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Cell Signalling and Neurochemistry > Lectures 1-17 > Flashcards

Lectures 1-17 Flashcards

1
Q

describe the reaction mechanism of glycogen synthesis and glucose formation

A

UDP-glucose —> glycogen by glycogen synthase

glycogen –> glucose-1-PO4– by phosphorylase

glucose-1-PO4– –> glycolysis

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2
Q

describe the resting state of phosphorylase (structure etc)

A
  • 3 allosteric regulators AMP (active) and ATP,G6P (inactive)
  • pyridoxal phosphate = required to catalyse (in AS)
  • phosphorylase is a dimer
  • 280s loop: regulates occludes AS in inactive
  • glycogen BS
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3
Q

describe the mechanism of glycogen phosphorylase

A
  1. Pyridoxal phosphate (prosthetic group) + glycogen –> α(1-4 linkage)
  2. Protonation –> intermediate formation
    1. Protonation of glycosidic oxygen results in cleavage of α-1,4-glycosidic linkage
    2. from Pi–>O, breaks bond
  3. Free cleaved oligosaccharide
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4
Q

describe what residues must be correctly positioned on glycogen phosphorylase in resting state.

A

Arg must be in right position for Pi to be active

Arg569 responsible for Pi binding (AMP –> allosteric site)

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5
Q

why is AMP an allosteric modulator of phosphorylase

A

AMP will accumulate as ATP is used to generate energy so AMP is the regulator

glycogen breakdown –> ATP

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6
Q

describe muscle contraction.

A
  1. nerve impulse triggers increase in IC Ca2+
  2. binds troponin C –> contraction
  3. actin-myosine sliding (ATP hyolysis)
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7
Q

describe activation of phosphorylase

A
  • Ca2+ binds phosphorylase kinase –> active form
  • phosphorylase kinase phosphorylates phosphorylase b
    • PO4 to Thr, Tyr, Ser
    • upon phosphorylation at Ser-14 - locked in ACTIVE regardless of allosterics
  • becomes phosphorylase a
  • Ser-14 undergoes shift, induces Arg569 orientated properly
  • 280s loop movement, doesnt occlude BS
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8
Q

describe activation of phosphorylase in flight or fight situation.

A
  1. Adr binds AdrR
  2. Guanine nucleotide exchange in α subunit allows the exchange GDP–>GTP which is bound to transducer
  3. GTP binding causes dissoc. and activation of GP subunits
  4. GTP hydrolysis allow adenylate cyclase to produce cAMP
  5. 4 cAMP activate PKA (coop binding)
  6. phosphorylase kinase is activated by PKA
  7. phosphorylase kinase then phosphorylates phosphoylase
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9
Q

what are the different ways phosphorylase kinase and phosphorylase can be activated?

A

phosphorylase kinase:

  1. ALLOSTERIC activated by Ca2+ when ACh triggers influx (can be quickly reversed)
  2. COVALENT activated by phosphorylation when Adr activates a GPCR which –>PKA activation (req. enzymatic inactivation)

phosphorylase

  1. ALLOSTERIC binding of AMP
  2. COVALENT phosphorylation by phosphorylase kinase
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10
Q

describe the signal amplification when hormone binds receptor.

A
  • Hormone-bound receptor can activate many GPs through GEF
  • adenylate cyclase can produce many cAMPs
  • PKA can stimulate activation of many phosphorylase kinases
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11
Q

too much cAMP –> tumours.

Describe the modulation of cAMP signals.

A
  1. switching off signal by GTPase of G-alpha
    1. GTP hydrolysis switches off
  2. degradation of cycloc nucleotide PDE
    1. cAMP degraded by PDE (cleaves it)
  3. desensitisation of β-adrenergic receptor by phosphorylation (built in mechanism from sustain stimulation)
    1. homo/heterologous
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12
Q

what is the difference between heter and homologous desensitisation?

A

hetero:affect various receptors responding to diff agonists

  • β-adrenergic receptor - phosphor of H-R (by PKA), decreases activity
  • receptors with other lig’s can be phosphor by PKA from β-adrenergic R

homo:only those receptors activated by same agonist

  • will not affect other unbound R’s
  • β-ARK phosphor H-R so they no longer activate GPs
  • only bind H-R as site is exposed
  • arrestin can then bind > internalisation > less R’s
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13
Q

what does arrestin do?

A

acts as adapter for internalisation

no affinity for unphosphorylated receptors

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14
Q

describe the role of Ga and Gβγ subunits?

A

Ga

  • GTPase, interacts with effector and agonist-bound receptors

Gβγ

  • ensure desen and localisation, some interact with downstream effects (e.g. Gi)
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15
Q

what is Epac?

A

IC receptor for cAMP (like PKA)

activates GEF and allows binding to Rap

low affinity for cAMP

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16
Q

what are the 6 ways in general cytosolic Ca2+ concentrations are regulated?

A
  1. Phosphorylase kinase activated by Ca2+ (CaM)
  2. Ca2+ channels (ON mech)
  3. Ca2+ pumps (OFF mech)
  4. couple the release of Ca2+ with generation of 2nd messengers e.g. PLC…
  5. sensing fluctuation of cytosolic [Ca2+]
  6. Ca2+ buffering proteins
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17
Q

describe the features of CaM.

