Chap. 16 Signaling Pathways That Control Gene Expression

1
Chap. 16 Signaling Pathways That Control Gene
Expression

• Topics
• Receptor Tyrosine Kinases (RTKs)
• The Ras/MAP Kinase Pathway
• Phosphoinositide Signaling
• TGFß Receptors and Smad Signaling

• Goals
• Learn the properties of RTKs.
• Learn about RTK signaling via the Ras/MAP kinase
  signaling pathway.
• Learn the general features of the PI-3 kinase
  signaling pathway.
• Learn about the TGFß/Smad signaling pathway.

EGF receptor bound to EGF
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Major Classes of Cell-surface Receptors
About one dozen classes of cell-surface receptors
occur in human cells. An overview of signaling by
the two receptor systems that are covered in this
chapter (receptor tyrosine kinases, RTKs, and
TGF-ß receptors) is shown in (Fig. 16.1a). The
signal transduction pathways used by RTKs are
summarized in (Fig. 16.2).
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Activation of RTKs via Ligand Binding
Receptor tyrosine kinases (RTKs) regulate cell
differentiation and proliferation. Ligands of
RTKs include nerve growth factor (NGF),
fibroblast growth factor (FGF), and insulin. In
some cases (e.g., the epidermal growth factor
(EGF) RTK), ligand binding causes receptor
dimerization (Fig. 16.3). In other cases (e.g.,
the insulin RTK), binding occurs to pre-existing
dimers. RTKs exhibit intrinsic tyrosine kinase
activity located within their cytosolic domains.
The binding of ligand activates the kinase
domains which cross-phosphorylate the two
monomers of the dimeric receptor. Phosphorylation
first occurs at a regulatory site known as the
activation lip. Phosphorylation of the lip causes
conformational changes that allow the kinase
domain to phosphorylate other tyrosine residues
in the receptor and in signal transduction
proteins.
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Recruitment of Signal Transduction Proteins to
Activated Receptors
Signal transduction system proteins interact with
phosphorylated RTKs via phosphotyrosine binding
domains. Two main binding domains--PTB and SH2
(Src homology domain-2)--within signal
transduction proteins such as the multi-docking
protein known as the insulin receptor substrate-1
(IRS-1) perform this function (Fig. 16.12). The
binding of signaling
proteins either directly to the receptor or to
IRS-1 allows them to be phosphorylated by the
receptor. Some of these signaling proteins are
involved in activation of the Ras GTPase (next
slides). Some such as phosphatidylinositol-3
kinase (PI-3 kinase) participate in
lipid-mediated signaling pathways. RTK signaling
typically is down-regulated by endocytosis of
receptors from the cytoplasmic membrane.
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RTKs and Ras/MAP Kinase Signaling
Nearly all RTKs signal via Ras/MAP kinase
pathways. They also may signal via other
pathways. For example, the insulin receptor uses
the Ras/MAP kinase pathway to regulate gene
expression and the PI-3 kinase pathway to
regulate enzyme activity (e.g., glycogen
synthase). RTK-Ras/MAP kinase signaling controls
cell division, differentiation, and metabolism.
Ras is a monomeric (small) GTPase switch protein
that unlike trimeric G proteins does not directly
bind to receptors. Ras typically relies on
guanine nucleotide-exchange factors (GEFs) for
binding GTP, and on GTPase-activating proteins
(GAPs) for stimulation of GTP hydrolysis. Once
activated, Ras propagates signaling further
inside the cell via a kinase cascade that
culminates in the activation of members of the
MAP kinase family. MAP kinases phosphorylate TFs
that regulate genes involved in the cell cycle
and in differentiation. Mutant RTKs or Ras/MAP
kinase signaling proteins are associated with
nearly all cancers. Dominant Ras mutations that
block GAP binding and lock Ras in the "on" state
promote cancer.
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RTK Activation of Ras
The mechanism by which EGF activates Ras is
illustrated in Fig. 16.17. In Step 1, EGF binding
causes receptor dimerization and
autophosphorylation on cytosolic tyrosines. In
Step 2, the adaptor protein GRB2 binds receptor
phosphotyrosine residues via its SH2 domain. GRB2
contains SH3 domains that allow the GEF protein
known as Sos to bind to the membrane complex. Sos
then recruits Ras to the complex. In the last
step of Ras activation (Step 3), Sos promotes GTP
exchange for GDP on Ras. The activated Ras-GTP
complex then dissociates from Sos, but remains
