Signal Transduction Flashcards
cell surface v intracellular receptors
cell surface most common
ligand does not enter cell
signal transduction inside cell from PM receptor
intracellular
more evolutionarily ancient
singal molecules diffues through PM freely
receptor located intracellularly (often in cytoplasm or nucleus)
activated receptor complex
intracellular signals
DNA damage
noxious chemicals
^^can be produced by radiation
monitored by cell
also:
-pH
-O2 conc
-cAMP conc
-ATP conc
in these cases call them sensors usually instead of receptors
Juxtacrine signalling
v close range
neighbours contacting neighbours
-ligand expressed on one cell
-receptor on neighbour
-signal not soluble but is displayed on cell surface
eg delta/notch
Paracrine signalling
Mid range
mediated by proteins
typically growth factors
protein secreted by one cell type
diffuse short distance in organ/tissue
recevied by other cells surface receptors
can be diff cell type
or same one (autocrine signalling)
Endocrine
Hormones eg
requires a carrier of the signal (eg blood)
gland secretes soluble hormones
one molecule can carry many different messages to many diff cell types in body
Synaptic signalling
between neurons
requires direct interaction like juxtacrine
but neuron cells grow v long axons
has reach of endocrine
relies on diffusible signals
but only through the synaptic cleft
signal reception by different cell types
same signal molecule can be decoded into different signals depending on cell context
different transduction of same signalling molecule
something to do w second messengers
signal integration and cellular decision making
the action a specific cell takes depends on integration of the combo of many signals it experiences
mammal cells need to receive survival signal to not apoptose
in addition to this
cell receives other signals which sould affect its decision to re-enter into cell cyce/divide or differentiate
Need for signal transduction
Decisions made in nucleus where gene expression changed, affecting protein production etc…
signal comes form outside
need to transduce signal binding receptor to effect in nucleus
fast v slow cellular responses
slow:
by altering gene expression
signal from outside
transduction into nucleus
affects gene expression
-minutes to hours
>5mins response = slow response
fast:
many things can happen by bypassing nucleus/gene expression
-fast decision making with no time available to activate genes, synthesise proteins: eg chemotaxis
-signal alters function of already present proteins instead of changing expression to make new ones
-seconds to minutes
mechanisms of intracellular signal transduction
signal transduction is achieved via:
-reversible signal-dependent modulation of protein-protein interaction networks within cells
protein-protein direct contact to make complex
complex makes response
often involves going to nucleus (slow)
responses that need to be quicker than gene expression
usually done through post-translational modification
-acetylation
-phosphorylation (inc. autophosphorylation)
-addition of proteins (ubiquitin, SUMO…)
second messengers
secondary signal released all around inside cell
eg cAMP cascade
Allosteric regulation
basis of signal transduction
proteins large structures w many domains/conformations
functional domains (enzymatic, protein interaction sites)
and regulatory domains - where signal is received
many proteins found in autoinhibition state where they are in conformation that obscures the functional domain
allosteric regulation - something interacts with regulatory domain
protein changes conformation in response
-as a result the functional domain is allowed to become available for its function
allosteric regulation - cAMP and Epac
Epac active conformation:
functional domain can interact w small GTPases
regulatory domain can swing like door
blocks functional domain
cAMP binds the regulatory region
caused protein to swing open
leaving functional domain open to interact w small GTPase target
Phosphorylation and protein state
addition of Pi from an ATP molecule
ATP->ADP + Pi
Pi usually added to OH group
by kinases
reverse does not require energy
hydrolysed off target by Phosphatases
Phosphorylation can result in allosteric regulation
-changes electric charge of protein (as Pi can carry btwn 2-4 -ve charges)
if ionic interaction is what keeps the conformation the way it is
can interrupt that by phosphorylating residue here
neutralise +ve charge
releases functional domain
protein kinases types
tyrosine kinases
-specifically phosphorylate OH on tyrosine
serine/threonine kinases
-phosphorylate OH on serine and threonine
Protein kinase general structure
kinase domain
-tyrosine
-serine/threonine
has two lobes
-n terminal lobe
-c terminal lobe
-phosphorylation of residue in the cleft between the lobes
-have an activation loop that is phosphorylated on the specific residues
some kinases are constitutively active and so dont need phopshorylation on active loop
Domain organisation of proteins
all higher eukaryote proteins made of domains (not so much in bacteria - chromosome organisation - all one long ORF)
-individual exons can correspond to a domain (sometimes domain is multiple exons)
-3D folding is important - similar folding can result in similar domain activity, certain shapes correspond certain functions
kinases in humans and fungi look v similar
however regulatory domains differ more
domain shuffling
can lead to changes in proteins in evolution
by changing diff combos of domains
Protein-Protein interaction domains
SH2, PTB: interact with phosphorylated tyrosines
other domains recognsie phosphoryalted serines, threonines
