Signal processing pathways Flashcards
Describe the signal processing pathway
Signal molecule, signal receptor, signal transduction cascade, effector proteins, altered cellular behaviour
Describe GPCRs
- Serpentine receptors with 7 TM domains
- Specific for external signals
- Over 700 in humans (sight, smell, taste)
- Change 3D structure upon ligand binding
- Act as GEFs to induce exchange of GDP for GTP
on a series of specific trimeric G proteins
Describe heterotrimeric G proteins
- Each is composed of an alpha, a beta and a gamma subunit
- The alpha and gamma subunits are membrane-bound by covalently attached lipid tails
- The alpha subunit can bind either GDP (inactive form of receptor) or GTP (activated form of
receptor) - In the activated form, the alpha subunit dissociates from the beta-gamma complex
Describe the process by which heterotrimeric G proteins work
- Upon activation, the GPCR induces the G-protein to exchange its GDP for GTP
- This causes the G-protein to dissociate, releasing the activated alpha subunit
- The activated alpha subunit can then bind an independent target, activating it
- The alpha subunit is an inefficient GTPase and hydrolyses the GTP
- This causes the alpha subunit to dissociate from the target protein and recombine with
the beta-gamma complex
Describe GPCRs, Gs and cAMP
- Many GPCRs are coupled to stimulatory trimeric G proteins (Gs
) which activate adenylyl
cyclase - Adenylyl cyclase is a membrane bound enzyme that produces the second messenger
cAMP from ATP - Cytoplasmic cAMP concentration is normally very low
- Within the space of a few seconds, this can increase to 5-10,000 fold
- Phosphodiesterases convert cAMP to AMP to turn off signal
Describe protein kinase A
- cAMP mediates its effects mainly via Protein
Kinase A (PKA) - PKA in a resting cell is a tetramer of two catalytic
subunits, and two inhibitory subunits - Binding of cAMP releases inhibitory subunits, thus
activating the kinase - PKA is normally localised to specific places in the
cell via AKAPs, to provide rapid response to signals - PKA phosphorylates target proteins:
– Fast ones (e.g. activating the
phosphodiesterase to ensure cAMP acts as a
“switch”
– Slow ones (e.g. CREB, which then binds CBP
and CRE elements on DNA upstream of target
genes, to target transcriptio
Describe GPCRs, Gq and PLC
Other GPCRs are coupled to
trimeric Gq proteins which
activate membrane-bound
Phospholipase C-
* Phospholipase C- then acts on
phosphatidylinositol 4,5-
bisphosphate PI(4,5)P2
*PI(4,5)P2
is the least abundant
phosphoinositide in the PM
(<10% of phospholipids and >1%
of total lipids)
* Is cleaved to inositol 1,4,5-
triphosphate (IP3) and
diacylglycerol (DAG)
Describe IP3, DAG and Protein Kinase C
- Cleaved DAG remains at the plasma membrane and immediately binds Protein Kinase C (PKC),
bringing it to the membrane. - IP3 binds a gated Ca2+ ion channel in the ER membrane, causing an increase in Ca2+ concentration
in the cytosol - Ca2+ binds and activates PKC, which phosphorylates specific target proteins
Describe Ca2+
Ca2+ is also used as a signal during
fertilisation (for egg activation), in muscle
cells (for contraction) and in nerve cells
(triggering secretion of neurotransmitters
*Concentrations of cytosolic Ca2+ normally
kept low (10-7
) by 3 main mechanisms
*When a Ca2+ channel is transiently opened,
Ca2+ rushes out
* Propagation of a local Ca2+ signal can result
in a series of waves, or spikes.
*When a Ca2+ channel is transiently opened,
Ca2+ rushes out
* Propagation of a local Ca2+ signal can result
in a series of waves, or spikes.
*These depend on a combination of +ve and
–ve feedback: released Ca2+ stimulates
further release but, at a high enough
concentration, inhibits release and resequesters Ca2+
*The frequency of the spikes is recognised by
CaM kinases
What is Ca2+ also used as a signal for?
