L7: TGF-B 1 Flashcards
1
Q
TGF-beta ligands: Overview of role and family
A
- TGF-beta 1, 2 and 3 in humans
- Part of a large superfamily of cytokines including TGF-betas, activins, nodal, BMPs etc (connected by a large degree of structural homology)
- Control gene expression, especially in development and morphogenesis
2
Q
Pleiotropic role of TGF-beta in humans:
A
- Mainly inhibiting proliferation
- Can also control wound healing, ECM formation, apoptosis, differentiation and immunosuppression
- Affecting expression of caretaker, gatekeeper and landscaper genes
3
Q
TGF-beta structure and expression:
A
- Ubiquitous; one or more isoforms expressed in most cells
- Synthesised as precursor proteins -> sequestered in E-C matric where inactive form binds ECM components
- Provides a reservoir for rapid signalling events
- Very stable structure due to cysteine knots (3x disulphide bonds) which are hard to denature
- Other key domains: LAP (Pro-domain), LSKL/RPKP in LAP and TGFB respectively which bind together, finger turn domains (beta sheets able to interact with receptor)
4
Q
How are TGF-betas activated?
A
- Pro-protein: prodomain and TGF-beta domain
- Cleaved by proteolysis (e.g. furin)
- LAP dimers and prodomain dimers formed (disulphide bridges)
- LTBP binds prodomain
- Thrombospondin binds LSKL sequence
- To activate, TSP-1 competitively inhibits for prodomain -> proteolysis -> mature dimeric ligand formed
5
Q
TGFB-RI vs TGFB-RII:
A
- Both TM homodimers
- RI: Inactive until ligand binds
- RII: constitutively active kinase activity
- Both serine/threonine kinases
- TGB-RIII specifically required for TGFB2 signalling, increasing the local concentration of ligand
6
Q
Receptor binding in TGF-beta pathway:
A
- Constitutively active RII recruits TGFB dimer
- Stimulates recruitment of RI dimer to complex
- RII transphosphorylates RI (at GSGS sequence in GS domain; Ser165 crucial) -> determines intensity of signalling
- Conformational change in RI displaces immunophilin (FKBP12 site)
7
Q
What are Smads?
A
- Mediate signal transduction from receptor to nucleus
- R-smads: Receptor -> Smads 2 and 3 which can be phosphorylated
- Co-smads: common mediator aka Smad 4 -> not phosphorylated
8
Q
Smad signal transduction: Nuclear translocation
A
- MH2 domain of Smad2/3 binds P-ser (adjacent to kinase domain of RI) -> phosphorylation by RI at MH2
- -> dissociation of MH1 and MH2 domain within molecule, revealing the nuclear localisation sequence (NLS)
- MH1/NLS able to bind importin-beta
- Simultaneously, MH2 of Smad4 binds pMH2 domain of Smad2/3 and association of a second Smad 2/3 at MH2
9
Q
Smad signalling within nucleus:
A
- Smad/importin complex encounters Ran GTP
- When activated, Ran binds / displaces importin-B from complex
- Complex then able to bind TFs
- MH1 domains of Smads and TF recognise DNA target sequence
10
Q
How is specificity conferred in Smad signalling within the nucleus:
A
- Co-activators and co-repressors also bind smads -> control accessibility of DNA (HATs/HDACs)
- The right sequences / order / spacing in smads confers specificity of regulation
11
Q
Smad structure:
A
- MH1, linker and MH2 domains
- NLS (binds imp B) and DNA binding part of MH1
- Other binding sites: Smurf (at PPXY in linker) and SARA (Smad anchor of receptor activation)
- Smurfs: E3 ubiquitin ligases
12
Q
Receptor internalisation in TGF-beta signalling;
A
- TGF-beta receptors continuously internalise and recycle
- Smad signalling requires internalisation by clathrin-mediated endocytosis
- They can, however, enter caveolae
- Here, SARA interacts with PLs in PM and stabilises Smad 2/3 binding to complex
- Targets complex to CCPs
13
Q
+ Example of TF that Smads interact with and impact on gene expression (bone formation)
A
- Runx 2 transcription factor
14
Q
+ How are TGFB-receptors post translationally modified?
