MODULE 5: cell signalling Flashcards
signal transduction: general principles (4) and features (5)
principles:
1) shape change - interior receptor changes conformation in response to signal, allows functional change, allows response from interior
2) signal relayed until received by effector protein - allows amplification of signal
3) effector protein causes biological response
4) signal shutdown - allows other signals to be heard
features:
a) SPECIFICITY - not every ligand can bind every receptor
b) AMPLIFICATION - hard for nucleus to hear signal unless amplified
c) MODULARITY - activation of one signalling molecule has predictable response - organised into modules
d) ADAPTION/DESENSITISATION - triggers negative feedback - ensures that signal is turned off after being heard
e) INTEGRATION - two signals integrated to achieve average of both response
allosteric vs covalent modification
allosteric modification:
- the ability of a molecule to alter the conformation of a protein when it binds non covalently to that protein
- e.g. calmodulin
covalent modification:
- modification of the chemical structure of the target protein
- can be reversible
- e.g. phosphorylation, ubiquitination, lipidation, SUMOylation
protein phosphorylation/dephosphorylation
phosphorylation:
- achieved by kinases
- act on serine, threonine and tyrosine amino acid residues
- can activate or deactivate proteins
dephosphorylation:
- achieved by phosphatases
- dephosphorylation by protein phosphatases allows phosphorylation to operate against a loq background
- makes system precise and sensitive
- eg. SHP1
modular domains
proteins contain small domains that can interact with each other —> essential for signal transduction
examples:
- C1 –> binds diacylglycerol –> recruitment to membranes
- EF hand –> binds calcium –> calcium dependent mechanism
- SH2/PTB –> binds phospho-tyrosine –> tyrosine kinase pathways
- PH –> binds phospho-inositides –> recruitment to membranes + motility
some modular domains contain enzymatic activity (e.g. kinase, phosphatase, protease)
some proteins (signalling adaptors) do not contain any functional activity but possess multiple interaction domains that allow them to function as a bridge between other proteins
polyubiquitination: K48 mechanism
Ub proteins must be made ready for target protein (2 steps)
1) attaches to E1 Ub ligase changes protein conformation
2) now binds E2
E3 Ub ligase binds to specific E2 and target protein - gives specific interaction —> covalent attachment
Repeats for multiple rounds of ubiquitination
K48 linkage tells cell that protein needs to be destroyed polyubiquitination must be K48-linked for recognition by the proteasome
Ub chain binds to lid of proteasome, triggering unfolding of protein
Protein enters proteasome and is degraded
polyubiquitination: TRAF 6 mechanism
1) TRAF 6 auto-ubiquinates to generate K63 polyUb chains on itself
2) these form a scaffold to recruit TAK1 kinase and the I-κB Kinase complex
3) TAK1 activates I-κB Kinase by phosphorylation
4) I-κB Kinase then phosphorylates I-κB (inhibitor of NF-kB)
IL-1 receptor signalling: turning signal off
1) turning off receptor
- IL-1RA (receptor antagonist) made in signalling pathway of IL-1
- IL-1RA binds to IL-1 receptor, prevents signalling cascade activating (neg. feedback inhibition)
2) Turning off NFκB
- I-κB re-synthesised to inhibit NF-κB
- restore signal silence
GPCR: adenylate cyclase activity
(follow the a’s)
adenylate cyclase = effector = trans-membrane protein with two cytosolic catalytic domains
cAMP = 2nd messenger
Gαs = activate adenylate cyclase = increase cAMP
Gαi = deactivate adenylate cyclase = decrease cAMP
PKA = protein kinase A = in inactive state consists of 2 catalytic subunits that are inhibited by 2 regulatory subunits
activation:
1) adenylate cyclase interacts with Gαs
2) conformational change –> two domains interact to form active site
3) adenylate cyclase cleaves the γ and β phosphates of ATP to generate cAMP at active site
4) cAMP binds the two regulatory subunits of PKA –> causing them to release catalytic subunits –> PKA activated
* ** signal amplified at each step
deactivation:
1) adenylate cyclase interacts with Gαi
2) conformational change –> two domains forced further away
3) no active site —> no cAMP production
4) signal is silenced
GPCR: phospholipase C activity
(follow the p’s)
1) GPCRs interact with Gαq or Gαo to activate phospholipase C (membrane-bound enzyme)
2) phospholipase C hydrolyses the phosphoester bond in PIP2
