Transcript MAP Kinase Pathways

Lodish  Berk  Kaiser  Krieger  scott  Bretscher  Ploegh  Matsudaira
MOLECULAR CELL BIOLOGY
SEVENTH EDITION
CHAPTER 16
Signaling Pathways That Control
Gene Expression
2015-10-13
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Chapter Opener
A molecular valentine-dimerized extracellular domain of the
epidermal growth factor receptor (red, yellow, and green) bound to
two molecules of epidermal growth factor (magenta).
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Extracellular signals can have both short- and long-term
effects on cells.
Short-term effects are usually triggered by modification of existing
proteins or enzymes, as we saw in Chapter 15.
Many extracellular signals also affect gene expression and thus
induce long-term changes in cell function. Long-term changes
include alterations in cell division and differentiation, such as
occur during development and cell fate determination.
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No signaling pathway acts in isolation.
Many cells respond to multiple types of hormones and other
signaling molecules; some mammalian cells express ~100
different types of cell-surface receptors, each of which binds a
different ligand.
Since many genes are regulated by multiple transcription factors
that in turn are activated or repressed by different intracellular
signaling pathways, expression of anyone gene can be
regulated by multiple extracellular signals.
Especially during early development, such "cross talk" between
signaling pathways and the resultant sequential alterations in the
pattern of gene expression eventually can become so extensive
that the cell assumes a different developmental fate.
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Figure 16.1 Several common cell-surface receptors and signal transduction pathways.
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16.1 Receptors That Activate Protein Tyrosine Kinases
Figure 16.2 Overview of signal transduction pathways triggered by receptors that activate protein
tyrosine kinases.
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Numerous Factors Regulating Cell Division and Metabolism
Are Ligands for Receptor Tyrosine Kinases (RTK)
These RTK ligands include many, such as
nerve growth factor (NGF),
platelet-derived growth factor (PDGF),
fibroblast growth factor (FGF), and
Epidermal growth factor (EGF),
that stimulate proliferation and differentiation
of specific cell types.
Others, such as insulin, regulate expression of multiple genes
that control sugar and lipid metabolism in liver, muscle, and
adipose (fat) cells.
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Binding of Ligand Promotes Dimerization of an RTK and
Leads to Activation of Its Intrinsic Kinase
Figure 16.3 General structure and activation of receptor tyrosine kinases (RTKs).
Phosphorylation causes the lip to move out of the kinase catalytic
site, thus increasing the ability of ATP and the protein substrate to
bind.
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Figure 16.4 Ligand-induced dimerization of HER1, a human receptor for epidermal growth factor
(EGF).
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Homo- and Hetero-oligomers of Epidermal Growth Factor Receptors
Bind Members of the Epidermal Growth Factor Superfamily
Figure 16.7 The HER family of receptors and their ligands.
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Understanding the HERs has helped explain why a particular
form of breast cancer is so dangerous and has led to an
important drug therapy.
Amplification of the HER2 gene occurs in approximately 25
percent of breast cancers, resulting in overexpression of HER2
protein in the tumor cells. Breast cancer patients with HER2
overexpression have a worse prognosis, including shortened
survival, than do patients without this abnormality.
Discovery of the role of HER2 overexpression in certain breast
cancers led researchers to develop monoclonal antibodies
specific for the HER2 protein. These have proved to be
effective therapies for those breast cancer patients in which
HER2 is overexpressed, reducing recurrence by about 50
percent in these patients.
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Cytokines Influence Development of Many
Cell Types
• Erythropoietin (Epo)
• Granulocyte Colony Stimulating
Factor (G-CSF)
• Thrombopoietin (Tpo)
• Prolactin (Prl)
• Growth Hormone (GH)
interleukin 2
IL-4
interferon
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Cytokine Receptors and the JAK-STAT Pathway
Cytokine Receptors and Receptor Tyrosine Kinases Share Many
Signaling Features
• Hormone- induced receptor dimerization
• Activation of JAK protein tyrosine kinase
• Phosphorylation of tyrosine residues on the receptor
• Receptor phosphotyrosine residues bind to SH2 domains on several
signal transduction proteins
• Activation of Stat transcription factors
• Partnering of Stats with other transcription factors
• Termination of signaling by activation of protein
tyrosine phosphatases
• Inhibition of signaling by CIS proteins containing only SH2
domains
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STAT:
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th
HEMATOPOIESIS
Epo acts to stimulate
the proliferation
and differentiation
of erythroid
progenitor
cells to mature
red cells
Myeloid
G-CSF
CFU-GM
Granulocytes
IL-3, GM-CSF, SCF
IL-6
M -CSF
CFU-MEG
SCF
Monocytes
TPO
IL-3, GM-CSF
CFU-GEMM
Platelets
BFU-E
Epo
SCF
CFU-E
Epo
GM -CSF
IL-3
Erythrocytes
Pluripotent
Stem Cell
CFU-Eo
IL-3, GM-CSF
Eosinophils
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Lymphoid
Progenitor
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Cytokines influence development of many cell types
Prevent apoptosis
Erythropoietin
Kidney
Oxygen
HIF-1
3 to 5 terminal cell
division
Colony-forming unit
(CFU)
Semi-solid medium (methylcellulose)
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Receptor-Associated JAK Kinases Activate STAT Transcription
Factors Bound to a Cytokine Receptor
The JAK2 kinase is tightly bound to the cytosolic domain of
the erythropoietin receptor (EpoR).
