Signaling Pathways

1. Signaling Pathways that Control
the Expression of Gene Activity

2. Transforming Growth Factor β
Signals (TGFβ)
•A large class of molecular signals involved
in regulating development (e.g., bone
growth, mesoderm formation, formation of
cell-adhesion molecules, anti-proliferation
effects).
•The activated TGFβ receptor directly
phosphorylates a transcription factor.

3. Human TGFβ Signal Molecules
•Three isoforms that are tissue specific:
•TGFβ - 1
•TGFβ - 2
•TGFβ - 3
•Dimeric protein (homo- or hetero-)

4. TGFβ Receptors
•Cell surface proteoglycans
•Three types: Types RI, RII, and RIII
•Have serine/threonine kinase activity
•Activation of these receptors results in the
activation of transcription factors called
Smads.

5. Types of Smads
•Receptor-regulated Smads (R-Smads)
•Co-Smads
•Inhibitory or antagonistic Smads (I-Smads)

6. The Structure of R-Smads
N C
DNA-
binding
segment
Nuclear
Localization
Signal
MH1 DOMAIN Linker MH2 DOMAIN

7. TGFβ Signaling Pathway -I
•TGFβ binds to the RIII or RII receptors.
•If binding is to RIII, then TGFβ is presented to
RII.
•Ligand-bound RII recruits and phosphorylates
RI, causing its activation.
•Activated RI phosphorylates Smad3 (an R-
Smad), exposing the NLS.
•Two phosphorylated Smad3 molecules interact
with Smad4 (a co-Smad) and importin-β.
•The complex is translocated to the nucleus.

8. TGFβ Signaling Pathway -II
•Importin dissociates from the complex.
•Smad complex then associates with other
transcription factors to activate
transcription. Often, growth-inhibitory
proteins are produced form these
transcription events.
•Dephosphorylation of the Smads within
the nucleus results in their translocation to
the cytoplasm.

10. Oncoproteins and I-Smads
Regulate Smad Signaling
•Oncoproteins cause abnormal cell growth.
Two such proteins, SnoN and Ski, block
transcription activation by the DNA bound
Smad complexes. The growth-inhibitory
proteins normally produced are not.
•I-Smads (e.g., Smad 7) block the ability of
RI to phosphorylate R-Smads.

11. Cancer and TGFβ Signals
•Many cancers are caused by mutations to
proteins in the TGFβ signaling pathway.
•In most human pancreatic cancers, a
deletion to the Smad 4 gene occurs. The
Smad 4 protein is not produced (or is non-
functional), and proteins that inhibit cell
proliferation upon stimulation by TGFβ are
not synthesized.

12. Cytokine Receptors
•Inactive cytokine receptors consist of two
monomeric transmembrane polypeptides.
•Each polypeptide is associated with a separate
cytosolic kinase.
•Ligand binding to the receptor results in
dimerization of the polypeptides (formation of a
dimer). The kinases then phosphorylate a
tyrosine residue on each other, which causes
each to phosphorylate tyrosine residues on the
cytosolic regions of each sub-unit. [Figure 14-5]
•The receptor is then active.

13. What happens after activation of
the cytokine receptor?
•Amino acid sequences on the activated receptor
that contain a phosphotyrosine residue recruit
myriad proteins possessing either SH2 or PTB
domains. [See figure 14-6]
•The recruited proteins are then phosphorylated,
which enhances their activity.
•The proteins go on to cause transcriptional
activation.

14. What are cytokines?
•Group of relatively small (~160 aa) secreted proteins that
control growth and differentiation of different cell types.
•Responses to cytokines include increasing or decreasing
expression of membrane proteins (including cytokine
receptors), cell proliferation, and secretion of effector
molecules.
•Cytokines may act on the cells that secrete them
(autocrine action), on nearby cells (paracrine action), or
in some instances on distant cells (endocrine action).
•Examples include prolactin, interleukin-2 (T-cell
proliferation), interleukin-4 (B-cell proliferation),
erythropoietin (Epo), thrombopoietin (platelet formation)
and growth hormone.
•All of these molecules bind to cytokine receptors.

15. Cytokines and the JAK/STAT
Pathway
•JAKs are cytosolic kinases associated with cytokine
receptors.
•Four JAKs exist (JAK1-JAK4).
•Ligand binding to a JAK-associated cytokine receptor
causes the JAK to be phosphorylated and activated.
•JAK phosphorylates tyrosine residues on the receptor,
which then recruits SH2 containing STAT proteins.
•STATs are transcription factors.
•STATs are phosphorylated by JAK.
•The active STATs dissociate from the receptor and
dimerize, exposing two NLS.
•Translocation of the STAT dimer to the nucleus results in
binding to enhancer sequences and activation of target
genes.

17. How is the active cytokine receptor
down-regulated?
•If activation entails phosphorylation, then
inactivation must involve the removal of
the phosphate groups from the tyrosine
residues.
•SHP-I phosphatase binds to a
phosphotyrosine on the receptor and
removes the P from JAK, preventing
further activation.
•This occurs in a period of a few minutes.

18. Receptor Tyrosine Kinases (RTKs)
•These receptors are similar to the cytokine
receptors already discussed, except that the
cytosolic domain has intrinsic protein kinase
activity.
•Binding of ligand is associated with dimerization
and activation of the cytosolic domains of each
polypeptide by the phosphorylation of tyrosine
residues.
•The phosphotyrosines recruit adapter proteins
with SH2, SH3 and PTB domains. The adapters
couple activated RTKs to other components of
signal-transduction pathways. One such
pathway involves Ras protein.

20. Ligands that bind to TRKs
•Nerve Growth Factor (NGF)
•Platelet-derived Growth Factor (PDGF)
•Fibroblast Growth Factor (FGF)
•Epidermal Growth Factor (EGF)
•Insulin

21. Ras Protein
•Ras is a monomeric, GTP-binding switch
protein.
•Ras alternates between an inactive state
with bound GDP and an active state with
bound GTP.
•Ras is not directly linked to cell-surface
receptors.
•Ras is anchored to the plasma membrane
by a hydrophobic anchor.

22. Activation of Ras
•Binding of ligand to RTK causes
dimerization and activation of inherent
kinase activity.
•Activated receptor recruits GRB2 adapter
protein.
•GRB2 recruits SOS protein (which has
guanine-nucleotide exchange activity -
GEF), that causes the replacement of
GDP by GTP on RAS.

23. What occurs after Ras is activated?
•MAP kinase pathway is activated:
•Ras activates Raf protein, a serine/
threonine kinase, by binding to it.
• Hydrolysis of RasGTP causes the release
of active Raf.
•Raf activates MEK, another kinase.
•MEK activates MAP Kinase (MAPK).
•MAPK translocates to the nucleus ,
causing induction of gene transcription.

24. Role of Scaffold Proteins
•Scaffold proteins associate the different
kinases of one signaling pathway to
prevent accidental phosphorylation of
other substrates.
•They accomplish this by allowing the
kinases of one pathway to interact with
one another, but not with kinases in other
pathways.

25. Insulin and Protein Kinase B (PKB)
•Insulin binds to a tyrosine kinase receptor that
may activate the Ras-MAPK pathway or can
lead to the activation of protein kinase B .
•In adipose and muscle cells, PKB causes the
movement of GLUT4 transporter from
intracellular membranes to the plasma
membrane.
•In liver and muscle cells, PKB also stimulates
glycogen synthesis from UDP-glucose by
causing the activation of glycogen synthase.

26. Mutations to proteins in the MAP
kinase pathway can cause cancer.