some recognise methyl/acetylated residues - important in histone interactions
others to ubiquitination - important in protein regulaiton and degradation
SH2 domain
Src homology domain 2
binds phosphorylate tyrosines
most common eukaryotic domain
SH3 domain
also v common
binds PXXP motif
two prolines separated by 2 other AAs
-PXXP causes a kink in the protein - SH3 recognised this kink motif
PH domain
recognises highly negatively charged phosphoinositide ligands
bind these signalling lipids (PIP2, PIP3)
Src
protein that has SH3 and SH2 recognition domains
Scaffolds and adapters
proteins made up entirely of protein-protein interaction domains
adapter - binds one protein at one end and another at the other end
-bridges two proteins
scaffold:
-important for holding proteins together in reactions
eg assembling important signalling complex and tethering it to where it needs to be eg a calcium channel
-domain 3 binding Ca2+ channel
-domain 1 with PLC
-domain 2 with PKC kinase
-brings them all tohether at channel
-calcium enters through channel and activates PKC
>enriching necessary protein in pathway where they need to be
2 parts of a receptor
Discriminator domain
-tells what ligand it is
effector domain
-part which transduces the signal
-many divergent ways
transition btwn inactive and active frequently an example of allosteric regulation
(most?) receptors found in inactive conformation when no ligand present
if it is active without ligand then problems
antagonist mechanism
ligands which stabilise the inactive conformation
agonists
the actual signal
stabilises the receptor’s active conformation
Cell surface receptors activity
some have intrinsic enzymatic activity involved in signalling
the others that do not instead co-opt other proteins with enzymatic activity to signal
enzymatic activity through cell surface receptors
-large class of receptors that are coupled with G-protein
-receptor kinases, RKs:
>more ancient serine/threonine
>Receptor tyrosine kinases found in animals (higher eukaryotes)
receptors w/out enzymatic activity
many different types
no clear classification
-ion channel coupled receptors
-Adhesion (ECM) sensing (includes integrins)
Intracellular receptors
includes steroid hormone receptors.
Are often transcription factors
Intracellular receptors: bacterial transcription regulators
either:
-ligand activated
-ligand inhibited
Monomeric proteins consisting of two domains
-DNA motif recognition domain
-signal binding domain
Ligand activated bacterial TRs:
require ligand to dimerise from monomers in cytoplasm
-requires sufficient signalling molecule conc inside cell
-due to symmetric nature of dimer - needs to bind divergent identical sequences that are palindromic on the DNA
-can be activators or inhibitors of transcription
-need to bind ligand to dimerise
-need to dimerise to bind DNA
eg TraR: bacterial quorum sensing TF:
Ligand inhibited bacterial TRs
opposite of ligand activated ones
-structurally similar to activated ones
-DNA binding motif
-ligand binding motif
-form stable dimer in absence of ligand
eg TetR
Tetracycline binds receptor
dimer doesnt dissociate
but loses DNA affinity when bound to ligand
Intracellular receptors in humans
nuclear receptors
DNA binding motif
ligand binding motif
sense small molecules that penetrate membrane
-Steroid hormones:
>cortisol
>retinoic acid
>thyroxine
principle similar to bacteria
BUT can be Monomeric OR Dimeric
also can coopt a lot of coactivators and coinhibitors - form complexes w other TFs in eukaryotic nucleus
TGF beta pathway basic
Based on serine threonine receptor kinases
sense important growth horomones that are also important morphogens
TGF beta pathway signalling molecule
Large protein Dimer
2 identical subunits - antiparallel dimer
Class II cell surface receptor (TGF beta pathway stuff)
Class II:
Constitutively active TM kinases
-Recognise the antiparallel dimer ligand
-2 Type II receptors bind across the ligand
-now have affinity to recruit the Type I receptor
-enables protein-protein interaction btwn type I and II receptors
-Type II is constitutively active kinase that phosphorylates and activates Type I
Type I receptor signalling after phosphorylation by Type II/Ligand complex
Type I receptor is the Effector part of receptor complex
responsible for further signalling
SMADs
-recognise phosphorylated type I receptor
-the phosphorylation allows them to bind type I
-Type I phosphoryaltes SMAD on c terminal tail (2 serines)
-changes SMAD conformation, comes off receptor complex
SMAD signalling complexes
after phosphorylation by Type I receptor and coming off the receptor complex:
-3 SMADs to form signalling complex
-phosphorylation allows them to assemble
assemble into heterotrimer:
-2 SMADs
-1 CoSMAD
heterotrimer exposes on its surface a nuclear localisation signal
-imported
-in nucleus it can bind resident TFs and influence gene expression
nuclear negative regulation of SMAD signalling
phosphatases in the nucleus remove the phosphate from SMADs
individual SMAD monomers exported out of nucleus
this process of SMAD activation - nucleus - phosphatase - export cycles as long as there is signalling from receptor
phosphatase disassembles all the SMAD complexes eventually when signalling stops
Receptor endocytic dynamics
important factor in signalling
-receptors in eg class II system are clustered by binding the ligand
-this complex is endocytosed
by clathrin coated pits:
-pathway where complexes signal and receptors get recycled back to PM
-maintains sensitivity by continuously recycling receptors
by Caveolin pathway:
-receptors endocytosed
-go to degradation pathway
-cell can downregulate signalling by removing receptors into degradation pathway