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ERYTHROPOIETIN (EPO)
THE PROTEIN THAT CONTROLS
RED BLOOD CELL PRODUCTION
165 AMINO ACIDS
~ 40% CARBOHYDRATE
• PRODUCED BY THE KIDNEY IN RESPONSE TO LOW O2PRESSURE IN THE
BLOOD
• BINDS TO EPO RECEPTORS ON THE SURFACE OF ERYTHROCYTE
PROGENITOR CELLS IN THE BONE MARROW
• STIMULATES THESE CELLS TO DIVIDE 5 TO 7 TIMES; EACH OF THE ~30
TO 100 DAUGHTERS THEN DIFFERENTIATES INTO A RED BLOOD CELL
• USED CLINICALLY TO TREAT ANEMIA CAUSED BY KIDNEY FAILURE OR
BY DISEASES SUCH AS AIDS
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Epo
synthesis
induced
in response
EPO REGULATES
REDis
CELL
MASS IN RESPONSE
TO TISSUEto
HYPOXIA
hypoxia
RED CELL MASS
Tissue pO2
+
Epo
By means of the oxygen-sensitive transcription factor HIF-1, the kidney
cells respond to low oxygen by synthesizing more erythropoietin and
secreting it into the blood.
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Signal Transduction Proteins
that Bind to the Erythropoietin
Receptor
JAK2
130 kDa CYTOSOLIC PROTEIN
TYROSINE KINASE
HOMOLOGOUS TO JAK1 AND
TYK2
WIDELY EXPRESSED IN
HEMATOPOIETIC CELLS AND
FIBROBLASTS
NO SH2 OR SH3 DOMAINS
N-T ERMINAL
CON SERVED
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PSEUDO-KINASE
DOMAIN
MODULATOR Y
KIN ASE D OMAIN
T YROSINE KINASE
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Figure 16.11 Surface model of an SH2 domain bound to a phosphotyrosine-containing peptide.
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Figure 16.12 Recruitment of intracellular signal transduction proteins to the cell membrane by
binding to phosphotyrosine residues in receptors or receptor-associated proteins.
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P
Dimerization of STAT proteins leads to
formation of a functionally active
transcription factor
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P
DIMER OF STAT
PROTEIN IS
FUNCTIONAL
TRANSCRIPTION
FACTOR: MOVES
INTO NUCLEUS,
BINDS TO DNA, AND
ACTIVATES
TRANSCRIPTION OF
THE BCL-X ANTIAPOPTOTIC PROTEIN
AMONG OTHERS
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P
DIMERIZATION OF
STAT PROTEIN
BY BINDING OF
PHOSPHOTYROSINE
TO THE SH2 DOMAIN
ON THE PARTNER
SUBUNIT
STAT
STAT
STAT
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Dominant negative mutant
Tyr  Phe
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FIGURE JAK-STAT signaling
pathway. Because the
STAT homodimer has two
phosphotyrosine–SH2 domain
interactions, whereas the receptor-STAT
complex is stabilized by
only one such interaction,
phosphorylated STATs tend not to
rebind to the receptor. The STAT dimer,
which has two exposed
nuclear-localization signals (NLS),
moves into the nucleus, where
it can bind to promoter sequences and
activate transcription of
target genes.
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Different STATs activate different genes in different cells.
In erythroid progenitors:erythropoietin: STAT5Bcl-xL
Indeed, mice lacking STAT5 are highly anemic because many of the
erythroid progenitors undergo apoptosis even in the presence of high
erythropoietin levels.
Such mutant mice produce some erythrocytes and thus
survive, because the erythropoietin receptor is linked to other
anti-apoptotic pathways that do not involve STAT proteins (PI-3 kinase,
PLC, MAP kinase…).
Because different cell types have unique complements of transcription
factors and unique epigenetic modifications on their chromatin, the
genes that are available to the activated by any STAT are also different.
