Lecture 8: TGFbeta (2) Flashcards
What are the receptor Smads of TGF-beta signalling?
Smad2/3
How are the receptor Smads involved in nucleocytoplasmic shuttling?
- MH2 domain of Smad2/3 recognise the phosphorylated residues of GS domain of TGF-beta receptor I
- brings Smad2/3 close to kinase domain of receptor 1 so Smad2/3 is phosphorylated on MH2
- conformation change results in dissociation of Smad2/3 from receptor into cytoplasm
- MH1 and MH2 domains of Smad dissociate and nuclear localisation sequence is recognised by beta-importin
- co-Smad4 MH2 domain binds to phosphorylated Smad2/3 - this complex is moved into the nucleus
- in nucleus, Smad complex interacts with transcription factors - ‘feet’ recognise different nucleotide sequences in the DNA for attachement
Why can the transcriptional response initiated by TGF-beta signalling differ between cell types?
Different cell types have different complement of transcription factors that determine DNA binding of Smad complex
After transcriptional activation of target genes, how is the smad complex transported back to the cytoplasm?
Protein phosphatases M1A in nucleus dephosphorylates smad2/3 resulting in dissociation of the smad complex
- the dissociated smads are exported from the nucleus and used in another round of signalling
How is co-smad4 exported from the nucleus?
co-smad4 has a nuclear export signal in linker region between MH1 and MH2 domains that is recognised by CRM1
- binding of RAN-GTP to CRM1 results in conformational change to allowing binding to Smad4
- complex exported out of nucleus
- GTP hydrolysed to RAN-GDP causing dissociation of smad4 from CRM1 (release into cytoplasm)
- CRM1 and RAN-GDP translocate back into the nucleus where GDP is exchanged for GTP on the RAN
Under normal conditions, where are the majority of Smads located?
cytoplasm
What was the result of treatment of cells with leptomycin B on Smad4 export?
Leptomycin B inhibits exportin mediated trasnfer out of the nucleus
- treatment with leptomycin B Smad accumulated in the nucleus (nuclear fluorescence)
How are the receptor smads2/3 exported from the nucleus?
Interaction with karyopherins (Kaps) that recognise nucleoporins of the nuclear pore complex so target Smad2/3 to the nuclear pore complex (NPC) for export
How is the TGF-beta signalling pathway regulated?
i-Smads (Smads6/7)
transcriptional co-repressors (SnoN and Ski)
SMURFs
i-Smad6 inhibits which signalling pathway and how?
BMP signalling
by competing with Co-Smad4 for binding to the R-Smad1
i-Smad7 inhibits which signalling pathway and how?
TGF-beta and BMP signalling
by:
- competitively binding to TGF-beta receptor I preventing phosphorylation of receptor smads
- recruits protein phosphatase 1 to dephosphorylate TGF-beta receptor I - not able to be recognised by R-Smads
- recruits SMURFS or NEDD4-2 to Receptor I, targeting it for degradation
i-Smad7 an example of what type of feedback mechanism?
negative feedback (action is induced by TGF-beta signalling but acts to inhibit TGF-beta signalling)
What are the receptor Smads for TGF-beta and BMP?
TGF-beta = Smad2/3
BMP = Smad1/5/8
How do transcriptional co-repressors Ski and SnoN regulate the TGF-beta pathway?
- bind to the R-Co-Smads when they form the triplet in the cytosol and imported into the nucleus with the triplet
- initiates the binding of histone deacetylase that results in more tightly packed DNA so no longer accessible and blocks transcriptional activation of TGF-beta target genes
The transcriptional upregulation of Ski and SnoN is an example of what type of feedback mechanism?
Negative feedback - Ski and SnoN are targets of TGF-beta signalling
How are Ski and SnoN linked to the development/progression of cancer?
They are oncoproteins because they inhibit TGF-beta signalling that normally promotes expression of growth inhibitory proteins (E.g. p21 cell cycle inhibitor)
- inhibit TGF-beta signalling at transcriptional level = inhibit expression of growth inhibitory proteins = uncontrolled cell proliferation
What is Arkadia?