Ca2+ is also used as a signal during
fertilisation (for egg activation), in muscle
cells (for contraction) and in nerve cells
(triggering secretion of neurotransmitters
*Concentrations of cytosolic Ca2+ normally
kept low (10-7
) by 3 main mechanisms
Describe Ca2+/CaM dependent protein kinases
- Calmodulin is a flexible protein which,
when bound to Ca2+, undergoes a
conformational change - The change is allosteric (2 or more Ca2+
ions must bind to change conformation)
causing a switch-like activation - Its flexible activated structure allows it to
interact with many proteins, activating them - One important class of targets are the
Ca2+/CaM kinases - Ca2+/CaM kinase II is initially activated by
Ca2+/CaM, causing auto-phosphorylation - Thus, even when Ca2+ signal is lost,
activity of the kinase remains until
phosphatases overwhelm it
Describe enzyme-coupled receptors
- Transmembrane proteins which are either directly, or indirectly coupled to
enzymes, usually kinases on their cytosolic side:
– Receptor tyrosine kinases
– Tyrosine kinase-associated receptors
– Receptor Ser/Thr kinases
– Histidine kinase-associated receptors
– Receptor guanylyl cyclases
– Receptorlike tyrosine phosphatases
Describe tyrosine kinases
- Very widely used to transmit signals from hormones and growth factors such as
Insulin, IGF, PDGF, NGF, FGF etc. - Ligand binding usually causes dimerisation of RTKs in the plasma membrane,
resulting in each phosphorylating and activating each other –
transautophosphorylation - Docking proteins can then bind phosphorylated tyrosines and signal downstream
- Different RTKs possess slightly different docking domains so will activate different
combinations of downstream targets
Describe RTK targets I - small GTPases
- Two classes transmit signals from RTKs –
Ras and Rho - Ras is anchored to the cytoplasmic face
of the PM (so in the right place) - RTK docking proteins, such as Grb2 (IR) or
Drk (SevR) bind the pTyr on the receptors - This brings a Ras-GEF to the PM, activating
Ras - Ras associates with and activates a kinase,
triggering activation of the MAP kinase cascade - Ultimately results in phosphorylation of
many target proteins and a cellular response - Negative feedback (activation of a
phosphatase and MAPK-dependent
inactivation of Raf) exists to regulate the cascade
Describe RTK targets II - PI 3-Kinase
- PI is the only lipid that can undergo
reversible phosphorylation at multiple sites
on its inositol head group - PI 3-kinase is able to produce a variety of
intermediates – all with a phosphorylated 3
carbon - Essentially diverts some of the PI(4,5)P2
from the PLC pathway, to generate
PI(3,4,5)P3 - PI(3,4,5)P3 is then able to interact with a
variety of target proteins (through
Pleckstrin Homology domains, and other
motifs) - These include two kinases, PDK1 and Akt
- PDK1 phosphorylates and activates Akt on
two Ser/Thr residues - Akt then targets proteins, both at the PM
and elsewhere
Integrating cell signalling
- Signalling pathways must co-ordinate with each other to produce an appropriate cellular response
- Downstream molecules from one pathway might act upon molecules from another, depending on
the cell type
Describe the mTOR signalling pathway components
- mTOR – mammalian Target Of Rapamycin
- Ser/Thr Protein Kinase
- Central element in the control of cell
growth and proliferation - Regulated by a variety of cellular signals
(growth factors, insulin, nutrients such as
amino acids and glucose, cellular energy
levels, stress…) - Two complexes, mTORC1 and mTORC2
- RHEB – Ras Homolog Enriched in Brain
- Ras family small GTPase
- RHEB-GTP activates mTOR
- TSC1/TSC2 – Tuberous Sclerosis Complex
- Inhibits mTOR (growth suppressors)
- TSC2 inactivates RHEB
- TSC2 has GAP activity towards RHEB
(GAP: GTPase activating protein) - Activated Akt phosphorylates
and inhibits TSC2
Describe the steps of the mTOR signalling pathway
- Activated Akt phosphorylates and inhibits TSC2
- Without growth factor: no cell growth; with growth factor: cell growth (look at slides)
Describe aktivation of mTOR with the phosphorylation of downstream targets
- mTORC1:
- Promotes protein synthesis
Control of mRNA translation,
Initiation of translation - Promotes ribosome production
Synthesis of components of the protein
translation system (ribosomal proteins,
initiation factors, rRNAs, tRNAs…) - Inhibits protein degradation
- mTORC2:
- actin cytoskeleton/cell shape
Describe the Aktivation of mTOR phosphorylation of downstream targets
- S6 kinase
- phosphorylates ribosomal protein S6
- increases translation of ribosomal
components - elF4E
Translation initiation factor - indirectly activated by mTOR
by inhibition of 4E-BP (an inhibitor of elF4E)
Continue explaining the mTOR signalling pathway
mTOR – central modulator of proliferative
signal transduction
- Integrates external signals with internal
signals
- Coordinates cellular growth &
proliferation
* Ideal therapeutic target against cancer
Multiple components of pathways that
signal through mTOR are dysregulated
in numerous cancer types
* Clinical importance of INHIBITORS of mTOR
rapamycin (bacterial toxin)
immunosuppressant
anti-cancer drug
Describe tuberous sclerosis
- Multi-system genetic disease
- Mutations in TSC1 or TSC2 (hamartin and tuberin)
- non-malignant tumours in the brain and other
vital organs (kidneys, heart, eyes, lung, skin) with
enlarged cells - loss of control of cell growth and cell division,
predisposition to forming tumours - Symptoms: developmental delay, behavioural
problems, skin abnormalities, lung & kidney disease
Describe polarisation of yeast cells by mating factor signalling
Cdc42-WASp-ARP - local actin nucleation at the site of mating
factor binding
actin filament growth – cytoskeletal polarity
actin cable formation – serve as tracks for directed transport
and exocytosis of new cell wall material – tip growth
Describe the insulin signalling pathway
- A receptor Tyr kinase present on the
surface of insulin responsive cells:
– Muscle
– Liver
– Fat - Consists of 2 subunits, held together by
disulphide bonds:
– alpha subunits (outside)
– 2 beta subunits (transmembrane and
intracellular) - Binding of insulin brings together
intracellular domains, allowing crossphosphorylation - This leads to docking and
phosphorylation of other proteins
Draw out the insulin signalling pathway
[slides]