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FIGURE 16-8 Overview of signal-transduction pathways
triggered by ligand binding to the erythropoietin receptor
(EpoR),
a typical cytokine receptor.
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Multiple Mechanisms Down-Regulate Signaling from RTKs
and Cytokine Receptors
Receptor-Mediated Endocytosis
Prolonged treatment of cells with ligand
desensitization response
clathrin-coated pits into endosomes
HER1 receptors for this ligand are relatively long-lived,
with an average half-life of 10 to 15 hours.
HER1 mutants that lack kinase activity
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Down-regulation of RTK signaling occurs by endocytosis
and lysosomal degradation
lysosomal degradation
Ubiquitin
E3 ubiquitin ligase; c-Cbl; "tag“; proteasome
TGF-b: Ski
Cytokines: SOCS, SPH-1
Receptor-mediated endocytosis
Phospotyrosine phosphatases
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polyubiquitinated
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SOCS (CIS) is induced by STAT
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16.2 The Ras/MAP Kinase Pathway
Mutant Ras: many types of human cancer
bind but cannot hydrolyze GTP, are permanently in the
“on” state and contribute to neoplastic transformation.
Viral Ras: H(arvey)-ras, Ki-(rsten)-rasA, Ki-rasB and the
N(euroblastoma)-ras gene
Determination of the three-dimensional
structure of the Ras-GAP complex explained
the puzzling observation that most oncogenic, constitutively
active Ras proteins (RasD) contain a mutation at position 12.
Replacement of the normal glycine-12 with any
other amino acid (except proline) blocks the functional
binding of GAP, and in essence “locks” Ras in the active
GTP-bound state.
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An Adapter Protein and Guanine Nucleotide–
Exchange Factor Link Most Activated Receptor
Tyrosine Kinases to Ras
Fibroblast cells (3T3)  remove serum (growth factors)  arrest G0/G1
RasD
Proliferation (S phase)
arrest
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+ PDGF and EGF
+ anti-Ras
+ anti-Raf
How does binding of a growth factor (e.g., EGF) to
an RTK (e.g., the EGF receptor) lead to activation
of Ras?
Two cytosolic proteins— GRB2 and Sos —provide the key links
(Figure 14-16).
An SH2 domain in GRB2 binds to a specific phosphotyrosine residue in
the activated receptor.
GRB2 also contains two SH3 domains, which bind to and activate Sos.
GRB2 thus functions as an adapter protein for the EGF receptor.
Sos is a guanine nucleotide–exchange protein (GEF),
which catalyzes conversion of inactive GDP-bound Ras to
the active GTP-bound form.
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By use of this screen, researchers identified the genes encoding
three important proteins in the Sev pathway (see Figure
14-16):
an SH2-containing adapter protein exhibiting 64
percent identity to human GRB2;
a guanine nucleotide–exchange factor called Sos (Son of Sevenless)
exhibiting 45 percent identity with its mouse counterpart;
and a Ras protein exhibiting 80 percent identity with its mammalian
counterparts.
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Binding of Sos Protein to Inactive Ras Causes a Conformational
Change That Activates Ras
The adapter protein GRB2 contains two SH3 domains,
which bind to Sos, a guanine nucleotide–exchange factor, in addition to
an SH2 domain, which binds to phosphotyrosine residues in RTKs.
Formation of this complex depends on the ability of GRB2 to bind
simultaneously to the receptor and to Sos.
Thus receptor activation leads to relocalization of Sos from the cytosol
to the membrane, bringing Sos near to its substrate, namely,
membrane-bound RasGDP.
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MAP Kinase Pathways
Activated Ras promotes formation at the membrane of signaling
complexes containing three sequentially acting protein kinases that are
associated with a scaffold protein.
This kinase cascade culminates in activation of MAP (mitogenactivating protein) kinase, a serine/threonine kinase also known as ERK
(extracellular regulated kinase).
After translocating into the nucleus, MAP kinase can phosphorylate
many different proteins, including transcription factors that regulate
expression of important cell-cycle and differentiation-specific proteins.
Activation of MAP kinase in two different cells can lead to
similar or different cellular responses, as can its activation in
the same cell following stimulation by different hormones.
