BIOL 2056 - Signal transduction and intracellular signalling

methods of signal transduction: membrane translocation

• clustering of proteins at the cell membrane

• enzymatic reactions dependent on conc of substrates

• drives transformation of the cell

MECHANISMS CONTROLLING PROTEIN TRANSLOCATION

1. PROTEIN INTERACTION

   • receptor binds to ligand —> conf change

   • increased ability of protein to interact with receptor

2. LIPID INTERACTION

   • generates new surfaces of CSM which allows protein to bind to CSM

3. LIPID THETHER

   • fatty acid tail linked to a protein

   • help to tether protein to the membrane

   • often one lipid tail not enough to lipid tails modified to stabilise protein in memb.

lipid tethering

MYRISTOLATION

• activation pathway for fatty acids

• activate w CoA then remove met and couple myristific acid —> N terminal glycine residue can now attach to CSM

• cotranslational modification

• mediates cell apoptosis

• highly controlled —> only active when caspase activated and allows Bid to attach to CSM —> bingd in other proteins —> cyt c release —> cell death

PRENYLATION

• occurs in CAAX motifs on carboxyl terminus

• C residue is the isoprenoid attachment site

• A is any aliphatic AA

• X is any AA

• initiated by attachment of farnesyl or geranyl geranyl isoprenoid lipid to Cys residue farnesyl transferase or geranylgeranyl transferase

• E.g: the prenylation of Ras results in the active form

Ras association w CSM

• 3 isoforms of Ras: H, N, R

• H ras has 2 prenyl groups which forms a more stable association with the membrane than the R ras which only has 1 prenyl group and a polybasic region

• Serine residue in the middle of the R ras’ polybasic region can be phosphorylated causing a negative charge—> repels from the CSM and falls off

• when it falls off it translocates to teh other organelles and can induce apoptosis

• phosphorylation regulated by PKc

methods of signal transduction: protein phosphorylation

• requires an Oh group therefore occurs at serine, threonine and tyrosine

• increases size and surface charge of molecule

• protein kinase - writer, phosphatase - eraser

METHODS OF INCUDING CHANGE:

1. conformational change

• can disrupt to promote molecular interaction

• leads to shape change

2. protein readers

• reader proteins may only interact with the protein when its in the phosphorylated form

• E.g: PIN1 —> proline isomerase that switches proline from cis —> trans which causes downstream signalling

what can phosphorylation do?

1. conf shape change —> activation of kinases

2. induce new interactions

3. prevent protein binding

4. change subcellular localisation of proteins

5. activation of degradation pathway

sequestration

• sequestration to membranes can be used to inhibit processes

• E.g: hormones/notch signalling which requires the translocation to the nucleus

adrenergic stimulation

ADRENERGIC STIMULTION

activation of both a adreno receptors on smooth muscle cells and b adreno receptors on cardiac, liver, adipose and skeletal muscle

• increased cardiac output

• increased O2 supply

• glucose to the blood

• lipolysis for energy

• increased muscle tension

• decreased stomch/intestinal activity

• decreased peripheral blood flow

• adrenal release into blood from adrenal gland

• nervous adrenaline at postganglionic synapse

adrenal gland

ADRENAL GLAND ADRENALINE

• similar to nervous adrenaline

• chromatin cells release epinephrine in an endocrine manner

• adrenaline secreted from the medulla

OTHER HORMONES

• cortex of the medulla secretes cortisol, andonesterone and androgens

• cortisol: stress response, metabolism and immune regulation

• Aldosterone (mineralocorticoid): Regulates sodium and potassium balance, and blood pressure

• Adrenal androgens: Contribute to sex hormone balance.

adrenal pathway

• adrenaline binding to the receptor activates the heterotrimeric G protein which is tethered to the membrane

• activation of GPCR causes a conformational change so that the intracellular domain can interact with the a subunit of the G protein which binds GDP

• GDP replaced with GTP which causes the dissociation of the a subunit with the bg subunit.

• the a subunit and GTP bound stimulates the formation of cAMP by binding to adenylyl cyclase

• cAMP acts as a 2nd messenger and binds to the regulatory subunits of pKA, revealing the catalytic subunits whcih leads to the phosphorylation of glycogen phosphorylase

pKA

• 4 subunits, 2 regulatory, 2 catalytic

• allosteric activator

• when cAMP increase becomes active

• phosphorylates at serine and threonine residues

• the pseudo substrate keeps the enzyme in the inactive conformation —> looks the same as the substrate except for a glycine residue

• phosphorylation of phosphorylase causes a twist in the structure which causes activation —> phosphorylated at serine 14

