L8: TGFbeta II

Question 1

How does nucleocytoplasmic shuttling occur?

Answer 1

• 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

Question 2

RanGTPase export system:

Answer 2

• 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

Question 3

i-Smads: (role, 2 examples)

Answer 3

• 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

Question 4

How does Smad7 operate in a negative feedback mechanism?

Answer 4

• 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

Question 5

BMP R-Smads:

Answer 5

• 1
• 5
• 8

Question 6

How do transcriptional corepressors operate in TGFB signalling (SnoN and Ski):

Answer 6

• 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)

Question 7

How are Ski and SnoN opposed?

Answer 7

• Arkadia is a E3 ubiquitin ligase
• It targets SnoN/Ski for degradation
• Possible role as a tumour suppressor
• Additionally, targeted by Smurfs in nucleus

Question 8

How do Smurfs regulate TGFB and BMP signalling?

Answer 8

• 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!)

Question 9

Smurf degradation of lipid rafts:

Answer 9

• 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

Question 10

Role of caveolin:

Answer 10

• 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)

Question 11

Summarise the different levels of diversity in TGF-beta signalling (conferring specificity):

Answer 11

• 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

Question 12

+ Other TGF-B signalling interactions conferring specificity

Answer 12

• Point to explore

Question 13

Overview of role of TGB signalling in cancer:

Answer 13

• Early stage: suppression
• Later stage: promotes progression
• Biphasic

Question 14

Examples of therapeutic approaches to block TGFB signalling:

Answer 14

• 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

Question 15

Juvenile Polyposis Syndrome (JPS): Features, role of GFB in pathology

Answer 15

• 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)

Question 16

Marfan syndrome: Features, involvement of TGFB

Answer 16

• 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

Question 17

What type of kinase activity do TGFBRs display?

Answer 17

• Serine/threonine kinase activity
• Only known family with this type of activity
• Type II: Constitutively active
• Type I: phosph. by Type II

Question 18

+ Name the 3 subfamilies of BMPs antagonists:

Answer 18

• CAN
• Twisted gastrulation
• Chordin/noggin families
• Groupings by size of cysteine knot
• Crucial for regulation of BMP signalling in development

Question 19

+ How do the co-receptors operate in TGB signalling? Name them both:

Answer 19

• Regulating cell surface localisation, internalisation etc through interaction with scaffolding proteins (e.g. GIPC)
• e.g, TBRIII and endoglin

Question 20

+ TGFB signalling in DMD:

Answer 20

• 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

Question 21

+ Dpp is a TGF family member in humans; what is its role in development?

Answer 21

• Participates in dorsal-ventral patterning in fly embryos
• Links to PG signalling (dally mutants have abnormal Dpp gradients)

Question 22

+ What is the relevance of the ECM to Marfan phenotypes?

Answer 22

• 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