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An Adapter Protein and Guanine Nucleotide–
Exchange Factor Link Most Activated Receptor
Tyrosine Kinases to Ras
Fibroblast cells (3T3)  remove serum (growth factors)  arrest G0/G1
RasD
Proliferation (S phase)
arrest
GF   …….. Ribosomal S6*
ribosomal S6 kinase* (RSK)
(mitogen-activating protein) MAP kinase*
(P-Y and P-S MAPK)
MEK* (MAPKK)
Raf (MAPKKK)
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+ PDGF and EGF
+ anti-Ras
+ anti-Raf
14.4 MAP kinase pathways
Growth factors
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Signals Pass from Activated Ras to a Cascade of Protein Kinases
A remarkable convergence of biochemical and genetic studies in yeast, C.
elegans, Drosophila, and mammals has revealed a highly conserved
cascade of protein kinases that operate in sequential fashion downstream
from activated Ras as follows:
1. Activated Ras binds to the N-terminal domain of Raf, a
serine/threonine kinase.
2. Raf binds to and phosphorylates MEK, a dual-specificity protein
kinase that phosphorylates both tyrosine and serine residues.
3. MEK phosphorylates and activates MAP kinase, another
serine/threonine kinase.
4. MAP kinase phosphorylates many different proteins, including
nuclear transcription factors, that mediate cellular responses.
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Activation of Raf Kinase
This inactive conformation is stabilized by a dimer of the 143-3 protein, which binds phosphoserine residues in a number
of important signaling proteins.
Each 14-3-3 monomer binds to a phosphoserine residue in Raf, one to
phosphoserine-259 in the N-terminal domain and the other to
phosphoserine-621 (see Figure 14-21). These interactions are thought to
be essential for Raf to achieve a conformational state such that it can
bind to activated Ras.
The binding of RasGTP, which is anchored to the
membrane, to the N-terminal domain of Raf relieves the inhibition
of Raf’s kinase activity and also induces a conformational change in Raf
that disrupts its association with 14-3-3. Raf phosphoserine-259 then is
dephosphorylated (by an unknown phosphatase) and other serine or
threonine residues on Raf become phosphorylated by yet other kinases.
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MAP Kinase Regulates the Activity of Many Transcription Factors
Controlling Early-Response Genes
Addition of a growth factor (e.g., EGF or PDGF) to quiescent
cultured mammalian cells in G0 causes a rapid increase
in the expression of as many as 100 different genes. ?????
These are called early-response genes because they are induced well
before cells enter the S phase and replicate their DNA.
One important early-response gene encodes the transcription factor c-Fos.
Together with other transcription factors, such as c-Jun (AP-1) c-Fos
induces expression of many genes encoding proteins necessary for cells
to progress through the cell cycle.
Most RTKs that bind growth factors
utilize the MAP kinase pathway to activate genes encoding
proteins like c-Fos that propel the cell through the cell cycle.
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Figure 16.22 Induction of gene transcription by MAP kinase.
The enhancer that regulates the c-fos
gene contains a serum-response
element (SRE), so named because it is
activated by many growth factors in
serum. This complex enhancer contains
DNA sequences that bind multiple
transcription factors.
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As evidence for this model, abundant expression in cultured mammalian
cells of a mutant dominant negative TCF that lacks the serine residues
phosphorylated by MAP kinase blocks the ability of MAP kinase to
activate gene expression driven by the SRE enhancer.
Moreover, biochemical studies showed directly that phosphorylation
of SRF by active p90RSK increases the rate and affinity of its binding to
SRE sequences in DNA, accounting for the increase in the frequency of
transcription initiation.
Thus both transcription factors are required for maximal growth factor–
induced stimulation of gene expression via the MAP kinase pathway,
although only TCF is directly activated by MAP kinase.
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Scaffold Proteins Isolate Multiple MAP Kinase Pathways in
Eukaryotic Cells
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d) You overexpress Stat that cannot be phosphorylated because its critical
tyrosine has been mutated to a phenylanaline.
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You are studying the response of a fibroblast cell line to treatment with fibroblast
growth factor which you know binds to and acts through a receptor tyrosine
kinase. The cell line grows and divides when treated with growth factor, but do
not in absence of growth factor.
A) You express a mutant form of the fibroblast growth factor receptor (FGFR)
lacking its entire cytoplasmic domain to a level similar to the wild type receptor.
You observe that the cells grow much more slowly than normal when treated
with growth factor. Explain.
B) You inject anti-Ras antibodies that prevent Ras from binding Raf into a few
cells. What is the growth and division phenotype of these cells? Why?
C) You express a constitutively active Ras that remains in its GTP bound form.
You observe that the cells divide and grow even in the absence of growth factor.
Explain.
D) You treat the cells with tyrosine kinase inhibitor, and observe the effect on the
cell growth and division. What do you see and why?
E) FGFR contains a tyrosine that involves in the desensitization of FGFR. You
overexpress FGFR that cannot be phosphorylated the tyrosine because the tyrosine has
been mutated to a phenylanaline. You observe that the cells grow much more faster
than normal when treated with growth factor. Explain.
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