A
- Phosphorylation -> Smad activation, MAPK pathways
- Ubiquitylation -> Receptor degradation
- Sumoylation -> Facilitating Smad activation
- (All enzymatically reversible)
15
Q
+ How might receptor PTMs facilitate MAPK activation?
A
- TGFBRII phosphorylation by Src (on tyrosine)
- Enables TGBRII to recruit Grb2 and Shc through their SH2 domains
- TGFB-induced activation o fp38 mitogen-activated proteins kinase pathway (MAPK)
16
Q
+ How are Smurfs activated?
A
- N-terminal domain of Smad7 stimulates activity of Smurf1 or Smurf2 by recruiting the ubiquitin-conjugating E2 enzyme ‘UbcH7’ to the HECT domain of the E3 ligase (smurf)
17
Q
+ What is sumoylation?
A
- Covalent attachment of a SUMO polypeptide that resembles a ubiquitin
- Process requires sequential activation of E1, E2 and E3 SUMO ligases
- Regulates subcellular localisation of perinuclear and nuclear proteins (i.e. TFs)
18
Q
+ How is ubiquitylation-induced degradation inhibited in TGFBRI?
A
- UCH37 (deubiquitylation enzyme) associates
- Site is distinct from Smurf interacting motif (PY)
19
Q
+ Additional function of iSmads beyond direct inhibition:
A
- Acting as a scaffold for recruitment for further inhibitory factors
- e.g. PP1 (via GADD34 subunit) -> dephosphorylation and subsequent inactivation of TGFBRI
- Also recruit HECT-type E3 ubiquitin ligases (Smurfs are a member of this family) -> degradation via ubiquitination
20
Q
+ What is the normal role of Dapper proteins? Extra role in TGFB signalling?
A
- First identified as suppressors or activators of Wnt signalling
- Dapper2 (Dpr2) is able to inhibit TGFB and nodal signalling by binding type I receptors and promoting degradation in lysosomes (not via ubiquitylation)
21
Q
+ Purported role of WD-repeat containing proteins on TGFB signalling (name 2):
A
- Several WD-repeat containing proteins have been implicated as negative regulators of TGFB signalling
- e.g. STRAP
- e.g. B-alpha subunit of PP2A
22
Q
+ Purported role of FYVE-domain containing proteins on TGFB signalling (name 2):
A
- Facilitating Smad interactions with receptors and activation
- e.g. SARA (stabilises Smad2, 3 binding to receptor complex; also directs the activated receptor to clathrin-mediated endocytosis)
- e.g. Endofin can interact with Smad1 (as in BMP signalling); possibly fulfilling a similar role to SARA. Thought to have multiple complex interactions since it also interacts with type I receptors and Smad4
23
Q
+ Give 2 examples of non-Smad signalling by TGFB with physiological effects:
A
- TGBRI-induced, dissociation of tight junctions -> Par6-Smurf1 complex forms, confers ubiquitylation of RhoA -> key role in EMT
- Activation of MAPK signalling (typically weaker than that of the Smad leg of the pathway) (requires association of Grb2 and pShc)
24
Q
+ What are the 2 routes for endocytosis of TGFB-bound TGFBR complexes?
A
- Clathrin-mediated endocytosis
- Caveolin-1 and lipid-raft mediated-endocytosis
25
Q
+ Outline clathrin-mediated endocytosis (key domain):
A
- Receptors internalised constitutively through clathrin coated pits
- Mediated by a di-leucine internalisation motif in TBRII cytoplasmic domain
- They then enter EEA-1 and Rab5-positive early endosomes
- Able to recycle back to PM (Rab11 dependent)
