.
From: Yun et al. Cancer Cell 11: 217-227, 2007
Freely accessible at
www.sciencedirect.com/science/article/pii/S1535610807000281
Activation of EGFR kinase
Drugs designed to inhibit the EGFR kinase (and other
ErbB-family members) are used to treat cancer
Erlotinib and gefitinib are
EGFR kinase inhibitors used
To treat lung cancer
Lapatinib is an ErbB2
inhibitor used to treat breast
cancer.
These small-molecule
tyrosine kinase inhibitors
(TKIs) work by
Blocking binding of ATP
Protein kinase inhibitors approved for clinical
use (in US)
Table 1.
From: Johnson LN. Q. Rev. Biophys. 42: 1-40,
2009
Available at http://www.esaimcocv.org/action/displayIssue?decade=2000&jid=
QRB&volumeId=42&issueId=01&iid=5827576
Monoclonal antibodies directed against the extracellular domain of the EGFR
are also used to treat cancer, especially cancers that involve over expression of
the receptor. A quick look at three different modes of antibody inhibition: compare
cetuximab/EGFR, Traztuzumab (herceptin)/ErbB2, and pertuzumab/ErbB2
Figures 3A & 4A
From: Li et al. Cancer Cell 7: 301-311, 2005
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S1535610805
000905
Blocks EGF binding site
Three different modes of antibody inhibition: compare cetuximab/EGFR,
Traztuzumab (herceptin)/ErbB2, and pertuzumab/Erbb2
Figure 1.
From: Cho et al. Nature 421: 756-760, 2003
Freely accessible at
http://www.nature.com/nature/journal/v421/n6924/full/
nature01392.html
Blocks dimerization (indirectly)
Three different modes of antibody inhibition: compare cetuximab/EGFR,
Traztuzumab (herceptin)/ErbB2, and pertuzumab/ErbB2
Figure 6.
From: Franklin et al. Cancer Cell 5: 317-328, 2004
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S1535610804000832
Pertuzumab binds Domain II and directly blocks heterodimerization
EGFR is just one type of Receptor Tyrosine kinase…
Figure 1.
From: Lemmon and Schlessinger Cell:141:1117-1134, 2010
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S00928674
10006653
Other tyrosine kinases are entirely cytoplasmic;
these are called non-receptor tyrosine kinases…
Small protein domains/modules important for protein-protein interactions
in signal transduction. They form adaptors to create a signaling network.
They can also regulate catalytic activity when they are coupled to kinases
And other signaling enzymes (phosphatases, lipid kinases, phospholipases…)
Figure 13.25
From: Branden and Tooze. Introduction to Protein Structure (2nd
ed.). 1999. Garland Science. ISBN: 9780815323051
Domain architecture of Src-family tyrosine kinases
REGION
FUNCTION
(SH1: kinase)
N-terminal sequence
Unique domain
Src-homology domain 3 (SH3)
anchors the protein to cell membranes
Src-homology domain 2 (SH2)
binds phosphorylated tyrosine containing sequences
SH2-CD linker
binds intramolecularly to SH3, associates with CD
Catalytic domain (CD)
has enzymatic activity, divided into two lobes
Activation loop
participates in regulation, found between two lobes of CD
C-terminal tail
function not clear
binds proline-rich ligands
when phosphorylated, binds to the SH2 domain
Autoinhibited Src kinase
Figure 3.
From: Xu et al. Mol. Cell 3: 629-638, 1999
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S10972
76500803561
A model for Src activation
Figure 7.
From: Xu et al. Molec. Cell 3: 629-638, 1999
Freely accessible at
http://www.sciencedirect.com/science/article/pii/
S1097276500803561
Common themes, variations...
A multidomain regulatory architecture reused.
Figure 1.
From: Harrison. Cell 112: 737-740, 2003
Freely accessible at
http://crystal.harvard.edu/lib-sch/Harrison-2003-CELL112-737.pdf
Figure 2A (right)
From Nagar et al. Cell 112:
859-871, 2003
Freely accessible at
http://www.sciencedirect.
com/science/article/pii/S0
092867403001946
Chronic myelogenous leukemia (CML) is caused
A 9:22 chromosomal translocation – the Philadelphia
Chromosome. This results in expression of a fusion
Between two proteins, BCR and the Abl tyrosine
kinase
In BCR-Abl, the “cap” region and
N-terminal myristoylation are
absent…yielding a de-repressed
kinase…
Figure 1A.
From Nagar et al. Cell 112: 859-871, 2003
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S0092867403001946
Pak Domain Structure Couples Cdc42
Binding motif with a kinase domain
Figure 1.
From: Lei et al. Cell 102: 387-397, 2000
Freely accessible at https://crystal.harvard.edu/libsch/LeiM-00-Cell-102-387.pdf
Direct activation of a kinase by a guanine nucleotide
binding protein…
Pak autoinhibition and release of inhibition by Cdc42
Figure 6.
From: Lei et al. Cell 102: 387-397, 2000
Freely accessible at https://crystal.harvard.edu/libsch/LeiM-00-Cell-102-387.pdf
A mechanism for intercommunication in signaling pathways…
Protein phosphatases reverse the effects of kinases.
They catalyze the removal of the phosphate group:
-Ser-OH or Thr-OH for serine/threonine
kinases
-Tyr-OH for Tyrosine kinases
Figure 1.
From: Tonks and Neel. Curr. Opin. Cell Biol.
13: 182-195, 2001
Available at
http://www.sciencedirect.com/science/article/
pii/S0955067400001964
The autoinhibited structure of SHP2
reveals steric block of PTPase active site
Adapted from Fig. 2 in Hof et al. Cell 92: 441-450, 1998.
Figure 4.
From: Hof et al. Cell 92: 441-450, 1998
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S009286740
0809381
Figure 6A.
From: Hof et al. Cell 92: 441-450, 1998
Freely accessible at
http://www.sciencedirect.com/science/article/pii/S009286740
0809381
Activation by phosphoprotein binding
to both SH2 domains
Adapted from Fig. 2 in Hof et al. Cell 92: 441-450, 1998.
SHP-2 Tyrosine Phosphatase:
N-terminal SH2 domain blocks active site
==> catalytically inactive
Phosphopeptide binding changes the
shape of the N-SH2 domain so that it no
longer “fits” the PTPase
==> inhibition is released
N + C binding contributes entropically
Common themes...
• Separate domains for recognition and
catalysis
• In Src, SHP2 see how targeting domains
also serve as inhibitors of enzyme activity
• Proper intermolecular contacts release
inhibitory intramolecular interactions
• Result: intrinsic coupling of activity with
targeting (the essence of signal
transduction).
The ErbB signaling network
Figure 1.
From: Yarden and Sliwkowski, Nat. Rev. Mol.
Cell Biol. 2: 127-137, 2001
Available at
http://www.nature.com/nrm/journal/v2/n2/fu
ll/nrm0201_127a.html