-drops sensitivity to ligand (adaptation)
Receptor tyrosine kinases
evolutionarily novel
large extracellular domain involved in ligand sensing:
-discriminator
beneath membrane is kinase domain:
-tyrosine kinase
-C terminal tail has role in signalling
allosteric regulation in RTKs
though to not be present at first
idea that binding ligand brough kinase domains of two receptors close enough to cross-phosphorylate
but this is not true
even tho both kinases are identical
-one serves as allosteric regulator of other
-only one gets kinase activity activated
-then this Kinase phosphorylates BOTH c terminal tails to activate receptors
-Each pY on these tails is a binding slot for a protein with an SH2 domain
allows complex to grow and grow
creating almost a solid phase of protein underneath membrane that connect individual receptors through multivalent binders
Non-receptor tyrosine kinases
soluble cytoplasmic proteins
can still interact w PM
eg SFK - Src Family Kinases
viral protein co-opted for cellular use
Src domains
SH1: Tyrosine kinase domain
SH2: Binds phosphorylated Tyrosine
SH3: Binds PXXP kink strucrture
SH4: N-terminal lipidated fragment allowing binding to PM
Src activation
Tyrosine residue at C terminal domain
phosphorylated in inactive form
SH2 domain loops back to bind this
puts protein in “latched” closed conformation
closing in on itself inhibiting the phosphorylation
this c terminal phosphate needs to be removed to activate Src kinase activity
this allows protein to open up
SH2 and SH3 domains can also find external ligands now (SH2 - pY, SH2 - PXXP)
complex stage wise activation process which eventually leads to phosphorylation at activation loop of kinase
Src plays big role in signalling of many receptors
Src inactivation
Phosphorylate C terminal tail
dephosphorylate activation loop
let molecule fold back in on itself and diffuse back into cytoplasm
activity of proteins in RTK downstream signalling complexes
Src kinase
-SH2 and SH3 domains
-recognise pY in on RTK and contribute to phosphorylating other proteins in vicinity
ZAP70
-similar organisation kinase
-2 SH2 domains, can bind 2 pY residues
-carries tyrosine kinase so can contribute to making more pY in the complex
Grb2
-scaffold
-has SH2 and SH3 domains
PI3 kinase:
-lipid kinase
-generates PIP3 - a second messenger (adds another phosphate to PIP2
PI3 kinase
lipid kinase
generates PIP3 second messenger from PIP2
is a complex of 2 diff proteins
>P110 -catalytic
>P85 - regulatorty
if this complex is not bound to pY then the regulatory subunit inhibits the caralytic one
inhibited conformation
2 SH2 domains - binding pY on c terminal tails of RTK: de inhibits the catalytic subunit
can start generating PIP3 lipid
connects RTK signalling to 2nd messenger PIP3 generation
Signalling complexes in receptors without enzymatic activity
eg cytokine receptors (erythropoietin receptor eg)
looks similar to RTK
-EC discriminator
-TM region
-C terminal tail that can be phosphorylated
1st part of complex activation is different
requires specific class of non-receptor TKs that recognise active bound receptors (allosteric regulation :0)
These kinases are JAKs
recognise active receptor
bind it
they themselves are allosterically activated by this
then similar to RTK
activated JAKs can phosphorylate C-terminal tails
generate lots of pY
proteins eg Src can bind
signalling into nucleus after JAK activation and c terminal tail phosphorylation
requires signal transducer: STATs
also are transcription activators
has SH2 domain
binds pY on receptors C terminal tail
STAT is phosphorylated by JAKs
unbinds C terminal tail
two STAT subunits complex by binding each others’ pY with their SH2 domain
-dimerisation exposes their nuclear localisation signal
-go to nucleus
-then are disassembled by phosphatase in the nucleus
-in some cases: after the STATs are dephosphorylated the nuclear localisation signal becomes an export signal, exported by CRM1 protein
cycling continues as long as receptor active
Fast Negative feedback of JAK/STAT signalling complexes
Fast negative feedback:
-cytoplasmic tyrosine phosphatases (SHP1/2)
-recruited into signalling complex
-while the ligand is present activation prevails over inactivation
-overall signalling of complex
-once signal is removed
-phosphatases prevail, quickly remove phosphate from the pY residues
-signalling complex disassembles in seconds
Slow negative feedback on JAK/STAT signalling complexes
requires change in gene expression (transcription and translation takes time)
phosphorylated STATs transcriptional target is a molecule called SOCs
has SH2 domain and a domain that recruits SOC to signalling complex
ubiquitinates complex resulting in endocytosis of receptors and depletion of signal
Integrins - adhesion sensing receptors
sense adhesion of cells to environment
-integrin binds ECM
-alpha and beta integrins
-different kinds in each class so can generate diff dimer combos
normally in inactive conformation
folded over
inside out signalling and outside in signalling
inside out integrin signalling
cell making itself adhesive to environment ECM on request
eg leukocytes sensing signal from inflammation site
activates integrins
molecular basis for integrin activation
eg talin, kindlin, vinculin
adapters between cytoskeleton and c terminal tails of integrin receptors
integrin opens up and can now grab ECM
also causes them to cluster on cell surface
eg focal adhesions
outside in signalling from integrins
integrins have no tyrosine kinase domains
require help of non receptor tyrosine kinases
-Src kinase
-Focal adhesion kinase
different mechanisms of engaging them based on integrin class
but idea is same
