Lecture 14

Question 1

Explain the TGF-? signal transduction pathway following ligand binding

Answer 1

The activated Type I receptor phosphorylates Smad proteins. The type of TGF-? ligand that binds to the receptor determines the specific smads phosphorylated by the activated receptor. BMP ligand binding leads to phosphorylation of Smad1,5 and 8 whilst TGF-? binding results in Smad2 and 3 phosphorylation. Smads1,2,3,5 and 8 are collectively known as receptor-regulated or R-Smads. R-Smads are maintained near the plasma membrane close the kinase domains of the receptors. When the pathway is inactive the R-Smads are anchored at the cells membrane by Smad anchor for receptor activation proteins (SARA). Once R-Smads have been phosphorylated by the activated Type I receptor they oligomerise with Smad4 to form heterodimers. This Smad complex then migrates to the nucleus where its binds to the DNA to regulate gene expression

Question 2

What kind of bonding hols the LAP and TGF-? dimers together in a complex

Answer 2

Disulphide bridges between adjacent cysteine residues

Question 3

Explain the processes that lead to the release of the active ligand from the complex

Answer 3

Release of the TGF-?/LAP/LTBP complex from the extracellular matrix involves cleaves by the proteases plasmin and calpain that releases the complex from the extracellular matrix. Subsequent binding of thrombospondin to LAP releases the active TGF-? ligand

Question 4

Which TGF-? ligand family have an important role in left/right patterning and splitting of bilateral symmetry

Answer 4

Nodal

Question 5

What does TGF-? stand for

Answer 5

Transforming growth factor-?

Question 6

How can Smads lead to a decrease in gene transcription

Answer 6

Smads can also bind to co-repressors and recruit histone deacetylases (HDACs) which remove acetyl groups from the histones and decrease gene transcription by tightening the chromatin structure.

Question 7

The BMP family of TGF-? ligands are negatively inhibited by several different proteins that act as extracellular antagonists. List 5 of these such BMP antagonists

Answer 7

Noggin, Chordin, Follistatin, Cerberus, Gremlin

Question 8

What type of mutations related to TGF-? signalling are often found in patients with certain cancers

Answer 8

Hypomorphic (partial loss of function) mutations

Question 9

What are the four ligand subfamilies of TGF-? signalling

Answer 9

Activin, Nodal, BMPs/GDFs and TGF-?

Question 10

The TGF-? precursor is cleaved into two parts by the action of a protease, what are these two regions and what is their role

Answer 10

The C-terminal region is responsible for the formation of the active ligand by dimerisation with other TGF-?s. The N-terminal part binds to TGF-? and is called Latency Associated Protein (LAP)

Question 11

Decoy receptors can also inhibit TGF-? signalling, give an example of this

Answer 11

The BAMBI pseudo-receptor resembles the Type I receptor but lacks the intracellular kinase domain. It forms an inactive complex with the ligand and the Type II receptor and inactivates BMP, activins and TGF-? signalling

Question 12

Structurally, Type I and Type II TGF-? receptors are very similar. What action do they both have

Answer 12

They are both serine-threonine kinases but have different functions in the pathway

Question 13

How many classes are there of general TGF-? antagonists and what is the purpose of these different families

Answer 13

There are five different families of TGF-? negative regulators these allow for distinct expression profiles allowing for greater refinement of the signal by binding of different subsets with different affinities.

Question 14

What is significant about Smad4

Answer 14

Smad4 is constitituvely expressed and unlike the other Smads, it is common to all TGF-? signalling pathways. It is therefore referred to as the common-mediator or Co-Smad

Question 15

Lefty and antivin are TGF-? antagonists that inhibit Nodal signalling, explain how these pseudo-ligands act

Answer 15

These antagonists bind to the Type II receptors but lack an ?-helix loop regions that allows for dimerisation and the cysteine resides responsible for disulphide bridge formation. This ultimately results in the inability of the Type II receptor to bind to the Type I receptor

Question 16

What is the role of Latent TGF-? binding protein (LTBP)

Answer 16

LTBP tethers the LAP part of the complex to components of the extracellular matrix to increase the effective concentration of the ligand by restricting its diffusion

Question 17

What are the two ways in which TGF-? signalling antagonists can act

Answer 17

They can either compete for binding at one site on the ligand (I.e. gremlin, noggin, cerberus and DAN on BMP2) or, antagonists can bind to independent sites allowing for multiple inhibitor binding

Question 18

Which TGF-? ligand family have an important role in hormonal regulation

Answer 18

Activins

Question 19

What are the three key domains of an R-Smad and what is their function

Answer 19

The MH1 domain in the amino terminus is responsible for binding to the DNA. There is an MH2 interaction domain that controls protein-protein interactions and regulates the homo/heterodimerisation of Smads. Finally, R-Smads also contain a domain in the carboxyl terminus that acts as a recognition site for phosphorylation by the activated Type I receptor