A
  • delta subunit of phosphorylase kinase
  • allosterically activated many enzymes
  • cooperative binding - 4 Ca2+ BS
    • helix-loop-helix motif
  • undergoes conformaitonal change when Ca2+ bound
  • homologous with troponin
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18
Q

what are the Ca2+ ON mechanisms?

A
  1. Voltage-gated Ca2+ channel
    1. activated by depolarisation
    2. dihydropyridine = voltage-gated channel
  2. receptor-opoerated Ca2+ (NMDAR)
    1. R is channel
    2. glu channel
  3. Ca2+ store-operated channel
    1. senses Ca2+ levels in store and replenishes
    2. interactes with IP3 channels and Ca2+ re-enters via IP3
  4. ryanodine receptors - only excitable cells, IC
    1. Ca2+ induced-Ca2+ channel
  5. IP3 receptor, IC, 2nd messenger
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19
Q

desribe the dihydropyridine and ryanodine receptors.

A
  • depolarised by ACh receptor
  • propagation of AP to t-tubule
  • activation of receptor - Ca2+ influx > stimulation of ryanodine
  • efflux of Ca2+ from SR
  • conformational changes in dihydropyrindine R inducec by voltage change are propagated to ryanodine receptor, activating it
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20
Q

describe the IC Ca2+ channels

A
  • Phosphatidylinositol breakdown –> DAG and IP3
  • PLCβ activated by GPCR, PLCγ activated by phosphorylation by TK
  • Ca2+-induced Ca2+ release: both IP3 and ryanodine receptor can further be activated by Ca2+ (induces opening)
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21
Q

how does PKC –> tumour growth?

A
  • DAG activates PKA and translocates to mem > phosphorylates proteins and promotes cell proliferation
  • PKC is target of tumour promotor - phorbal ester
    • phorbal ester mimics DAG in binding and activating PKC
    • promotes tumour formation
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22
Q

describe the Ca2+ OFF mechanisms and the reaction that accompanies them

A
  • SR and plasma membrane ATPase pump
  • ATP hydrolysis:
    • AspCOO- + ATP –> intermediate
    • intermed + H2O –> AspCOO- + Pi
  • phospho-asp = intermed’s of ATPase
    • induces conformation change after ATP hydrolysis and Ca2+ binds with low affinity (so released)
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23
Q

how are SERCA and PMCA regulated?

A
  • SERCA - reg by phosphorylation of regulatory protein called phosphorlamban
    • kinases = PKA and CAMKII
    • cytoplasmic portion of phospholamban blocks ATP BS, upon phosphor it no longer inhibits
  • PMCA- reg by phosphorylationof C-terminal
    • kinase = PKC
    • cytoplasmic tail blocks BS
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24
Q

what are the 2 types of Ca2+ binding proteins and their features?

A

Ca2+ sensors - CaM, tropnin

  • bind with high affinity but low capacity

Ca2+ buffering proteins - in ER/SR

  • affinity but high capacity: “storage proteins”, can relase Ca2+ if store is low

all have helix-loop-helix binding motifs

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25
Q

in the Phosphotidylinositol (PPT) pathway, how is PPT phosphorylated/dephosphorylated?

A
  • PPT is phosphorylated by PPT kinases
    • PI-3 kinase, PI-4 kinase etc
  • some isoforms prefer to by phosphor at 3 position only if 4’ and 5’ are too
  • it is dephosphorylated by PTEN at the 3’ position but only if 4’ and/or 5’ is phosphorylated
  • myotubulanin - dephosphorylates only at 3’ position
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26
Q

describe the signal transduction pathway of EGF-receptor in cell growth

A
  1. lig binding > oligermisation (dimer)
    1. allows activation of TK domain
  2. autophosphorylation by TK domain
  3. stimulate multiple signalling pathways
    1. PI-3 kinase converts PI4P
  4. binding recruits 2 enzymes to membrane
  5. PH-domain recruits them together
  6. PDK1 activates PKB, PKB phosphor’s many molecules
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27
Q

describe the features of Ras

A
  • 3 types of Ras
  • Ras = enzyme
    • Ras-GDP –> Ras-GTP (active) catalysed by Sos

promotes cell growth:

  • binds GTP –> activates
  • mutation so no GTPase (always active)
  • binds plasma mem
  • req assistance of GAP to GTPase and therefore deactivate
  • GEF (a.k.a. Sos) alone cannot activate Ras: Grb-2 (adapter) required
    • Grb-2 has 2 SH3 and SH2 domains
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28
Q

describe the features of Grb-2 and how it binds Sos

A

adapter in activating Ras with Sos, it binds EGF>Sos

SH2 domain binds phosphor-tyr sites (from autophosphorylation of Sos)

SG3 domains bind -PXXP- motif in Sos

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29
Q

what are some signalling mechanisms of EGF-receptor?