activated integrins clustering results in formation of bound kinases which cross phosphorylate due to being at high density
Monomeric G-proteins - Ras superfamily
Ras small GTPases
can bind GTP - use as fuel for work
involved in allosteric regulation
have switch I and II
mobile elements
position of these elements depends on if bound to GTP or GDP
Small GTPase regulation mechanism
nucleotide cycles
active when bound to GTP
can bind effectors that they activate
effectors have high affinity for GTP form
low for GDP form
GEFs and GAPs activate and deactivate them
GEFs
G protein exchange factors
stabilise free of nucleotide conformation and release GDP
GTP can then bind to nucleotide free state and releases the GEF
GAPs
G protein deactivators
activate hydrolysis of GTP by the GTPase
GDIs
GDP dissociation inhibitors
certain families of GTPases possess them
proteins that transport the GTPases through the cytoplasm through various membrane compartments
GTPase conformation in active and inactive
Gamma phosphate of GTP stabilises interactions with G protein
folds in switch I and II
GAP has arginine residues with +ve charge
force open switches
now H2O can get in and hydrolysis of GTP is sped up 1000x from GTP on its own
Gamma phosphate removed from GTP -> GDP
causes inactive conformation
-switches open up due to no negative charge from phosphate group
-conformation no longer recognised by effectors
GEF can bind
residues open up the switches - allosteric regulation
lets GDP unbind from protein
-lots of GTP in cytoplasm much 10x GDP conc
-GTP much more likely to come in and bind
-Grabs the switches and pulls them back in
-G protein no linger has affinity for GEF
back to beginning of cycle
Effectors of Small GTPases
GTPases play 2 roles
-recruit effectors - GTPases usually membrane bound so bring enriches effectors from cytoplasm to membrane
-providing biological work for the reaction
-enzymes
-scaffolds
Scaffold interaction w GTPase
inactive scaffold conformation only has one open site for GTPase
small GTPase binds in active form
allosteric regulation of scaffold effector
can now bind its interaction partners (eg kinases in cascades)
GTPase allows the chemical reaction on the scaffold to take place
Small GTPases as membrane proteins
most of them are functional as membrane proteins
except Ran
– plamitoylated (ingolgi) = reversible
– prenylated = permanent
increase strength of interaction with membrane - can no longer dissociate from it
are packed in vesicles
transported to PM on cytoskeleton
GTPases end up on PM
activated here by upstream receptors
at enzyme there is enzyme that cleaves Palmitoyl - allows dissolving into cytoplasm
can float around until reach golgi again
cycles
Large G-proteins
Heterotrimeric complexes
3 subunits
Alpha - Binds and hydrolyses GTP
Beta - one large fold - tangled up with small peptide: the Gamma subunit
beta and gamma assembled at time of protein translation - unbreakable interaction
until they are both disassembled together later on
Alpha and gamma attached directly to membrane
relationship with beta subunit is more dynamic
coupled to receptors
receptors play the role of GEFs for Small GTPases
they activate the Alpha subunit
Large G-protein activation
assembled as trimer when inactive
alpha sub is bound to GDP
high affinity of alpha for beta/gamma
receptor activation:
-receptor acts like GEF
-widens GTPase domain cleft
-GDP falls out
-GTP now binds (10x GDP conc)
-3 things happen when EC signal binds receptor:
>exchange of nucleotide in G-alpha subunit
>as a result - Beta/gamma and G-alpha subunit complex breaks up
>both dissociate from receptor
active G alpha and Beta/gamma subunits can signal by binding partners
but only G-alpha has GTPase activity - can provide work
Large G-protein Deactivation
RGS proteins - Regulators of G-protein Signalling
have affinity for large G-proteins
help them hydrolyse GTP quickly
once inactivated - reverse process
-inactive G-alpha rebinds beta/gamma
-rebind receptor (alpha only bit that interacts w it)
G protein coupled receptors - GPCRs
7 pass strand membrane proteins
7 alpha helices incorporated into PM
3 loops in extracellular part
3 loops intracellular
N terminus - V diverse - usuallly EC
C teminus - IC
G proteins bind the intracellular loops
interact w G-alpha subunit
Ligand binding to GPCR
either at N terminus or with the EC loops or woth the alpha helices
alpha helices conformational change key part of transmitting signal
C terminal tail can be phosphorylated to interact with signalling molecules
GPCRs as allosteric regulators
eg opioid receptor
Binding pocket for ligand
ligand sinks into hole btwn alpha receptors
causes conformational change
have 100s conformations - varying levels of activity
GPCRs unusual as can have base level of activity without ligand binding
ligand binding increases or decreases this
agonist - high binding ligand - activity goes higher
-partial agonists dont activate as much - usually pharmaceuticals - real molecule is full agonist
inverse agonist shuts down the basal activity
GPCR example: Rhodopsin light sensing
Rhodopsin GPCR detects photons
has no chemical ligands
-Retinal is bound already
-2 conformations of Retinal
>Cis - arms folded
>Trans - Arms open
Photon absorbed by Cis-retinal
changes conformation to trans-retinal
physically pushes on alpha helix
transfers conformational change to GPCR a-helices
-activates G protein Transducin
-active transducin activates cGMP phosphodiesterase
-which hydrolyses the cGMP in the cell
-causes cGMP activated ion channels to stop working
-causes membrane hyperpolarisation
-initiates nervous pulse
what determines specificity of GPCR signalling?