Question 20

Describe the activation of the TGF-? receptor following ligand binding

Answer 20

Activation of the receptors comes from the formation of a dimer receptor complex. The Type II TGF-? receptor binds to the TGF-? ligand homodimer and then recruits and phosphorylates the Type I receptor. This leads to the phosphorylation of Smads and further downstream signal transduction

Question 21

What are the results of mutations in Smad4

Answer 21

Disruption of all TGF-? signalling pathways

Question 22

How does the structure of an I-Smad differ from that of an R-Smad

Answer 22

Whilst I-Smads have retained the MH2 interaction domain they lack the functional MH1 DNA binding domain as well as the phosphorylation domain

Question 23

Explain how both hypomorphic and amorphic TGF-? signalling mutations impact cancer and tumorigenesis

Answer 23

Null/apomorphic mutations cause benign lesions which cannot go through EMT. Hypomorphic mutations increases rate of benign lesions, by decreasing inhibition of clonal expansion, with enough signalling still conserved to trigger later stage EMT and metastasis.

Question 24

Give an overview of the TGF-? signalling pathway

Answer 24

The TGF-? ligand binds to the extracellular domain of the Type II TGF-? receptor and promotes its further binding to the Type I TGF-? receptor via the ligand also. The activated TGF-? receptor dimer undergoes transphosphorylation of the Type I receptor by the action of the intracellular serine-threonine kinase domain of the Type II receptor. This transphosphorylation causes the binding of a cytosolic Smad proteins which then get phosphorylated, dimerise with other Smads and translocate to the nucleus where they regulate gene transcription.

Question 25

An in situ hybridisation experiment revealed the expression of TGF-? in a developing chick embryo, it was later determined however that these cells weren’t signalling. Explain how this could be

Answer 25

Expression of a ligand by a cell doesn’t necessarily mean that the cell can signal. If a cell fails to make the proteases for example, then it will fail to release the active ligand

Question 26

Which TGF-? ligand family have an important role in proliferation and differentiation of cells

Answer 26

TGF-?

Question 27

Explain the role of Smad6 and Smad7 in the Smad pathway

Answer 27

Whilst smad2 and smad3 are positive regulators of the TGF-? signalling pathway, Smad6 and 7 are negative regulators. These are also known as inhibitory I-Smads and antagonise TGF-? signalling by binding to the activated Type I receptor and activated R-Smads, blocking their activity.

Question 28

Smads are transcription factors, T or F

Answer 28

T – they can be transcriptional activators or transcriptional repressors

Question 29

What are the four main branches of TGF-? signalling

Answer 29

BMPs/GDFs, TGF-?, Activin and Nodal signalling

Question 30

It was later found that mutations that abolish TGF-? signalling (amorphic mutations) are less dangerous than hypomorphic mutations, why is this

Answer 30

Later in cancer progression TGF-? signalling promotes tumorigenesis

Question 31

Explain the dual role of TGF-? signalling in cancer progression

Answer 31

TGF-? signalling inhibits the over proliferation of mutated cells to form benign legions in early stages of cancer. However, later in the progression of the cancer TGF-? signalling promotes the EMT and angiogenesis ultimately resulting in metastasis

Question 32

TGF-? is expressed prior to apoptosis and acts as a pre-requisite to indicate the programmed cell death. Give an example of a role of TGF-? signalling in apoptosis

Answer 32

Mouse mammary glands upregulation TGF-?3 within 9 hours of inactivity after weening. This results in death of the milk-secreting cells. After 3 days these cells will begin to express genes characteristic of apoptotic cells

Question 33

How do Smads lead to an increase in gene transcription

Answer 33

Smads can recruit and bind to histone acetylases one the DNA. These histone acetylases add acetyl groups to the histones which loosens the structure of the chromatin and increasing gene transcription

Question 34

If mutations in TGF-? signalling lead to some cancers, what can we infer about the role of this pathway in tumour formation

Answer 34

It is likely that TGF-? signalling supresses tumour formation

Question 35

Some extracellular TGF-? signalling antagonists can bind to multiple ligands whilst others can only bind to one, T or F

Answer 35

T

Question 36

Explain the two attributes of I-Smads and how this influences TGF-? signalling

Answer 36

I-Smads still bind to type I receptors but are not substrates so aren’t release. They also still bind to R-Smads but do not work as transcription factors

Question 37

What is meant by the ligand and receptors involved in TGF-? signalling being referred to as promiscuous

Answer 37

There are multiple ligands and receptors that can interact in different combinations

Question 38

What is often seen in the cancer causing TGF-? signalling mutations

Answer 38

Often involve microsatellite instabilities characterised by increases or decreases in the length of microsatellite repeat sequences in the DNA. Unstable microsatellites near to or in genes is thought to affect their transcription and may cause problems during DNA replication. This may indicate that the DNA repair machinery is malfunctioning

Question 39

The extracellular TGF-? antagonists all have the same rate of diffusion, T or F

Answer 39

F – they all have different diffusion rates, for example chordin is the slowest as it’s the largest protein (120kDa)