An E3 ubiquitin ligase that targets SnoN and Ski for proteasomal degradation
= promote TGF-beta signalling = more cell cycle inhibition and control over proliferation (potential tumour suppressor)
- loss of Arkadia function would result in more TGF-beta inhibition and uncontrolled cell proliferation
How does the biphasic action of SMURF on TGF-beta and BMP signalling depend on its cellular localisation?
Cytoplasm:
They are E3 ubiquitin ligases
- ubiquitinate R-smads
- i-Smad7 recruits SMURF to RII = ubiquitinates RII component
(Smad7 recruits Smurf2 to cavaeloae-mediated endocytosed ligand-receptor complexes in the absence of SARA)
–> targeted for proteasomal degradation to dampen down the signalling
Nucleus:
- ubiquitinates SnoN for degradation
–> removes TGF-beta inhibition so results in upregulation of TGF-beta signalling.
What does SMURF stand for?
Small Ubiquitination Regulatory Factor
What happens to the receptor-ligand complex in the presence of SARA?
Internalisation of the receptor-ligand complex by clathrin-coated pits, the signalling pathway proceeds and the receptor is recycled to the cell surface
What happens to the receptor-ligand complex in the absence of SARA?
receptor-ligand complexes are internalised by caveolae (lipid rafts - a detergent insoluble membrane with decreased fluidity) mediated by endocytosis
- the endosomes have iSmad7, which acts as a recruitment site for Smurf2 which targets the receptor-ligand complex for proteasomal degradation.
What is the role of Caveolin-1?
presence of caveolin-1 within lipid raft results in invagination and endosome formation
What is the significance of caveloin upregulation in cancer?
more cavaeloe = more internalisation of receptor-ligand complexes via cavaeloe-mediated endocytosis = more degradation of receptor instead of recycling to surface = decrease in TGF-beta signalling = less inhibitors of cell cycle produced = more proliferation
In what ways can the specificity of the TGF-beta response be altered?
- Different ligands (1, 2, and 3)
- Homo/heterodimers/tetramers of ligands
- RI has 7 variants, RII has 5 variants
- Presence of absence of RIII
- Different R-Smads
- Different transcription factors in different cells
- Non-Smad TGF-beta signalling (MAPK, PI3K, Rho-like GTPase)
How does TGF-beta signalling function in early stage cancer?
suppresses tumorigenesis in pre-malignant tumours in early stages
How does TGF-beta signalling function in later stage cancer?
promotes progression of cancer:
- cell proliferation
- angiogenesis
- apoptosis
- immune evasion
–> promotes metastasis of cancer
What are some targets within the TGGF-beta signalling pathway that may be acted upon by therapeutic drugs?
- inhibit conversion of inactive ligand to active dimer
- block ligand-receptor binding (antibodies bind to receptor or ligands)
- inhibit phosphorylation of receptor complex
- soluble receptor decoys (receptor lacks internal signalling domain - external domain still binds and sequesters ligand)
- inhibit complex binding to DNA (using small molecule inhibitors)
What is JPS?
Juvenile Polyposis syndrome is an inherited autosomal dominant mutation (often in Smad4 gene) - decreases TGF-beta signalling so cell proliferation is unchecked
- results in formation of benign polyps in the digestive tract
- colon electively removed in a number of cases to reduce risk of cancer (increased rate of proliferation in polyps = higher chance of mutation leading to cancer formation)
True or false: individuals with JPS have increased chance of malignancy?
True (10-50% increased risk)
The majority of sporadic colon tumours show mutations in what components of TGF-beta signalling?
RII
Smad4
What is marfan syndrome?
Autosomal dominant disorder that affects connective tissues (such as mutation in fibrillin-1 gene that interacts with inactive TGF-beta in the ECM)
- inactive TGF-beta ligand normally sequestered in ECM but in Marfan syndrome, defects in ECM results in increased availability of active TGF-beta so increased TGF-beta signalling
Mutation in which gene affects sequestering of inactive TGF-beta ligand in the ECM (implemented in Marfan syndrome - mutation results in increased availability of active TGF-beta)?
Fibrillin-1
What are some of the clinical symptoms of Marfan syndrome?
- tall, long slender limbs/fingers/toes
- heart defects
- lens dislocation
- chest deformities
