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Molecular Cell Biology > Signal Transduction > Flashcards

Signal Transduction Flashcards

1
Q

cell surface v intracellular receptors

A

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

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2
Q

intracellular signals

A

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

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3
Q

Juxtacrine signalling

A

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

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4
Q

Paracrine signalling

A

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)

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5
Q

Endocrine

A

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

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6
Q

Synaptic signalling

A

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

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7
Q

signal reception by different cell types

A

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

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8
Q

signal integration and cellular decision making

A

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

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9
Q

Need for signal transduction

A

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

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10
Q

fast v slow cellular responses

A

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

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11
Q

mechanisms of intracellular signal transduction

A

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

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12
Q

Allosteric regulation

A

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

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13
Q

allosteric regulation - cAMP and Epac

A

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

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14
Q

Phosphorylation and protein state

A

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

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15
Q

protein kinases types

A

tyrosine kinases
-specifically phosphorylate OH on tyrosine

serine/threonine kinases
-phosphorylate OH on serine and threonine

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16
Q

Protein kinase general structure

A

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

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17
Q

Domain organisation of proteins

A

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

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18
Q

domain shuffling

A

can lead to changes in proteins in evolution
by changing diff combos of domains

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19
Q

Protein-Protein interaction domains

A

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

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20
Q

SH2 domain

A

Src homology domain 2
binds phosphorylate tyrosines

most common eukaryotic domain

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21
Q

SH3 domain

A

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

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22
Q

PH domain

A

recognises highly negatively charged phosphoinositide ligands
bind these signalling lipids (PIP2, PIP3)

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23
Q

Src

A

protein that has SH3 and SH2 recognition domains

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24
Q

Scaffolds and adapters

A

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

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25
Q
A
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26
Q

2 parts of a receptor

A

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

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27
Q

antagonist mechanism

A

ligands which stabilise the inactive conformation

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28
Q

agonists

A

the actual signal
stabilises the receptor’s active conformation

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29
Q

Cell surface receptors activity

A

some have intrinsic enzymatic activity involved in signalling
the others that do not instead co-opt other proteins with enzymatic activity to signal

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30
Q

enzymatic activity through cell surface receptors

A

-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)

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31
Q

receptors w/out enzymatic activity

A

many different types
no clear classification
-ion channel coupled receptors
-Adhesion (ECM) sensing (includes integrins)

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32
Q

Intracellular receptors

A

includes steroid hormone receptors.
Are often transcription factors

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33
Q

Intracellular receptors: bacterial transcription regulators

A

either:
-ligand activated
-ligand inhibited

Monomeric proteins consisting of two domains
-DNA motif recognition domain
-signal binding domain

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34
Q

Ligand activated bacterial TRs:

A

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:

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35
Q

Ligand inhibited bacterial TRs

A

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

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36
Q

Intracellular receptors in humans

A

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

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37
Q

TGF beta pathway basic

A

Based on serine threonine receptor kinases
sense important growth horomones that are also important morphogens

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38
Q

TGF beta pathway signalling molecule

A

Large protein Dimer
2 identical subunits - antiparallel dimer

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39
Q

Class II cell surface receptor (TGF beta pathway stuff)

A

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

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40
Q

Type I receptor signalling after phosphorylation by Type II/Ligand complex

A

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

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41
Q

SMAD signalling complexes

A

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

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42
Q

nuclear negative regulation of SMAD signalling

A

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

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43
Q

Receptor endocytic dynamics

A

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)

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44
Q

Receptor tyrosine kinases

A

evolutionarily novel

large extracellular domain involved in ligand sensing:
-discriminator

beneath membrane is kinase domain:
-tyrosine kinase
-C terminal tail has role in signalling

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45
Q

allosteric regulation in RTKs

A

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

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46
Q

Non-receptor tyrosine kinases

A

soluble cytoplasmic proteins
can still interact w PM
eg SFK - Src Family Kinases
viral protein co-opted for cellular use

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47
Q

Src domains

A

SH1: Tyrosine kinase domain
SH2: Binds phosphorylated Tyrosine
SH3: Binds PXXP kink strucrture
SH4: N-terminal lipidated fragment allowing binding to PM

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48
Q

Src activation

A

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

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49
Q

Src inactivation

A

Phosphorylate C terminal tail
dephosphorylate activation loop
let molecule fold back in on itself and diffuse back into cytoplasm

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50
Q

activity of proteins in RTK downstream signalling complexes

A

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

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51
Q

PI3 kinase

A

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

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52
Q

Signalling complexes in receptors without enzymatic activity

A

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

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53
Q

signalling into nucleus after JAK activation and c terminal tail phosphorylation

A

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

54
Q

Fast Negative feedback of JAK/STAT signalling complexes

A

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

55
Q

Slow negative feedback on JAK/STAT signalling complexes

A

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

56
Q

Integrins - adhesion sensing receptors

A

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

57
Q

inside out integrin signalling

A

cell making itself adhesive to environment ECM on request
eg leukocytes sensing signal from inflammation site
activates integrins

58
Q

molecular basis for integrin activation

A

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

59
Q

outside in signalling from integrins

A

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

60
Q

Monomeric G-proteins - Ras superfamily

A

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

61
Q

Small GTPase regulation mechanism

A

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

62
Q

GEFs

A

G protein exchange factors

stabilise free of nucleotide conformation and release GDP
GTP can then bind to nucleotide free state and releases the GEF

63
Q

GAPs

A

G protein deactivators
activate hydrolysis of GTP by the GTPase

64
Q

GDIs

A

GDP dissociation inhibitors
certain families of GTPases possess them

proteins that transport the GTPases through the cytoplasm through various membrane compartments

65
Q

GTPase conformation in active and inactive

A

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

66
Q

Effectors of Small GTPases

A

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

67
Q

Scaffold interaction w GTPase

A

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

68
Q

Small GTPases as membrane proteins

A

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

69
Q

Large G-proteins

A

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

70
Q

Large G-protein activation

A

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

71
Q

Large G-protein Deactivation

A

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)

72
Q

G protein coupled receptors - GPCRs

A

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

73
Q

Ligand binding to GPCR

A

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

74
Q

GPCRs as allosteric regulators

A

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

75
Q

GPCR example: Rhodopsin light sensing

A

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

76
Q

what determines specificity of GPCR signalling?

