Signaling Pathways that

Control Gene Activity

 Overview:
Major Classes of Cell-surface Receptors
 Signal Transduction Pathways are
Complex
• Several classes of receptors can transduce signals by more
than one pathway

• Many genes are regulated by multiple TS factors

– each TS factor can be activated by one or more
extracellular signal

• Large amount of cross talk b/w signaling pathways

– especially seen during early development
Components of Major Signaling Pathways
Transforming Growth Factor β Signaling

• Transforming Growth Factor β (TGFβ) superfamily is a large

class of signals involved in a wide range of developmental
processes, including:
– induction of transformed phenotype in cultured cells
– induction of bone formation & growth
– formation of mesoderm
– expression of cell-adhesion & extracellular matrix molecules
– signals for synthesis and secretion of growth factors
– note: TGFβ has anti-proliferation effects on many mammalian cells
where TGFβ signaling generally inhibits cell proliferation

• Complex cellular effects, but fairly simple signaling pathway:

– TGFβ (signal) binds TGFβ-receptor, direct phosphorylation and TS
factor activation
 Human TGFβ Signals

• Human TGFβ isoforms (TGFβ1-3) are:

– encoded by unique genes
– tissue specific
– developmentally regulated
• TGFβ is synthesized as a part of a larger precursor that is
cleaved after secretion from the cell:
– Pro-domain is cleaved, but remains non-covalently associated
– Mature domain has cysteine residues that form disulfide bonds
• Latent TGFβ complex in ECM: Pro-domain & mature domain
stored in complex with latent TGFβ -binding protein (LTBP)
• Mature TGFβ is released as active homo- or hetero-dimer after
proteolysis or LTBP conformational change
– integrin or matrix proteins binding to LTBP can cause this
 TGFβ Receptors
• 3 types of receptors: TGFβ RI, RII, & RIII
– TGFβ-RIII: most abundant TGFβ receptor that binds and
concentrates TGFβ at the cell surface, but does not have
kinase activity
– TGFβ-RII: dimeric transmembrane proteins that can bind
TGFβ on the cell surface and has cytosolic S/T kinase activity
for TGFβ-RI
– TGFβ-RI: dimeric transmembrane proteins that does not bind
TGFβ outside the cell, but has cytosolic S/T kinase activity for
R-Smads
• TGFβ binding induces complex formation with 2 copies of
TGFβ-RII & TGFβ-RI
– TGFβ-RII phosphorylates & activates TGFβ-RI kinase activity
– activated TGFβ-RI directly phosphorylate & activate TS
factors called Smads
 Smads
• Three types of Smads:
– R-Smads: Receptor-regulated Smads (Smad2, Smad3)
– Co-Smads (Smad4)
– I-Smads: Inhibitory or antagonistic Smads (Smad7)
Structure of R-Smads:
•R-Smad inactive when C
unphosphorylated MH2
Linker
DBD NLS
– MH1 & MH2 domains associated MH1
– NLS is masked N
– Can’t bind Co-Smad or DNA TGFβ-RI
• TGFβ-RI phosphorylates 3 kinase
S residues on R-Smad near DBD NLS
PPP
C-term which separates the MH1 MH2
N C
MH1 & MH2 domains Linker
TGFβ Signaling Pathway
• TGFβ binds to TGFβ-RIII or TGFβ-RII
– if binding is to RIII, TGFβ presented to RII
– ligand-bound RII recruits and
phosphorylates RI, causing its activation
• Activated TGFβ -RI phosphorylates
Smad3 (R-Smad), exposing NLS
• Two phosphorylated Smad3 interact with
Smad4 (Co-Smad) & importin in cytosol,
forming large complex
• Entire complex translocates to nucleus
– Ran-GTP causes dissociation of importin
• Smad complex associates w/ other TS
factors forming activation complex
• TS begins (often growth-inhibitory genes)
• Dephosphorylation of Smads in nucleus
results in their translocation to cytoplasm
 I-Smads Regulate Smad Signaling

• I-Smads (Smad7) block the ability of TGFβ-RI to

phosphorylate R-Smads
– Induction of Smad7 inhibits intracellular signaling
 Oncoproteins Regulate Smad Signaling
• Smad signaling also regulated by other proteins, including
SnoN & Ski
– Identified as oncoproteins: cause abnormal cell proliferation
• SnoN & Ski bind to Smad2/Smad4 or Smad3/Smad4
complexes after TGFβ stimulation
– Smad complexes can still
bind to DNA control regions
– block TS activation by DNA
bound Smad complexes
– Blocks TS of growth-
inhibitory genes normally
made by TGFβ pathway
– How does that happen in
this example?
 Cancer & Loss of TGFβ Signals

• TGFβ signaling generally inhibits cell proliferation

• Loss of various components of the signaling pathways
contributes to abnormal cell proliferation & malignancy.
– Many cancers are caused by gene mutations in the TGFβ
signaling pathway (either TGFβ receptors or Smad proteins)
and are therefore resistant to growth inhibition by TGFβ.

