Cell Bio - Intracellular signalling Flashcards
what does FRET show
shows that proteins tethered to the lipid membrane interact more
what does FRET (fluorescent resonance energy transfer) dependant on
depends on the distance of the flurophores
must be within approx. 10 nm
what drives cell transformation
forced membrane localisation of PKB
concerning protein localisation at the membrane what are the phosphobinding motifs and what is the other type of binding motif
SH2
PTB
ubiquitin binding motif
what is AKAP interaction domain
interaction between a cytosolic and membrane bound protein
concerning lipid interaction motifs, what are the phosphoinositide interacting motifs
PH
FYVE
PX
PHD
lysine - arginine (K-R) rich patches
in terms of protein-membrane localisation, what are the mechanisms used to control lipid tether at the membrane
myristoylation
prenylation
single lipid tethers are often not stable enough, how is this problem rectified
lipid tether is compined with lipid interaction motifs to induce stable localisation
what is fatty acylation
mainly consists of the addition of myristic or palmitic fatty acids to a protein
how is myristate bound to the protein
bound to the N-terminal glycine residue via an amide bond
irreversible
what is step 1 in myristoylation
removal of the N-terminal methionine
step 2 of myristoylation
activation of myristic acid via CoA
step 3 of myristoylation
coupling of myristic acid to glycine
what is the role of myristoylation in apoptosis
1 - caspase mediated cleavage of Bid exposes a glycine
2 - Bid is myristoylated
3 - lipid tether induces insertion into mitochondrial membrane
4 - recruitment of BAK to the mitochondrial membrane
5 - cytochrome-C is released
6 - apoptosis downstream
to what does prenylation occur
to proteins containing a CAAX motif on their C-terminus
what is a CAAX motif and what does it generate
C - cystine residue, functions as isoprenoid attachment
A - signifies any aliphatic amino acid
X - any of several amino acids
generated a thioether linkage
how is prenylation initiated
by the attachment of farnesyl/geranylgeranyl to the cystine residue via farnesyltransferase or garenylgeranyltransferase
how can a prenylated protein be further processed
RAS-converting CAAX endopeptidase 1 (RCE1) which removes the -AAX residues
what is the function of isoprenylcysteine carboxylmethyltransferase (ICMT)
caps the carboxy group on the now C-terminal isoprenoid-modified cystine residue
using a methyl group
what is Kras4B targeted to and how
targeted to PM
via interactions with its polybasic region with phosphoinositides
how often is Ras mutated in human tumours and where
16%
mutation in codon 12,13 and 61
why do H and N Ras undergo prenylation and palmitoylation
to target them stably to the membrane
how does Kras get targeted stably to the membrane
only undergoes prenylation
uses PRB for stable association to the membrane
what is progeria syndrome such as Hutchinson-Gilford (HGPS) caused by
abnormal processing of the CAAX protein
prelamin A
what can be used to treat HGPS
Ftase inhibitors
effects of adrenergic activation
Increase strength and heart rate
Increased blood flow to muscle
Increased systemic and cellular energy supply
Increased skeletal muscle force
decreased gut motility
decreased peripheral blood flow
what releases norepinephrine
sympathetic nervous system post-ganglionic fibres
where do alpha-adrenoceptors occupy
smooth muscle cells
where do beta-adrenoceptors occupy
adipose tissue
skeletal muscle
liver
cardiac cells
role of cAMP in the activation of phosphorylase
adrenaline induced secondary messenger that leads to the activation of phosphorylase
what is the function of the N-terminus and c1 and c2 regions of adenyl-cyclase
important for regulation by Galpha-GTP Complex signalling in mammalian cells
which forms of adenylate cyclase are activated by calmodium/Ca2+ and inhibited by Ca2+
activated:
- III
- V
- VIII
inhibited:
- I
- VI
what does the GPCR-ligand stimulate
conversion of GDP to GDP at the Gα subunit
what do the GTP conversion stimulate
dissociation of Gα from the beta/gamma subunits
what stimulates adenylate cyclase
a dissociated Gα subunit
what does intrinsic or stimulated GTPase activity cause
return of Gα to resting state and association with beta/gamma subunits
structure of protein kinase A (PKA)
consists of 2 regulatory subunits - inactive form (R2-C2)
2 catalytic subunits - active form - (C2)
what is allosteric activation
when cAMP activates PKA by dissociating the R subunits from the R2-C2 complex
what does the R-subunit of PKA contain
pseudosubstrate-sequence
what does cAMP dependant PKA target and what does it lead to
phosphorylase kinase
leads to glycogenolysis
what precisely causes the conformational change of phosphorylase kinase
phosphorylation of serine 14
how are receptors desensitised to switch of GPCR and cAMP signalling
GPCR N-terminal is phosphorylated by beta-adrenergic receptor kinase
what do phosphorylated GPCR N-terminal tails recruit and what do they do
beta-arrestin
attenuates heterotrimeric G-protein activation
induces receptor internalisation
how is Gα deactivation achieved
intrinsic or GAP stimulated GTPase activity
what does GTP hydrolysis cause
stops activation of downstream targets
causes re-association of Gα with beta/gamma subunits
how is cAMP deactivation achieved
activation of phosphodiesterase (PDE)
hydrolysis of cAMP to generate AMP
what do PKA phosphorylates signal to in terms of fatty acids
hormone sensitive lipase (HSL)
perilipin
what is the function of HSL
leads to translocation to lipid droplet and its activation
function of perilipin and what inhibits it
acts as a barrier to lipid hydrolysis
phosphorylation
how does beta-adrenergic signalling stimulate increased heart rate and force
step 1
the Ca1.2 channel increases Ca2+ influx in response to depolarisation
how does beta-adrenergic signalling stimulate increased heart rate and force
step 2
the ryanodine receptor (RYR) stimulated increased calcium induced calcium released from the sarcoplasmic reticulum
increased heart force
function of phospholambam and what does it lead to
prevents it from inhibiting SERCA mediated Ca2+ uptake
enables faster relaxation for next contraction
faster HR
what is a-kinase anchoring protein (AKAP)
contribute to spatial and selective restriction PKA signalling
what does cholera toxin switch on
Gs-coupled proteins
what switches off Gi-coupled protiens
pertussis toxin
what adrenergic receptors does adrenaline act on for lipolysis
β3/α2 receptors in adipose
what does Gαq activate
phospholipase C (PLC) to regulate downstream smooth muscle cell contraction
Structure of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2)
Favoured chair structure has 5 equatorial OH and 1 axial oh.