OTHER EFFECTS OF PKA

• switches off glycogen synthesis

• pyruvate kinase inactivated and blocks glycolysis

• inactivates an enzyme that dephosphorylates glucose so that it is released into the blood

switching off of PKA

1. beta adrenergic receptor kinase phosphorylates the GPCR N terminus tail

2. the receptor is internalised causing G alpha inactivation

   • a low pH vesicle causes the ligand to fall off teh receptor

3. GTP is hydrolysed

   • receptors are back in teh GDP bound state

   • causes reassociation of the beta gamma subunit

4. activation of phosphodiesterase

   • hydrolyses cAMP to make AMP

other effects of adrenaline

• effects cardiac tissue

• effects adipose tissue

• effects smooth muscle tissue

alpha adrenoreceptors

BOTH

• gunaine nucleotide exchange factor

• 3 subunits

A1 ADRENRECEPTORS

• G alpha q type

• found in

A2 ADRENORECEPTORS

• G alpha i type receptor

• decreases concentration of cAMP

• blocks insulin release —> activation of PKA required for exocytosis of insulin

beta adrenoreceptors

• G alpha s type

• stimulates production of cAMP

• will cause glucose to be released into the blood but not insulin —> this helps to maintain high blood sugar levels

beta gamma subunit

• important in signalling too, not just the dissociation from the alpha subunit

• regulates the potassium channel which makes depolarisation more difficult

adrenalines effect on smooth muscle

SMOOTH MUSCLE

• binding of adrenaline to the a1 adrenoreceptor activates phospholipase C which hydrolyses PIP2 releasing IP3 and DAG

  • PIP2 is a lipid made up of an inositol head group

• IP3 diffuses into the cytoplasm and triggers the release of calcium from the sarcoplasmic reticulum

  • the IP3 receptor is a tetramer bound to the ER

  • the N terminus binds IP3

  • when calcium dependent protein calmodulin binds to cam kinase it can induce muscle contraction

• DAG stays in the membrane and recruits PKC to the membrane —> combined with the increase in intracellular calcium, DAG becomes active

• PKC is a serine threonine kinase that phosphorylates various target proteins with the following effects:

  • smooth muscle contraction

  • glycogenolysis

  • inhibition of glucose synthesis so that glucose released in the blood is not just taken up again

  • modulation of ion channels

adrenalines effect on cardia cells

INCREASE IN HEART RATE

• helps to get glucose and fatty acids to the muscles

• same pathway as pka but also acts on calcium channels

  • ion channel allows Ca2+ into the cell

  • ca2+ stimulates the release of more Ca from the RYR receptor

  • pka also phosphorylates phospholambam which prevents the RYR receptor from activating SERCA which will uptake calcium

  • increased Ca in the cell allows for faster relaxation for the next contraction

on adipose tissues

RELEASE OF FA

• in adipose

• adrenaline acts on a2 and b3 receptors

• same pathway activates adipose specific enzymes which metabolise lipid droplets

• activation of pKa phosphorylates perilipin and a conformational shape change causes it to fall off

• HSL is phosphorylated too and leads to translocation of lipid droplets as theyre broken down

• fatty acids in blood can now be used for energy

EPAC

• regulates cAMP levels in the cell as well as PKA

• activation of PKA can control transcriptional output through KREB

receptor tyrosine kinase structure

• insulin receptor is an example of a receptor tyrosine kinase

• approx 50 diff types of RTKs which control a range of outputs

• all single pass transmembrane proteins except the insulin receptor which is a dimer

• extracellular binding site which varies and an intracellular catalytic domain which is similar across receptor type —> a protein kinase that specifically phosphorylates tyrosine

activation of receptor tyrosine kinases

• ligand binding causes a conformational change

• receptors dimerise (can take different modes depending on the receptor) and it causes each receptor to phosphorylate one another —> caused by catalytic domain flipping from the inactive to the active state

• after cross phosphorylation it allows the receptors to phosphorylate the tails of the receptors on tyr residues

different modes of dimerisation

1. LIGAND DEPENDENT

   • no external receptor interaction

2. RECEPTOR DEPENDENT

   • not through ligand interaction

3. BI LIGAND INTERACTION

   • ligands bind with 2 interaction surfaces that bring the receptors together

4. ACCESSORY PROTEIN

   • ligand and receptor interaction which requires an accessory protein

SH2 domains

• domains present in around 100 proteins

• interact with specific phosphorylated tyrosine molecules

• PTB binds phosphorylated tyrosine doains but structurally different from SH2

• 2 interaction sites:

  • pocket domain drives specificity

  • pTyr pocket binds to pTyr

  • although the pTyr pocket fits any pTyr it will not bind unless it fit into the specificity pocket

PDGF receptor acts in 3 pathways:

