Signaling Pathways that Control the Expression of Gene Activity

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.

Human TGFβ Signal Molecules

• Three isoforms that are tissue specific:

• TGFβ - 1

• TGFβ - 2

• TGFβ - 3

• Dimeric protein (homo- or hetero-)

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.

Types of Smads

• Receptor-regulated Smads (R-Smads)

• Co-Smads

• Inhibitory or antagonistic Smads (I-Smads)

The Structure of R-Smads

N C

DNA-bindingsegment

NuclearLocalizationSignal

MH1 DOMAIN Linker MH2 DOMAIN

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.

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.

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.

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.

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.

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.

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.

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.

http://www.biomedcentral.com/1471-2202/7/S1/S10/figure/F3?highres=y

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.

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.

Ligands that bind to TRKs

• Nerve Growth Factor (NGF)

• Platelet-derived Growth Factor (PDGF)

• Fibroblast Growth Factor (FGF)

• Epidermal Growth Factor (EGF)

• Insulin

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.

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.

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.

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.

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.

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