3) PIP2 cleavage generates 2 second messengers: DAG and IP3
4) generation of IP3 opens channels in ER to increase cytosolic [Ca2+]
5) inactive protein kinase C (PKC) binds to Ca2+ and is relocalised to membrane
6) DAG + Ca2+ binds PKC to activate it
7) active PKC phosphorylates proteins, e.g. activating transcription factors/enzymes
GPCR: turning signalling off
1) turning off receptor:
- GPCR kinases (GRKs) phosphorylate C-terminus of receptor –> prevents binding of G proteins
- arrestins can now bind GPCR
- arrestins direct the GPCR for internalisation, resulting in:
- —–> recycling of the dephosphorylated inactive GPCR to the plasma membrane
- —–> degradation
2) turning off activated G-proteins:
- in inactive G-proteins, GTPase activity (GTP to inactive GDP) has low efficiency
- activated G-protein interacts with effector —> increases efficiency
- GTP —> GDP: returns G-protein to off-state
3) turning off 2nd messengers (cAMP/IP3)
- cAMP modified by enzyme cAMP phosphodiesterase
- returns to AMP (not signalling protein)
- IP3 reacts with phosphatase which removes phosphate
- returns to IP2 (not signalling protein)
cytokine receptors: mechanism
1) cytokine receptors (and RTKs) contain a dimer of two chains in cytosol
2) ligand binding triggers conformational change which brings two chains closer together —> activates signalling
3) each receptor chain bound to specific JAK kinase (far apart before activation)
4) receptor ligation causes conformational change which brings two JAKs close together
5) JAKS phosphorylate eachother —> activate kinase activity —> JAKs phosphorylate receptor chains
6) phosphoryated chains become docking site for a phosphotyrosine-binding motif (e.g. SH2)
7) STAT monomers are recruited to the receptor via their SH2 domain
8) JAKS phosphorylate STAT molecules
9) triggers release from receptor and self-interaction (SH2 domain of one monomer binds the phosphotyrosine of another)
10) STAT dimerisation reveals a nuclear localisation sequence, allowing nuclear entry
11) STATs bind to specific DNA motifs and regulate gene expression
receptor tyrosine kinases: mechanism
1) RTKs (and cytokine receptors) contain a dimer of two chains in cytosol
2) ligand binding triggers conformational change which brings two chains closer together —> activates signalling
3) kinase domains of the receptor then activate each other by phosphorylation, and phosphorylate other residues in the receptor tails (more efficient than cytokine, eliminates JAK)
4) phosphoryated chains become docking site for a phosphotyrosine-binding motif (e.g. SH2 / PTB)
5) can activate multiple pathways:
- IP3/DAG
- MAPK pathway
- PI3 kinase pathway
phospholipase Cγ signalling
RTKs and CRs commonly activate phospholipase Cγ
PLCβ and PLCγ perform same function
PLCβ localised in membrane and activated by G protein,
PLCγ is present in the cytosol and has an SH2 domain
PLCγ is recruited to activated (phosphorylated) receptors via its SH2 domain, which:
- enables PLCγ phosphorylation by the receptor –> activating it
- brings the PLCγ close to its substrate, PIP2
* now same pathway as GPCR * - PIP2 —> PIP3
- PIP 3 promotes release of calcium stores
- activation of calcium-sensitive proteins
activation of protein kinase B
- PKB binds to PI3Ps via its PH domain, unmasking its kinase activity
- other kinases (PDK1 and 2) then phosphorylate PKB, leading to full activation
- PKB leaves plasma membrane to regulate cell survival, glucose uptake, etc via phosphorylation
as PKB inhibits cell death, PKB dysregulation is often associated with cancer
MAPK pathway
- almost all RTKs and CRs activate the MAPK pathway
- ras is a G protein molecular switch, activated by a separate adaptor and a GEF (GPRC itself is GEF)
- ras mutations are frequently seen in cancers: locks Ras in GTP-bound ‘on’ state —> uncontrolled cell division
- binding of hormone triggers dimerisation –> forms binding sites
- forms binding sites
- GRB2 reacts with phosphotyrosine residues of receptor
- GRB2 then recruits SOS which recruits inactive RAS
- SOS tells RAS kick off GDP and bind GTP —> activates RAS (GRB2 = adapter, SOS = GEF)
- active RAS then activates RAF by removing inhibitory protein
- RAF instructs RAS to exchange GTP for GDP (GEF —> turns RAS off)
- RAF phosphorylates MEK
- MEK phosphorylates MAPK
- MAPK activates cellular programs via different mechanisms:
- —-> dimerise and phosphorylate cytosolic substrates
- —-> translocate to the nucleus and phosphorylate transcription factors