A

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

77
Q

integration of signals and flexibility

A

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

78
Q

second messengers basic

A

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)

79
Q

Hydrophilic/soluble second messengers

A

gases - NO, CO2 H2S

reactove oxygen species ROI

Ca2+ ion !!

cyclic nucleotides !!

inositol triphosphate !!

80
Q

Hyodrophobic/membrane diffusible second messengers

A

diffuse in membrane (PM, Golgi)

glycerol
phosphotidic acids
phosphoinositides

81
Q

2 major classes of lipid 2nd messengers

A

Sphygnolipids

Glycerophospholipids

82
Q

Sphygnolipids

A

processed from Sphygnomyelin
Sphygnoid base
2 lipid tails
Has positive choline attached to negative phosphate at head

83
Q

Sphygnolipid processing

A

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

84
Q

Glycerophospholipids

A

Phosphatidic acid - range of effectors, negative charge

diaglycerol - basic part of Phospholipids, can be dephosphorylated/phosphoryolated
neutral charge - binds and activates protein kinase C

85
Q

Phosphoinositide cycle

A

generates 8 v important lipids (phosphoinositides)
produced out of Phosphatidylinositol by sequential PI kinase and phosphate action

86
Q

Phosphoinositol qualities

A

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

87
Q

Phosphatidylinositol structure and phosphorylation

A

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

88
Q

Phosphatidylinositol biphosphates

A

the most important
3 diff ones
PI3,4P2
PI3,5P2
PI4,5P2

produced in small bursts

89
Q

Phosphatidylinositol triphosphate

A

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

90
Q

Phospholipids as source of soluble Second messengers

A

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

91
Q

Calcium ion as a major second messenger

A

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

92
Q

Ca2+ storage in cells

A

in 2 types of organelle

> the ER, 0.1-1mM
large conc gradient w cytoplasm

> mitochondria
can store higher conc than extracellular environment, 10mM

93
Q

Ca2+ signalling, channel activity

A

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)-

94
Q

Ca2+ toxicity in cell

A

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

95
Q

how is Ca2+ sensed in cell? - Calmodulin

A

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

95
Q

GEF and calmodulin example

A

Ca2+ floods into cell
binds calmodulin
calmodulin wraps around IQ motifs in GEF
GEF then changes to bind PM where they activate Ras GTPase

95
Q

Ca2+ influx regulation at channels by calmodulin

A

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

96
Q

the 5 types of Ca2+ channel in cell

A

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

97
Q

Calcium effectors examples:

A

Calmodulin kinase

Calcineurin Phosphatase - PP2B

98
Q

Calmodulin kinase structure and activation

A

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

99
Q

Calmodulin kinase use

A

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

100
Q

Calcineurin (PP2B) structure

A

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

101
Q

Calcineurin (PP2B) activation

A

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

102
Q

Cyclic AMP basic signalling properties

A

cAMP - organic molecule

signalling done by rapid generation and degradation of the molecule

generated by adenyl cyclase family of proteins

103
Q

cAMP generation

A

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

104
Q

cAMp degradation

A

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

105
Q

cAMP effectors

A

only know 3

nucleotide gated Ca2+ channels

protein Kinase A

GEF Epac - cAMP binding changes conformation so that it can bind Rab GTPase

106
Q

Protein kinase A and cAMP

A

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

107
Q

MAP kinase meaning

A

Mitogen activated protein kinases

108
Q

MAP kinase cascades

A

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

109
Q

MAP3K activation

A

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

110
Q

MAPK activation

A

needs both a serine and threonine phosphorylation
sites near each other on activation loop

111
Q

MAP kinase cascade opposition

A

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

112
Q

MAPK gene expression regulation

A

-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

113
Q

Yeast pheromone MAP kinase cascade pathway

A

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

114
Q

role of Scaffolds in MAPK signalling

A

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

115
Q

two yeast MAPK cascades controlling morphogenesis/growth

A
  1. Pheromone pathway (Ste/Fus stuff)
  2. Filamentous growth pathway
116
Q

yeast filamentous growth pathway control by MAPK cascade

A

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

117
Q

yeast pathways controlling stress response that respond to MAPK cascades

A

High osmolarity response

cell wall integrity response

118
Q

High osmolarity respones - Yeast MAPK cascade response to stress

A

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

119
Q

MAPK cascade response -yeast cell wall integrity

A

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

120
Q

Complexity of mammalian MAPK cascades (please make it stop)

A

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

121
Q

Activatory signals of mammal MAPK pathways

A

mitogenic signals/growth factors
eg the ERK pathway

can be involved in cancers so many drug targets within

122
Q

ERK pathway - downstream of growth factor receptors

A

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

123
Q

ERK pathway in cancer

A

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

124
Q

MAPK cascade generation of complex behaviour

A

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

125
Q

MAPK cascade dynamics and feedback loops

A

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

126
Q

MAPK cascade positive feedback

A

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

127
Q

MAPK cascade negative feedback

A

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

128
Q

Synthetic biology - building of MAPK cascades with new properties

A

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

Molecular Cell Biology (5 decks)

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