• Example: In most human pancreatic cancers, a deletion to

the Smad 4 gene occurs
– Smad 4 protein is not produced or is non-functional
– proteins that inhibit cell proliferation upon stimulation by TGFβ
are not synthesized
 Cytokine Signaling
• Cytokines are small secreted proteins that control growth and
differentiation of different cell types
– Cytokines can act in an autocrine, paracrine, and endocrine manner
• Cytokines bind to their cell surface cytokine receptor and turn on
intracellular signaling cascade resulting in various responses:
– increasing or decreasing expression of membrane proteins (including
cytokine receptors)
– cell proliferation
– secretion of effector molecules
• Examples of cytokines and responses:
– Interleukin-2: T cell & NK cell proliferation
– Interleukin-4: B cell proliferation
– Erythropoietin: regulates RBC production
– Thrombopoietin: platelet formation
 Cytokine Signaling
• Cytokine receptor: two monomeric transmembrane proteins each
associated with JAK, a separate cytosolic protein tyrosine kinase
• Cytokine binds to its receptor causes:
– Receptor dimerization
– Y phosphorylation and activation of JAKs
– Y phosphorylation of receptor tails
 Recruitment of Cytosolic Proteins to
Receptor Tails

• Phosphorylated Tyrosine on the

activated receptor tail recruit a
number of proteins that contain
SH2 or PTB domains
• The recruited proteins are then
phosphorylated (by the JAK)
which enhances their activity
• Those proteins go on to signal
and activate transcription.
 Cytokines & the JAK/STAT Pathway
• Ligand binds to cytokine receptor
• JAK to be phosphorylated & activated
• JAK phosphorylates Y residues on
the receptor tail
• Recruitment of SH2 domain
containing STAT proteins
– STAT: Signal Transducers and
Activators of Transcription
• STATs are phosphorylated by JAK
• Phosphorylated STATs dissociate
from the receptor and dimerize,
exposing NLS
• STAT dimer translocates to the
nucleus results in binding to enhancer
sequences & activation of TS of target
genes
 Short Term Regulation of Cytokine
Receptors
• Dephosphorylation of tyrosines
on JAK by phosphatase SHP1
– activation involves
phosphorylation, so
inactivation involves
dephosphorylation
• SHP1 binds to a
phosphotyrosine on the
receptor tail and removes
phosphate from JAK
• prevents further activation
• occurs w/in a few minutes
 Long Term Regulation of Cytokine
Receptors

• SOCS inhibit or terminate

long term signaling
– SOCS protein expression is
induced by STAT proteins in
Epo stimulated cells
• Mechanisms:
– Signal blocking: SOCS binds
to phosphotyrosine residues
on EpoR or JAK2
– Protein degradation: SOCS
target proteins for ubiquitin-
proteasome degradation
 Receptor Tyrosine Kinases (RTKs)
• RTKs are similar to cytokine receptors but the cytosolic domain has its own intrinsic Y
kinase activity
• Examples of RTK ligands include Growth factors and Insulin
– NGF (nerve), PDGF (platelet), FGF (fibroblast), EGF (epidermal)
• Ligand binding causes receptor dimerization, kinase activation, phosphorylation of Y on
receptor tails
 Ligand binding to RTK & Ras Activation

• Phosphorylated tyrosines on RTK tails recruit adapter proteins

w/ SH2 & PTB domains
• These adaptor proteins couple activated RTKs to various
components of signaling pathways, such as Ras activation

• Ras is a monomeric, GTP-binding switch protein

– Inactive Ras bound to GDP; Active Ras bound to GTP
• Ras is not directly linked to cell-surface receptors
• Ras is anchored to plasma membrane by hydrophobic anchor
Ligand binding to RTK & Ras Activation
• Activated RTK recruits SH2 domain containing GRB2
• GRB2 recruits SOS protein (which has GEF activity)
• SOS replaces GDP with GTP on RAS
 Active Ras Turns on MAPK Pathway

Ras/MAP kinase pathway:

•Ras binds to & activates Raf
(S/T kinase)
•Raf activates MEK (kinase)
•MEK activates MAPK (kinase)
•MAPK translocates to the
nucleus inducing TS
– MAPK regulates activity
of many TS factors
Ras/MAPK Pathway
Mutations to Components of MAPK
Pathway Linked to Cancer
 Role of Scaffold Proteins

• Upstream components of
MAPK cascades assemble
into large pathway-specific
complexes stabilized by
scaffold proteins
• Scaffold proteins associate
different kinases of a signaling
pathway to prevent accidental
phosphorylation of other
substrates
– allowing kinases of one
pathway to interact w/
each another, but not w/
kinases in other pathways
 Insulin & Protein Kinase B (PKB)
• Insulin binds to insulin receptor (RTK)
which can:
1. Activate Ras-MAPK pathway
leading to changes in gene
expression, or
2. Activate PKB/Akt, a S/T kinase
• How does Insulin lower blood glucose?
– In fat & muscle cells: PKB/Akt
causes movement of GLUT4
transporters from intracellular
vesicles to the plasma membrane
• GLUT4: increases import of
glucose by fat and muscle cells
– In liver & muscle cells: PKB/Akt
stimulates glycogen synthesis by
causing glycogen synthase
activation
• PKB/Akt phosphorylates and
inactivates an inhibitor of GS