1. PI3K pathway

2. PLC gamma pathway (DAG and IP3)

3. MAPK pathway

adaptor proteins

• some RTKs recruit an adaptor protein to the receptor induced by phosphotyrosine binding instead of phosphorylating their own tails

• adaptors are then post translationally modified or phosphorylated to mediate signalling

switching off

caused by internalisation of the receptor

• Casitas B lineage protein has an SH2 domain which recognises one of the phosphorylated tyrosine residues on the receptor

• Cbl will ubiquitinate the receptor for degradation

• the ubiquitinated receptor gets internalised to the endosomal system

• either goes to the lysosome or gets recycled into the membrane

• there are also phosphatases which can dephosphorylate the phosphorylated tyrosine kinase residues

PI3K

• PI3K is an enzyme which phosphorylates PI(4,5)P2 —> PI(3,4,5)P3 which cats as a second messenger

• there’s a dimer of the regulatory p85 domain and a catalytic domain

• the p85 domain contains the SH2 domain which binds RTKs

  • p85 domain also acts like a clamp for when ligand or receptor not bound, p85 domain will bind to the catalytic domain to suppress it

  • it also stops the enzyme from degradation and enables recruitment of the inactive enzyme to receptor through SH2

• middle t (an antigen which phosphorylates lipids instead of proteins), GPCRs and receptor tyrosine kinases all activate PI3K

PIP3 production

• phosphorylation of the YxxM motif on the receptor allows it to bind to the SH2 domain of the p85

• this brings the enzyme to the membrane and brings it to its substrate

• once the p110 (enzyme bound to p85) bought to the membrane it relieves the inhibition and allows it to produce PIP3

switching off of PIP3

• PTEN can remove the phosphate group at the 3 position

what does PIP3 do?

• the pH domain binds to PIP3 and are 2 beta sheets which mediate the protein interactions

• there is a famous pH domain that it binds to —> PKB which is a serine threonine kinase

PKB

• has a specific pH domain that binds to PIP3 only and becomes phosphorylated at the t308 and s473

• can now phosphorylate a number of serine/threonine

• works antagonistically against adrenaline pathway as it activates phosphodiesterase which hydrolyses cAMP

• can also activate GSK1 which increases glycogen synthesis as the PKB pathway increases the uptake of glycogen

  • it does this by putting glucose transporters into the membrane

  • this is because it drives other proliferative pathways which require glucose

different tissues

• receptor coupled PIP3 signalling can drive differential responses in different cell types

LIVER

• it inhibits glucose release and stimulates glycogen synthesis, decreasing gluconeogenesis

ADIPOSE

• it inhibits lipolysis

FIBROBLASTS

• it drives proliferation

oncogenic PIP3 pathway

• highly deregulated

• PI3K mutated

• the mutation/overexpression of tyrosine kinase drives more PI3K

• PTEN is deactivated

PLC pathway

• beta family activated by GPCR but gamma form activated by RTKs, as it has SH2 domains

• phosphorylated SH2 domain of PLC gamma allows it to be recruited to the receptor

• then it can hydrolyse PIP2 into DAG and IP3

RAS pathway

• small momomeric G protein

• inactive in the GDP bound state, active in the GTP bound state which allows it to interact with downstream proteins

• the Ras pathway controls proliferation

• 

CONTROL OF PROLIFERATION

• GRB2 is an adaptor protein with SH2 and SH3 domains

• the SH2 domain will bind specific phosphorylated tyrosine residues on the RTK

• GRB2 recruits SOS via SH3 domain interaction

• SOS is activated at the membrane and acts as a guanine nucleotide exchange factor so RAS has GTP not GDP

• when theres a conformational change in switch 1 and switch 2 domains RAS can bind many proteins including PI3K, RAF/MAPK

active RAS

• can bind a kinase called RAF kinase

• leads to a cascade which eventually leads to activation of MAPK

• MAPK can phosphorylate transcription factors and cause cells to proliferate

• active RAS can also bind to PIP3K to phosphorylate PIP2—> PIP3

• there are 3 isoforms of RAS in different tissue types:

  • HRAS in brain and muscle

  • NRAS in the gut the lung and the thymus

  • RRAS in the testis the thymus

• RAS is often mutated in tumors, most frequently at G12, G13, Q61 which stops RAS from hydrolysing GTP

• locks the RAS in the active state driving PI3K and MAPK pathways

switching off RAS

• RAS has its own GTPase activity but this is very slow so it relies on a GAP (eraser)

• however, these GAPs are mutated in tumors

• a mutation in the arginine finger will aid at pos G12, G13, Q61 will prevent the Arg finger from interacting with it and activating GTPase activity (via steric hindrance)