Variety of G-alpha subunits defines it
960 combinations of alpha, beta, gamma
from different isoforms
Gs - adenyl cyclase activator
Gi - adenyl cyclase inhibitor
Gq,11 - activates PLC-Beta
G12,13 - activate signalling via RhoA - specifically function upstream of small GTPases, in active conformation recruit GEFs for them - causes cascade of action
integration of signals and flexibility
integration of many signals provides additional flexibility
eg adenyl cyclase
generates cGMP from GTP
Gs-alpha subunits act upstream of it (from receptors receiving stimulatory hormones)
activates adenyl cyclase
inhibitory pathways activated by other hormones
compete for activation/inhibition of adenyl cyclase
integrates many signals into one response
second messengers basic
intracellular signalling molecules
of a non-protein nature
most are made intracellularly and are then free to diffuse in cytoplasm (hydrophilic) or in the membrane (hydrophobic)
Hydrophilic/soluble second messengers
gases - NO, CO2 H2S
reactove oxygen species ROI
Ca2+ ion !!
cyclic nucleotides !!
inositol triphosphate !!
Hyodrophobic/membrane diffusible second messengers
diffuse in membrane (PM, Golgi)
glycerol
phosphotidic acids
phosphoinositides
2 major classes of lipid 2nd messengers
Sphygnolipids
Glycerophospholipids
Sphygnolipids
processed from Sphygnomyelin
Sphygnoid base
2 lipid tails
Has positive choline attached to negative phosphate at head
Sphygnolipid processing
Sphygnomyelinase (a signalling enzyme) processes sphygnomyelin to remove the phosphocholine
ceramide molecule remains
-precursor for two diff signalling molecules
>tail at bottom: Arachidonic acid, precursor for others
>Sphignosine - poor affinity for lipid bilayer, is phosphorylated by sphignosine kinase to make Sphignosine-1-Pi (soluble in cytoplasm), so can diffuse between and within cells
Glycerophospholipids
Phosphatidic acid - range of effectors, negative charge
diaglycerol - basic part of Phospholipids, can be dephosphorylated/phosphoryolated
neutral charge - binds and activates protein kinase C
Phosphoinositide cycle
generates 8 v important lipids (phosphoinositides)
produced out of Phosphatidylinositol by sequential PI kinase and phosphate action
Phosphoinositol qualities
strongly negatively charged lipids
eg
PIP = -3
PIP2 = -4
PIP3 = -5
nonspecifically interact v strongly and non-specifically with positively charged proteins esp with lipid binding domains
many actin polymerisation regulator proteins recognise them in the membrane and bind them
Phosphatidylinositol structure and phosphorylation
Glycerol as base of lipid
3C with 3 OH groups
all 3 OH attached to something
-2 with carboxylic acids
-other one with a phosphate - importanf for connecting glycerol to head group
Head group is a sugar, inositol
– carbons are numbered
– C number one is attached to the phosphate
– 23456 around rest of ring
– different numbered carbons (3 4 or 5) phosphorylated to give diff Phosphotidylinsoitol
eg phosphorylate carbon 3 - PI3P
then carbon 5 to give - PI3,5P2
continuous flux of these molecules being modified into different ones by phosphorylation/phosphatases
Phosphatidylinositol biphosphates
the most important
3 diff ones
PI3,4P2
PI3,5P2
PI4,5P2
produced in small bursts
Phosphatidylinositol triphosphate
PI3,4,5P3
produced in small bursts
by Phosphatidylinositol triphosphate kinase (PI3 kinase - important with RTKs)
binds and is involved in activation of PKB kinase
important for cell survival
deregulation of this signal resuslts in cancer cells
quickly degraded by P10 phosphatases
Phospholipids as source of soluble Second messengers
some G-protein effectors are phospholipases
chop off the phosphatidylinositol phosphates
several classes
PLA (Phosphatidyl lipase A)
chops off the tails of the glycerol’
liberates the arachidonic acid making it a freely diffusible signalling molecule
PLC
chops the phosphate off the glycerol
releases the inositol sugar bound to the phosphates
a biphosphate would release inositol sugar with 3 phosphates
– results in IP3, v important signalling molecule as it activates calcium channels in ER and releases calcium from ER
– connects extracellular signals to release of intracellular calcium from ER
Calcium ion as a major second messenger
Inorganic ion
cell cannoth create or destroy it
can only manipulate by sequestration/release
Ca2+ is very naturally abindant
conc in environment - 1mM
cytoplasmic Ca2+ only 100nM (10,000x less)
-cells spend lots of energy pumping Ca2+ out
-no energy required for influx, just open channels
Ca2+ storage in cells