tethered to the inner leaflet of the cytoplasmic
membrane via a lipid anchor sequence. The active
form of Ras then activates the MAP kinase portion
of the signaling pathway (next two slides).
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Ras Activation of MAP Kinase
Ras activates MAP kinase via a phosphorylation
cascade that proceeds from Ras to Raf kinase, to
MEK kinase, and finally to MAP kinase (Fig.
16.20). MAP kinase then dimerizes and enters the
nucleus (next slide).
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MAP Kinase Activation of Transcription
In the final steps of RTK-Ras/MAP kinase
signaling, MAP kinase phosphorylates and
activates the p90RSK kinase in the cytoplasm
(Fig. 16.22). Both kinases enter the nucleus
where they phosphorylate ternary complex factor
(TCF) and serum response factor (SRF),
respectively. The
phosphorylated forms of these TFs bind to serum
response element (SRE) enhancer sequences that
control genes such as c-fos. c-fos activates the
expression of genes that propel cells through the
cell cycle. SREs occur in a number of genes that
are regulated by growth factors present in serum.
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Signaling via Phosphatidyl-inositol 3-phosphates
RTKs also can signal via formation of
phosphoinositide compounds. Like GPCRs, they
signal via the IP3/DAG pathway. However, RTKs
activate the PLCg isoform of phospholipase C, not
PLCß as occurs with GPCRs. PLCg binds to
activated RTKs via SH2 domains.
In addition, RTKs can signal via PI
3,4-bisphosphate and PI 3,4,5-trisphosphate
formed by the enzyme PI-3 kinase (Fig. 16.25).
PI-3 kinase is recruited to the membrane by SH2
domain-mediated binding to activated RTK
phosphotyrosine residues. The PI 3-phosphate
compounds synthesized by PI-3 kinase activate
protein kinase B (PKB) (next slide).
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Activation of Protein Kinase B
Signaling downstream of PI 3-phosphates is
conducted by PKB (Fig. 16.26). PKB is recruited
to the membrane via binding to PI 3-phosphates
via its PH domain. There it is phosphorylated and
activated by the PDK1 PDK2 kinases. PDK1 also
is recruited to the membrane via binding to PI
3-phosphates. Activated PKB then enters the
cytosol, where it phosphorylates target proteins.
In insulin receptor signaling, PKB phosphorylates
and inactivates glycogen synthase kinase,
stimulating glycogen synthesis. PKB also is a
potent inhibitor of apoptosis. PI 3-phosphate
signaling ultimately is terminated by cleavage of
3-phosphates from phosphoinositides by the PTEN
phosphatase. PTEN is inactive in many advanced
cancers.
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Biological Roles of TGFß
Growth factors are proteins that play important
roles in regulating cell differentiation,
division, and movement. Activating mutations in
growth factor receptors or their signaling
pathways commonly are associated with cancers.
Transforming growth factor ß (TGFß) plays
widespread roles in regulating development in
both vertebrates and invertebrates. Despite their
names, all three types of human TGFßs exert
anti-proliferative effects on target cells.
Therefore, the loss of a TGFß receptor can lead
to transformation of a cell to a cancerous state.
TGFß is secreted from cells as an inactive
precursor. It subsequently undergoes proteolytic
processing and attaches to the extracellular
matrix. It is released from the matrix after the
receipt of an appropriate signal, and then
carries out paracrine signaling on neighboring
cells.
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TGFß/Smad Pathway
The signal transduction pathway by which TGFß
regulates TF activity is illustrated in Fig.
16.28. The points to know for this pathway are 1)
TGFß binds to cell surface receptors causing
phosphorylation of these receptors, 2) the
activated receptors phosphorylate certain Smad
TFs exposing their nuclear localization signals,
and 3) Smad TFs enter the nucleus where they
combine with other TFs and activate the
transcription of target genes. Smad TF activity
ultimately is shut down via the action of
transcription repressors, whose activity is
induced by TGFß via a feedback loop. These
repressors bind to the Smad TFs, and recruit a
histone deacetylase to the activated promoter,
which then causes chromatin condensation and gene
silencing. Because TGFß has antiproliferative
effects on cells, overexpression of the
repressors results in cellular transformation and
cancer.