L9 Cell Signalling Flashcards
how does cAMP act through PKA
cAMP can directly activate certain types of ion channels in plasma membrane of highly specialised cells
but in most animals it exert its effects mainly by activating cAMP-dependent protein kinase A > catalyse transfer of terminal P group from ATP to specific serines or threonines > regulate activity of target proteins
structure and mechanism of PKA
PKA is a tetramer in inactive state with 2 regulatory and 2 catalytic subunits
binding of cAMP to regulatory subunits > dissociation from tetramer > catalytic domains released to phosphorylate downstream subunits
second messengers generated by phospholipase C
diacylglycerol: lipid that remains at plasma membrane to activate protein kinase C
IP3: found in cytoplasm to bind to receptors on ER to release Ca2+ into cytosol
Ca2+: found in ER to also help activate protein kinase C and other Ca2+ sensitive pathways
mechanism of phospholipase C
activated receptor stimulates plasma membrane bound phospholipase C-beta via a G protein
PI(4,5)P2 hydrolysed > IP3 and diacylglycerol produced
IP3 blind to IP3-gated Ca2+-release channels in ER membrane > large electrochemical gradient causes Ca2+ to escape into cytosol
diacylglycerol remains in plasma membrane and together with phosphatidylserine and Ca2+, helps activate PKC (recruited from cytosol to cytosolic face of plasma membrane)
three subfamilies of PKC
conventional PKC: require Ca2+, diacylglycerol and a phospholipid such as phosphatidylcholine for activation
novel PKC: do not require Ca2+ but require diacylglycerol
atypical PKC: neither dependent on Ca2+ nor diacylglycerol
PKC maturation
nascent PKC is found in inactive open conformation associated with membrane fractions
HSP90 binds in kinase domain while PDK-1 binds in carboxy-terminus and phosphorylates the activation loop > mTORC2 phosphorylates the turn motif and hydrophobic motif > fully matured enzyme localised to cytosol with pseudosubstrate blocking the substrate binding pocket
PKC activation
inactive state > PKC isozyme in close conformation with pseudo substrate blocking substrate binding region
agonist stimulated lipid hydrolysis > PKC binds Ca2+ and translocates to membranes where binding of diacylglycerol (DAG) occurs > promote activation and opening os isozyme > activated isozyme can now bind ATP and phosphorylate various substrates
substrates of PKC
major cellular substrate: myristolyated alanine-rich C kinase substrate (MARCKS) > involved in various cellular processed including rearrangement of actin cytoskeleton
other substrates: MAP kinase, transcription factor inhibitor lib, calpain, epidermal growth factor receptor
how does the cell maintain low concentration of free Ca2+ in their cytosol when Ca2+ not needed
Na+ driven Ca2+ exchanger: remove Ca2+ while transporting Na+ into cytosol
Ca2+ pump to actively pump out of cytosol
Ca2+ pump in ER membrane: actively pump Ca2+ from cytosol into ER which acts as storage
Ca2+ binding molecules in cytoplasm
active Ca2+ import in mitochondria (act as storage)
what is calcium calmodulin kinase II (CaMKII)
protein found mostly in brain to help translate calcium signals into actions
affects phosphorylation state and activity of target proteins involved in neurotransmitters synthesis and release, neuronal plasticity and gene expression
calcium levels rise > calmodulin grabs onto 4 calcium ions > Ca2+-calmodulin complex > complex switches on CaMKII
structure of CaM-kinase II
forms large oligomers made of alpha, beta, gamma and delta isoforms
each subunit has a N-terminal Ser/Thr kinase domain, regulatory Ca2+-calmodulin-binding segment and a C-terminal association domain that is responsible for oligomerization
activation of CaMKII
absence of Ca2+/calmodulin, enzyme is inactive due to interaction between inhibitory domain and catalytic domain
binding of Ca2+/calmodulin alters conformation of protein > allow catalytic domain to phosphorylate inhibitory domain of neighbouring subunits in complex and other proteins in cell
how does the autophosphorylation of CaMKII prolong the activity of enzyme
- traps bound Ca2+/calmodulin so that it does not dissociate from enzyme complex until cytosolic Ca2+ levels return otherwise basal values for at least 10 seconds
- converts enzyme to Ca2+ independent form so that the kinase remains active even after Ca2+/calmodulin dissociates from it > continues until autophosphorylation process is overridden by a protein phosphatase
what is protein kinase
enzyme that modifies other proteins by adding phosphate groups > functional change of target protein by changing enzyme activity, cellular location or association with other proteins
how is protein phosphorylation carried out
kinase removes a phosphate group from ATP > covalently attach to one of the free amino acids that have free hydroxyl group
most kinases act on serine, threonine and tyrosine
how is PKC inhibited
- Inhibitors of ATP binding to PKC > PKC cannot phosphorylate substrates > no cellular response
- PKC regulators that target DAG binding site
- inhibitors that prevent the anchoring of enzyme to receptor of activated C kinase (RACK) which was supposed to bring activated isozyme next to substrates > no function
- inhibitors of protein-protein interactions at a specific sub cellular location or with a specific substrate may provide unique inhibitors of the phosphorylation of one substrate and not others
two types of intracellular signalling complexes
preformed signalling complex on scaffold: scaffold protein hols several signalling proteins in place before signal arrives > faster and more specific
assembly of complex after receptor activation: signalling proteins only come together after receptor detects signal > more flexible
how does acetylcholine-induced opening of K+ channels in heart muscle plasma membrane work
binding of acetylcholine by muscarinic acetylcholine receptors triggers activation of transducing G protein by catalysing exchange of GDP for GTP on alpha subunit > released G beta-gamma subunit binds to and open K+ channel
K+ permeability hyperpolarises membrane > reduce frequency of heart muscle contraction
how is G protein used in yeast mating
alpha, beta and gamma subunits in G protein called GPA1, Ste4 and Ste18 respectively in yeast G protein
binding of pheromone to receptor on yeast cell > activate G protein > GPA1 dissociates > Ste4-Ste18 stay together to do the signalling
if Ste4-Ste18 subunits get deleted > sterile mutants that cannot respond to mating factor
what is important to achieve high fidelity intracellular signalling
spatial organization
if components of cell well mixed > signalling proteins hard to find each other > inefficient signalling
scaffold proteins reduces extent of cross talk > maintain pathway specificity and prevent irrelevant stimuli, and amplify signalling
types of molecular switches
phosphorylation: signals can be turned on or off by addition of removal of phosphate groups
GTPase: signals can be turned on by binding to GTP and turned off by hydrolysis of GTP to GDP
how can receptors be desensitised
can be temporarily moved to interior of cell so they no longer have access to their ligands
can be destroyed in lysosomes after internalisation
can be altered so they can no longer interact with G proteins or other enzymes coupled to them
how is GPCR desensitised
activated GPCR phosphorylated by GPCR kinase (GRK) > arrestin binds to phosphorylated GPCR > prevents GPCR from interacting with G protein and serves as an adapter for clathrin-dependent endocytosis of receptor