in 2 types of organelle
> the ER, 0.1-1mM
large conc gradient w cytoplasm
> mitochondria
can store higher conc than extracellular environment, 10mM
Ca2+ signalling, channel activity
extracellular stimulus on PM receptor
activates Phospholipase C PLC
-PLC processes phosphatidylinositol lipids
-releases Inositol triphosphate IP3 signalling molecule
-This opens Ca2+ channel
-Ca2+ floods cytoplasm
>low Ca2+ cellular conc - activates channels
>high Ca2+ cellular conc - inhibits channels
>excitable system - add a little - increases release even more
>then later they close down
Biphasic dynamics creating a wave of Ca2+ propagating in the cell
either:
>small flashes
>or complex wave patterns
causes quick violent reaction from cell
Ca2+ con decreases from active pumping out of Ca2+ (to mitochondria, extracellular, ER)-
Ca2+ toxicity in cell
Ca2+ is toxic to cell if too much in cytoplasm for too long
because it activates so many signalling molecules
also activates ancient response of ocean cells for breaking of the PM
-activates PM healing response in cells
how is Ca2+ sensed in cell? - Calmodulin
By Calmodulin
main Ca2+ sensor
Ca2+ binds to the EF-hand motif via negative charged residues
EF-hand can coordinate many Ca2+ ions (up to 4)
one EF-hand on each end of Calmodulin
responds to changing Ca2+ conc via allosteric regulation
2 diff spatial conformations
Ca2+ binds
changes Calmodulin conformation
then can bind other proteins that have an IQ motif/domain
that affects conformation of downstream protein
GEF and calmodulin example
Ca2+ floods into cell
binds calmodulin
calmodulin wraps around IQ motifs in GEF
GEF then changes to bind PM where they activate Ras GTPase
Ca2+ influx regulation at channels by calmodulin
All Ca2+ channels autoregulated by Ca2+
PM channel has calmodulin near it
-channel opens
-Ca2+ floods in from EC environment
-immediately binds calmodulin molecules nearby
-Bound calmodulin binds the channel and causes it to close
-only lets the channel signal for ~1sec
-around 1million ions let in
fast negative feedback on channel
the 5 types of Ca2+ channel in cell
Receptor operated
-respond to Extracellular agonist ligand (eg neurotransmitters)
cyclic nucleotides
-second messenger operated
allows cell to let in calcium based on intracellular activity
machinery for complex feedback loops
voltage operated
-voltage across membrane generates pulse
stretch activated mechanosensory channels
Piazza 1 and 2
-like bath plug
-bent in a way
-membrane stretches
-their conformation changes to open up
-lets Ca in
allows cell to sense mechanical tension in PM
Ca release activated channels
CRACs
-Ca in ER drops
-RI sensor protein
-forms membrane binding cluster
-interacts w Steam channnels
-steam/RI permits input of calcicum in response to Ca depletion in ER
Calcium effectors examples:
Calmodulin kinase
Calcineurin Phosphatase - PP2B
Calmodulin kinase structure and activation
Shuriken structure
2 hexamer layers of molecules
6 upper
6 lower
12 kinase domains overall
all independent - no cooperativity
all are autoinhibited :
-locked on something that resembles substrate but cannot be acted on so is stuck
-IQ motif on each kinase
-calmodulin binds it
-deinhibits
-kinase unfolds and can perform phosphorylation to activate neighbour domains
-other proteins dephosphorylate activation loop and inactivate kinase domains again
Calmodulin kinase use
can sense the frequency of the Ca2+ signal pulses
infrequent pulses activate fewer kinase domains (1-2)
more frequent pulses cause the kinase to keep activating
-every new pulse allows a new kinase domain to open up
-activity of the kinase grows proportionally to eg frequency of neural pulses allowing info processing in neuron cells
Calcineurin (PP2B) structure
Heterodimer
2 peptides - A and B subunit
autoinhibited in signal absence
B subunit binds Ca directly via EF-hand motifs
A subunit has flexible domain called the B subunit binding helix
Calcineurin (PP2B) activation
under normal circumstances A subunit bound to B subunit via the B subunit binding helix
in Ca2+ presence Calmodulin wraps around the helix and prevents it from binding B subunit
activation of this phosphatase requires both directly binding Ca2+ and binding Ca bound calmodulin
Cyclic AMP basic signalling properties
cAMP - organic molecule
signalling done by rapid generation and degradation of the molecule
generated by adenyl cyclase family of proteins
cAMP generation
by adenyl cyclase proteins
take molecule of ATP
hydrolyse the beta and gamma phosphates together as a pyrophosphate
