Lecture 14 - Signal Processing Pathways Flashcards
Signals are transduced by
Reversible signals causing conformational changes
Phosphorylation can
Switch on proteins
Phosphorylation is done by enzymes called
Kinases
Use ATP to phosphorylate at specific amino acids
Dephosphorylation can
Switch off proteins
Dephosphorylation is done by enzymes called
Phosphatases
Phosphorylation occurs at
Serine and Threonine amino acids
Why does phosphorylation cause conformational change?
Negative phosphate gives amino acid a negative charge which causes a conformational change
Conformational change in a protein is what causes
Signalling
e.g. opens up the protein to interact with a substrate
GPCRs are
Serpentine receptors (spaghetti) with 7 TM domains
GPCRs pick up
External signals
How many GPCRs in humans
700
When a ligand binds to a GCPR it causes
Conformational change
GPCR
G protein coupled recptor
GPCRs act as GEFs (guanine nucleotide exchange factors) to
cause exchange of GDP to GTP on a set of 3 G proteins (heterotrimeric)
Heterotrimeric G proteins are
A set of three proteins, alpha, beta, gamma
Alpha and gamma subunits of G proteins are
Membrane bound by covalently attached lipid tails
Alpha G protein subunits bind
GDP (inactive receptor) or GTP (active receptor)
When alpha subunits are activated
They dissociate from the beta-gamma subunits
Steps of G protein activation (3)
- Signalling molecule binds to the GPCR
- Receptor causes alpha subunit to bind GTP
- Active alpha subunit then binds a target and activates it
G proteins are inefficient
GTPases
Hydrolyse GTP, dissociate from target and bind with beta and gamma units again
Many GCPRs are coupled to
Stimulatory trimeric G proteins Gs
adenylyl cyclase
Stimulatory trimeric G proteins Gs activate
Adenylyl cyclase
Adenylyl cyclase is a
Membrane bound enzyme
Produces cAMP from ATP
cAMP
Is a second messenger
A derivative of ATP and used for intracellular signal transduction in many different organisms in the cAMP-dependent pathway
Second messengers are
Intracellular signalling molecules released after the extracellular first messengers
Cytoplasmic cAMP is normally
Very low
But can increase rapidly
What enzyme converts cAMP to AMP (turns off the signal?)
Phosphodiesterases
cAMP causes effects through
Protein Kinase A (PKA)
Structure of PKA
2 catalytic subunits and 2 inhibitory subunits
When cAMP binds to PKA
The inhibitory subunits are released and PKA is activated
PKA is
Localised in the cell by AKAPs (A kinase anchoring proteins)
Provides rapid response to signals
AKAPs
A kinase anchoring proteins
A group of proteins which bind the regulatory subunit of protein kinase A (PKA) and confine the holoenzyme to discrete locations within the cell
PKA phosphorylates (2)
Two kinds of target proteins:
Fast (phosphodiesterase switch)
Slow (CREB (dna transcription))
Phosphorylated CREB (by PKA)
Controls transcription
Binds to CBP and CRE portions of DNA upstream of target genes
Trimeric Gq proteins activate
Phospholipase C-beta (membrane bound)
Phospholipase C-beta acts on
phosphatidylinositol 4,5-bisphosphate
PI(4,5)P2
PI(4,5)P2 is the
least abundant phosphoinositide in the PM
Phosphatidylinositol
A family of lipids that form a minor component on the cytosolic side of eukaryotic cell membranes
The phosphate group gives the molecules a negative charge at physiological pH
PI(4,5)P2
Is cleaved to inositol 1,4,5-
triphosphate (IP3) and diacylglycerol (DAG)
Cleaved DAG
Remains at the plasma membrane and immediately binds Protein Kinase C (PKC)
IP3 binds
A gated Ca2+ ion channel in the ER membrane, causing an increase in Ca2+ concentration in cytosol
Ca2+ binds and activates
PKC, which phosphorylates specific target proteins
When a Ca2+ channel is transiently opened
Ca2+ rushes out
Propagation of a local Ca2+ signal
Results in waves or spikes
Ca2+ is used for
Egg activation
Muscle cell contraction
Neurotransmitter secretion
Ca2+ concentrations in the cytosol are
Low (10-7)
Low cytosolic Ca2+ is achieved by (3)
- Ca2+ pump in ER membrane
- Ca2+ importer in mitochondrial membrane
- Ca2+ binding molecules in the cytosol
Ca2+ waves and spikes are controlled by
Positive and negative feedback
Released Ca2+ propagates further release until v high concs then inhibits
Ca2+ spikes are recognised by
CAM kinases
Calmodulin is a
Protein which changes conformation allosterically when bound to Ca2+
Needs 2 Ca2+ to bind
Targets CAM kinases
The flexible structure of Ca2+ allows it to
Interact with many proteins, activating them
Calmodulin targets
CAM kinases
Ca2+ is a
Secondary messenger
Allosteric regulation is
The regulation of a protein by binding an effector molecule at a site other than the enzyme’s active site The site to which the effector binds is termed the allosteric site
Calmodulin and Haemoglobin
CAM kinases can
Autophosphorylate
So even when Ca2+ is lost, the signal is active until phosphatases overwhelm it
Enzyme coupled receptors are usually
Transmembrane proteins that are directly or indirectly coupled to enzymes on the cytosolic side
Examples of enzyme coupled receptors
Receptor tyrosine kinases Tyrosine kinase-associated receptors Receptor Ser/Thr kinases Histidine kinase-associated receptors Receptor guanylyl cyclases Receptorlike tyrosine phosphatases
Receptor tyrosine kinases (RTKs) transmit
Signals from growth hormones and growth factors
When a ligand binds to a RTK it
Dimerises (joins with another) resulting in transautophosphorylation
Transautophosphorylation is
RTKs dimerising, phosphorylating and activating each other
What binds to the phophorylated RTKs?
Docking proteins
Docking proteins
Signal downstream
Different RTKs possess
Different docking domains
so will activate combinations of downstream targets
RTKs target
Small GTPases Ras and Rho AND PI 3-Kinase
Ras is anchored to
The cytoplasmic side of the PM
RTK docking proteins bind the
pTyr on the GTP receptors
Binding of the RTK docking proteins to Ras
Brings a Ras-GEF to the PM which activates Ras
Ras activates a kinase
Causes a MAP kinase cascade
Results in phosphorylation of many target proteins
Cellular response
Activation of a phosphatase and MAPK-dependent
inactivation of Raf regulates the
MAP cascade
MAP kinase cascade is also regulated by
Negative feedback
PI (RTK target) 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
PI3 kinase diverts
Some of the PI(4,5)P2 from the PLC pathway, to generate PI(3,4,5)P3
PI(3,4,5)P3 interacts with
kinases: PDK1 and Akt
PDK1 phosphorylates and activates
Akt on two Ser/Thr residues
Akt targets
Proteins at the PM and elsewhere
Signalling pathways must
Co ordinate with each other to produce and appropriate cellular response
Downstream molecules from one pathway
Might act upon molecules from another
GPCRs and enzyme coupled receptors are the two main
Mechanisms that transduce information encoded in via an extracellular signal, into the cell
Second messengers are
Cyclic nucleotides, lipids and cations
Messages can be altered within the cell with
Intracellular regulation by upregulating, downregulating or integrating the signal with others
Signalling pathways work
Synergistically to produce the correct cellular response
Signalling steps (5)
- Signal molecule
- Signal receptor
- Signal transduction cascade
- Effector proteins
- Altered cellular behaviour
Signalling can result in
Altered gene expression, metabolism, cell shape or movement