Consider a turtle with its head as the axial OH then the glycerol is connected to the right flipper which is position 1.
what does α-1 adrenoceptors act as
act as GEF for Gαq
leads to PLC activation and Ptdlns4,5P2 hydrolysis
leads to contraction and vasoconstriction
how is PLCβ1/3 activated
PLCβ1 - activated by Gαq
PLCβ3 - activated by β and gamma subunits
how is PLCγ1 activated
Tyrosine phosphorylated receptor interacts with SH2 domain of PLCγ1. PLCγ1 is then tyrosine phosphorylated and activated
GPCR activate PLC mediated hydrolysis of PtdIns(4,5)P2 to produce what
to generate two new second messengers Diacylglycerol (DAG) and Ins(1,4,5)P3
features of lns(1,4,5)P3
water-soluble molecule and diffuses across the cytoplasm to the IP3 receptor which is found on the membrane of the endoplasmic reticulum
features of lns(1,4,5)P3-receptor
ligand-gated ion channel that allows the influx of calcium cations upon activation
3 isoforms exist
structure of lns(1,4,5)P3-receptor
made of a tetramer:
- N-terminal Ins(1,4,5)P3binding domain
- Coupling domain
- Transmembrane domain
- Gatekeeper domain
how is Ins(1,4,5)P3-Receptor
activated
IP3 binds to the binding domain on the cytosolic face
leads to conformational changes
channel is opened
calcium influx
what downstream target is activated in response to increase in diacylglycerol (DAG)
Protein Kinase C (PKC)
what relieves psuedosubstrate inhibition
DAG and Ca2+ at the membrane induce conformational changes
what is PKC regulated by and what is its function
receptor for activated C-kinase (RACK)
regulates downstream phosphorylation
what is EPAC (exchange protein directly activated by cAMP)
EPAC is a guanine nucleotide exchange factor (GEF) that binds cAMP which induces a conformational change to activate its GEF activity
which has a higher lower affinity for cAMP
PKA or EPAC and what does it mean
EPAC
only activated at high concentrations
what is RAP
a small molecular weight G-protein
acts as a molecular switch to regulate cytoskeletal dynamics, cell adhesion and secretion
what family does the insulin receptor belong to
tyrosine kinase receptor superfamily
what does ligand induced dimerisation of tyrosine receptor induce
cross phosphorylation
increased kinase activity
further receptor tail phosphorylation
purpose of the activation-loop phosphorylation
stabilises the active conformation of the insulin receptor tyrosine kinase (IRK)
what is P85 and what does it lead to
a regulatory subunit of PI3K
contains an SH2 domain
when bound to the receptor PI3K becomes active
generates PIP3
what does the SH2 domain of P85 also recruit
PLCγ to the phosphorylated receptor
activates PLCγ
generates DAG and IP3
function of the SH2 domain of Grb2
mediates recruitment and subsequent activation of the RAS/MAPK pathway
what does tyrosine phosphorylation lead to
receptor ubiquination and internalisation
how is ubiquitin coupled to tyrosine phosphorylation
via the SH2 domain of casitas B-lineage lymphoma (Cbl protein)
what is the agonist lipid secondary messenger
Ptdlns(3,4,5)P
what is PTEN and what does it do
a tumour suppressant
removes the 3 phosphate from Ptdlns3,4,5P3
what is SHIP1/2
a 5-phosphatase
removes the 5 phosphate from Ptdlns3,4,5P3
what does SHIP generate
Ptdlns3,4P2
has messenger functions
interaction of Class 1A p110 catalytic domain with p85 regulatory domain causes what
suppression of p110 subunit activity
prevents proteolytic degradation of p110
enables recruitment of holoenzyme to activated RTK receptor
what enables the coupling of tyrosine kinase receptor to class 1A PI3K
the SH2 domain of p85 interaction with specific tyrosine phosphorylated receptor peptide
what drives the synthesis of PIP3
SH2 mediated activation of PI3-kinase
function of GRB2
is an adapter protein
DH2 domain binds to specific tyrosine phosphorylated residues on an RTK
what does GRB2 recruit and how
recruits the SOS protein
via interaction with its SH3 domain with the proline-rich domain on the SOS protein
what is SOS and what enhances it
guanine nucleotide exchange factor for RAS
activity is enhanced by interaction with the PH domain of Ptdlns4,5P2
what leads to conformational changes in RAS and what does it allow
GTP exchange
enables reader interaction
what does the binary switch action of RAS cause (in terms of regulation)
converts GTP binding into the regulation of protein phosphorylation driven by the RAF/MAP kinase pathway
in terms of activation, what does the binary switch action of RAS cause
converts GTP binding into the activation of PI3K and PtdIns(3,4,5)P3 signalling
what does deregulation of RAS signalling lead to
cell proliferation and tumours
what specific mutations lead to RAS GTP accumulation
Gln61
Gly12