remaining alpha phosphate ends up bound to 2 groups on the same sugar
creating cyclic molecule
cAMp degradation
by phosphodiesterases
many of them present in cell on lookout for cAMP
gives v short range of cAMP signalling
gives v sharp spatial maxima
if inhibit phosphodiesterases - whole cell floods with cAMP
makes it useless for signalling by giving uniform distribution
cAMP effectors
only know 3
nucleotide gated Ca2+ channels
protein Kinase A
GEF Epac - cAMP binding changes conformation so that it can bind Rab GTPase
Protein kinase A and cAMP
PKA
heterotrimer
2 kinase domains - catalytic sububits
dimer of regulatory subunits
each reg subunit has 2 cAMP binding slots
when cAMP binds allosteric regulation makes them release the catalytic kinase domains as free floating molecules
cAMP molecules in fast equilibrium with regulatory site binding
so cAMP degradation in cytoplasm by phosphodiesterases:
-causes conforation of regulatory subunits to change back so that catalytic subunits are sequestered again
MAP kinase meaning
Mitogen activated protein kinases
MAP kinase cascades
consist of 3 kinases that sequentially activate each other via phosphorylation
MAP3K - Serine/Threonine
MAP2K - Double specificity: Tyrosine and Serine/threonine
MAPK - usually the effector - goes to nucleus and phosphorylates targets
cascade used for signalling
MAP3K activation
by a variety of input signals controlled by various pathways including:
-receptors
-small GTPases
-G-proteins…
all MAP3K require activation by an upstream kinase sometimes called MAP4K
BUT these ones belong to all sorts of classes of kinase so not so consistent to call them that
MAPK activation
needs both a serine and threonine phosphorylation
sites near each other on activation loop
MAP kinase cascade opposition
each phosphorylation step in the cascade is opposed by phosphatases
without the opposition o phosphatases the cascade would flare up one time and stay up all the time - useless for signalling
MAPK gene expression regulation
-MAPK phosphorylated by upstream kinases
-detaches from the cascade (suggests some allosteric regulation - change in affinity for scaffold proteins)
MAPK can phosphorylate cytoplasmic targets
eg p90 RSK
but generally signal by being imported into nucleus
>have no DNA binding domain
>but instead phosphorylate resident factors
>can do this via parallel pathways: activates two diff factors that work together to bind DNA and change gene expression
Yeast pheromone MAP kinase cascade pathway
in budding yeast
a produces chemoattractant for alpha
binds GPCR receptor:
-beta/gamma subunit of G-protein signals
-active b/g subunit binds and recruits scaffold from cytoplasm - Ste5
-recruits the three MAPKs:
>Ste11 -MAP3K
>Ste7 -MAP2K
>Fus3 -MAPK effector
>phosphorylates Ste12 TF
MAP4K initiator is Ste20 - phosphorylates Ste11
Ste20 activated by small GTPase Cdc42
Fus3 inhibits Tec1 binding to Ste12 in filamentous growth pathway
role of Scaffolds in MAPK signalling
thought before that MAPKs phosphorylated each other in cytoplasm
but in reality it occurs on scaffold that recruits necessary components and enriches them near receptor
scaffold originally thought to be one big protein that combines all the components together like a holder
-keeps them on the same structure so changes dynamics as no longer need to randomly bump into each other
however Ste5 (and other scaffolds) operate a bit differently:
-Ste7 MAP2K already coupled w Ste5 scaffold
-Fus3 (MAPK) is allosterically activated by Ste5 scaffold, its activation loop is revealed upon binding Ste5, allows access by Ste7 (MAP2K)
-phosphorylated Fus3 then released to nucleus
basically - all kinases dont simultaneously bind the scaffold
two yeast MAPK cascades controlling morphogenesis/growth
- Pheromone pathway (Ste/Fus stuff)
- Filamentous growth pathway
yeast filamentous growth pathway control by MAPK cascade
non sexual growth
doesnt result in fusion
instead produces protrusion that grows like Hypha (isnt one tho)
-shares elements w pheromone pathway
-difference is whether or not Ste5 (scaffold) is present
-Ste20 activated by Cdc42 small GTPase in both pathways
-filamentous growth pathway skips Ste5
-activates ONLY Kss1 protein (pheromone pathway activates both Fus3 and Kss1 MAPKs)
-because Fus3 requires binding to Ste5 to reveal activation loop for phosphorylation by Ste7
-changes Kss1 activity (also no Fus3 direct activity present either)
-so get different response
reuse of proteoins in diff context to give diff response
yeast pathways controlling stress response that respond to MAPK cascades
