L8: TGFbeta II Flashcards
1
Q
How does nucleocytoplasmic shuttling occur?
A
- Upon association with Smad4, nuclear import occurs
- Within nucleus, nuclear phosphatases (e.g. PPM1A) dephosphorylate MH2 domain of R-smads -> dissociation
- Smad4 binds exportin (Cmr1) and Ran GTPase -> exported (Cmr1 has nuclear export signal in linker)
- Smad2/3 (R) interact with karyopherins (Kaps) which bind nucleoporins (NUP214 and NUP153) in NPC
2
Q
RanGTPase export system:
A
- Against gradient of RanGTP
- Exportin (aka Cmr1) changes conformation when binding RanGTP -> able to bind cargo (Smad4)
- Export from nucleus occurs -> GTP on Ran hydrolysed
- Exportin reverts conformation and releases both cargo and RanGDP
- Exportin and RanGDP independently return to nucleus, Ran exchanges GDP for GTP
3
Q
i-Smads: (role, 2 examples)
A
- Inhibitory smads
- e.g. Smad6 (BMP) which competes for Smad4 for binding to Smad1
- e.g. Smad7 (TGFB/BMP) induced by TGB signalling, provides negative feedback
4
Q
How does Smad7 operate in a negative feedback mechanism?
A
- Smad7 (i-Smad) competitively binds to TGFB-RI and prevents phosphorylation of Smad2/3 or Smad1/5/8
- Recruits PP1 to dephosphorylate TGFBR1 (enhanced by SARA)
- Recruits SMURFs or NEDD4-2 to RI -> targets RI for degradation
5
Q
BMP R-Smads:
A
- 1
- 5
- 8
6
Q
How do transcriptional corepressors operate in TGFB signalling (SnoN and Ski):
A
- Both initially cytosolic but bind to Smads 2/3 and 4 -> carried into nucleus
- Causes formation of a complex including histone deacetylase, blocking transcription activation (epigenetically)
- Both proteins are transcriptionally upregulated by TGFB signalling (negative feedback)
- They can also act as oncoproteins (inhibiting the inhibition of proliferation)
7
Q
How are Ski and SnoN opposed?
A
- Arkadia is a E3 ubiquitin ligase
- It targets SnoN/Ski for degradation
- Possible role as a tumour suppressor
- Additionally, targeted by Smurfs in nucleus
8
Q
How do Smurfs regulate TGFB and BMP signalling?
A
- Smad Ubiquitination Regulatory Factors
- Examples of Ubiquitin ligases
- They damp down signalling via BMP and TGFB by ubiquitinating R-smad and recruited to RII by i-Smad
- Also able to promote signalling-mediated transcription by targeting SNON for degradation (nucleus only!)
9
Q
Smurf degradation of lipid rafts:
A
- In the absence of SARA, TGF-B-bound receptors are internalised via lipid rafts (cavaolae)-mediated endocytosis
- The resultant endosomes have Smad7 (i-Smad) which recurits Smurf2
- Smurf-2 is a ubiquitin ligase and leads to receptor degradation
10
Q
Role of caveolin:
A
- Coats internal face of caveolae
- Upregulated in many cancer types
- More caveolin = more caveolae = more receptor degradation
- Downregulating TGFB pathway, opposing the TSG effect (inhibiting proliferation)
11
Q
Summarise the different levels of diversity in TGF-beta signalling (conferring specificity):
A
- Ligands (1, 2, 3 + other superfamily members) -> homo/hetero/tetradimers
- RI (x7), RII (x5) + other superfamily members
- Presence / absence of RIII
- R-Smads (2, 3 + other superfamily members)
- Different groups of TFs (vary by cell type)
- Crosstalk with other pathways -> MAPK, PIK, Rho-like GTPase
12
Q
+ Other TGF-B signalling interactions conferring specificity
A
- Point to explore
13
Q
Overview of role of TGB signalling in cancer:
A
- Early stage: suppression
- Later stage: promotes progression
- Biphasic
14
Q
Examples of therapeutic approaches to block TGFB signalling:
A
- Inhibit conversion of ligand to active dimer
- Block ligand-receptor binding
- Inhibit phosphorylation of RI
- Soluble receptor decoys (act as a ‘sponge’ to soak up ligand but unable to transmit any signal themselves)
- Block synthesis of TGFB
- Inhibit complex binding to DNA
15
Q
Juvenile Polyposis Syndrome (JPS): Features, role of GFB in pathology
A
- AD inheritance
- Result of mutation in various elements of TGFB but predominantly Co-Smad
- Multiple benign polyps form in the digestive tract
- Increased chance of malignancy (10-50%)
- (The majority of sporadic colon tumours also show mutations in RII or Smad4)
- This is an issue for digestive tract because TGFB is so important for colonic epithelial homeostasis (frequently sloughing off and replacing cells)
16
Q
Marfan syndrome: Features, involvement of TGFB
A
- AD inheritance
- Affects connective tissues; one or more of… tall, long slender limbs/fingers/toes, heart defects, lens dislocation, chest deformities
- Mutations in fibrillin-1 (ECM formation), regulator of availability of TGFB -> increased TGFB signalling in patients -> maldevelopment
17
Q
What type of kinase activity do TGFBRs display?
A
- Serine/threonine kinase activity
- Only known family with this type of activity
- Type II: Constitutively active
- Type I: phosph. by Type II
18
Q
+ Name the 3 subfamilies of BMPs antagonists:
A
- CAN
- Twisted gastrulation
- Chordin/noggin families
- Groupings by size of cysteine knot
- Crucial for regulation of BMP signalling in development
19
Q
+ How do the co-receptors operate in TGB signalling? Name them both:
A
- Regulating cell surface localisation, internalisation etc through interaction with scaffolding proteins (e.g. GIPC)
- e.g, TBRIII and endoglin
20
Q
+ TGFB signalling in DMD:
A
- Inherited lethal muscular wasting disorder
- Great phenotypic variability (principally due to mutations in muscle dystrophin)
- Several missense mutations in GDF8 identified in DMD patients (encodes myostatin)
- Myostatin: muscle specific TGFB ligand that limits muscle growth -> possible avenue for DMD pathology
21
Q
+ Dpp is a TGF family member in humans; what is its role in development?
A
- Participates in dorsal-ventral patterning in fly embryos
- Links to PG signalling (dally mutants have abnormal Dpp gradients)
22
Q
+ What is the relevance of the ECM to Marfan phenotypes?
A
- TGFB family proteins are inactively stored in the ECM
- In marfan, the gene encoding Fibrillin-1 is mutated resulting in impairment of this process and overabundance of free TGFB ligands -> overactive signalling, growth inhibitive effects upon connective tissue