High osmolarity response
cell wall integrity response
High osmolarity respones - Yeast MAPK cascade response to stress
Sensors between cell membrane and cell wall
-Hypertonic stress (eg salinity increase from drying puddle)
-cess sense change in turgor pressure from water leaving cell, cell shrinking - sense mechanical change
-activates Cdc42
-activates Ste20 (MAP4K)
-This time Pbs2 protein is activated: is the MAP2K and Scaffold:
-Cdc42
-then Ste20 MAP4K
-Ste11 MAP3k
-Pbs2 Scaffold + MAP2K
-Activates Hog1 MAPK, activating its many nuclear factor targets
HOG pathway
MAPK cascade response -yeast cell wall integrity
Sensors also betwenn membrane and wall
-Rho1 small GTPase activated
-an effector is Pkc1 -the MAP4K
-MAP3,2,1Ks actiavted
-MAPK activates factors that stop budding growth
because as bud grows cell wall synth hasnt caught up fully - so is weaker at bud
-pathway acts as frrdback in normal growth so it doesnt break the wall
-can also act to stop cell cycle under stress
Complexity of mammalian MAPK cascades (please make it stop)
kinases at each step able to be replaced by diff homologues
many kinases can do same step
diff combos of kinases to make pathways
many of these cascades require input from small GTPase
the MAP4K is the effector of the small GTPase
Activatory signals of mammal MAPK pathways
mitogenic signals/growth factors
eg the ERK pathway
can be involved in cancers so many drug targets within
ERK pathway - downstream of growth factor receptors
growth factor receptor is an RTK
dimerises when ligand binds
-Adapter Grb2 has SH2 domains binding the C-terminal pY residues on the RTK dimer
-this adapter binds mSOS - a Ras GEF
-causes Ras to be activated on the PM down stream of the Grb2/mSOS complex
-Ras recruits the MAP3K - RAF
-the MAP4K PAK2 is recruited by Rac, another GTPase
many scaffolds involved
MP1 scaffold holds MEK1 (MAP2K?)
then MAPK ERK1 - has many targets in nucleus and on nuclear membrane
activates early respnse genes
ERK pathway in cancer
dysregulated in cancer
can try to target RAF(MAP3K)
or Ras GTPase
Ras cant take small molecule inhibitors
so instead artificially target it for degradation
MAPK cascade generation of complex behaviour
why not just have one kinase autoamplifying
scaffolds exist so not just a bunch of amplification in cytoplasm
instead having 3 kinases allows having a Non-linear response:
-eg Michaelis menten - describes a system where response is linear until a certain point
-If the Hill coefficient is >1 then response begins to look more non-linear
-as n gets bigger - more extreme non linear
-basically sets a threshold after which activity jumps on a lot
-allows the system to not respond to the small background noise in the simulus (as it would a small amount if response was linear proportionally)
Will only give response after a significant stimulus threshold has been passed
hill coefficient in MAPK pathway could be ~5
MAPK cascade dynamics and feedback loops
dynamics of the cascade shaped by feedback loops
system has fast positive feedback for activation
sharp rise
followed by slower negative feedback for a drop back to pre-stimulus levels
-called PERFECT ADAPTATION
MAPK cascade positive feedback
Positive feedback helps to provide fast and strong activation
many mechansims
phosphatases that inactivate the kinase targets are found already bound to the MAPK protecting it from noise signal
but activated MAPK phosphorylates the phosphatase and removes it
since the inhibitor is removed - MAPK activity can go up
MAPK also activates a GEF that gives the Small GTPases in teh response more GTPase activity
MAPK cascade negative feedback
MAPK slowly phosphorylates other components of cascade incl. the scaffold protein
After MAPK actovoty grows too high it disassembles the complex back down
until the phosphatases have time to remove the phosphorylations so complex can reassemble for more activation
Synthetic biology - building of MAPK cascades with new properties
using the principles of MAPK cascades
it is possible to control behaviour of signal transduction pathways at will:
experimental example:
-yeast pheromone pathway modified to encode Ste5 with a sticky bit and also additional proteins whose expression was controlled by Ste12 as either negative regulators (phosphatases with binding domain for the Ste5 sticky bit) or another version positive regulators (Ste50 with a binding domain)
these experiments demonstrated that the pathway with additional feedback loop demonstrated Hill coefficient 2.4 vs 1.2 for the WT cascade