A
  • one effector of Ras is Raf-1 (Ser/Thr PK)
    • MAP3 kinase = Raf-1 (kinase kinase kinase)
    • which phosphorylates (act) MEK
  • MEK = MAP 2 kinase which can phosphorylate Tyr and Thr at the same time on MAP kinase
    • duel specificity
    • upstream activating kinase
  • MAP kinase phosphorylated at Thr and Tyr (not a TK)
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30
Q

Describe the interaction of NO and PKG (cGMP-dependent protein)

A
  • cGMP is produced in response to stimulation of NO –> smooth muscle relaxation
  1. Bradykinin receptor stim (GPCR) –> 1P3 formation
  2. Ca2+ binds CaM
  3. NOS in endothelial cell = activated
  4. NO generated
  5. NO diffuses into smooth muscle cells
  6. NO bind guanylate cycle
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31
Q

describe the process of phosphorylation by PKA

A
  • requires proper alignment of PKA
  • chelation of ATP helps align proper
  • γ-phosphate of ATP attacks O: on substrate
  • catalytic base (Asp) required to facilitate reaction
  • AspCOO- + H+ –> COOH
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32
Q

what are the functions of the PK AS?

A

binds and positions ATP properly for reaction

binds and positions the target Ser/Thr/Tyr residue

positions the catalytic base properly for reaction

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33
Q

what is the general structure and features of PKA?

A
  • small lobe -high abundance of B-sheet
  • large lobe - lots of a-helices
  • ATP BS in between lobes

features:

  • Gly-rich loop: essential for anchoring PO4–
  • alpha-helixC (in small loop) contains essential glutamic acid
  • catalytic loop: contains Asp (base)
  • Activation loop: governs accessibility of substrate to AS
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34
Q

what are the catalytically critical motifs and residues of PKA?

A
  • Gly-rich loop (ATP binding)
  • Lys72 (ATP binding a/b subunits)
  • Lys72 and Glu91 (electrostatic interactions)
  • Asp for Mg2+ binding loop (req chelation of β and γ ATP subunits for proper positioning)
  • pThr197 (inactivation loop for entry of sybstrate)
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35
Q

what are the 2 main functions of the activation loop?

A

positioning Asp166 in cat loop properly

binding of substrate protein

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36
Q

what is the PKA phosphorylation sequence?

A

Arg-Arg-X-Ser-Leu

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37
Q

how is PKA regulated?

A

the R subunit and PKI both mimic protein substrate in binding AS of PKA

Ser replaced with Ala in PKI

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38
Q

how does PKI bind with high affinity?

A
  • co-localisation
  • scan residue for high affinity binding sequence
  • structural basis:
    • Phe binds hydrophobic pocket on C-subunit
    • Arg forms electrostatic interactions with C-sub
  • flexible loop on PKI adopt structure when bound to AS
    • can move in/out with high adffin + efficiency
    • recognise loop in region
39
Q

describe cAMP binding PKA

A
  • when cAMP binds, R undergoes significant conformation changes which decrese affinity for C-sub
  • part of R binds Trp196 and Tyr247 binding pocket in C
  • once cAMP binds, conformation change deforms Trp196 and Tyr247 binding region
  1. binding of cAMP –> domain B
    1. Arg366 moves to top, allowing cAMP to bind
  2. capping of domain B by Y371, breakage of R366-E261 int
  3. conformational changes enhances alignment of residues in domain A
    1. movt of glu261
  4. binding of cAMP to dom A
  5. R2C2 dissociation
40
Q

describe the structure of the R-subunit in PKA.

A
  • each subunit contains 2 cAMP binding domains with
    • hydrophobic cap: Trp binds Adenin ring
    • loop-helix-loop motif with an Arg (electostatic int with phosphate group in cAMP)
  • in free R: Arg366 + Glu261 electostatic interaction disrupted
41
Q

describe the R/C complex in PKA

A
  • pseudo-substrate site enters AS of enzyme
  • C-sub utilises similar determinant to interact with pseudo-substrate motifs of both R-sub and PKI
  • R wraps around C - sig interface
42
Q

how is PKA recruited to its substrate and what is the substrate specificity governed by?

A
  • A-kinase anchoring protein (AKAP) - co-localises PK with substrate proteins
  • substrate specificity governed by:
    • recognition of specific residues near the target phosphorylation site
    • co-localisation/ direct docking of PK to its substrate
43
Q

how does the R subunit of PKA dimerise?

A

2 disulphide bonds link regulatory subunits to form the D/D domain

44
Q

what are the general features of AKAP?

A
  • subcellular targeting motif and BS of target proteins e.g. NMDAR
  • BS for other signalling proteins e.g. PP1
  • BS of R-sub of PKA (always R-sub)
  • AKAP = scaffolding proteins that assemble PKA with other sig proteins to provide fine-tuned reg of targets
  • the D/D domain of 2 R-subs form hydrophobic cradle for AKAP
    • extensive Hb interactions
    • binding is very specific
45
Q

what is special about D-AKAP2?

A

dual specific AKAP-2

can bind 2 different forms of R-sub

46
Q

describe the general regulation system of PK/PP1 (phosphotase) functions.

A
  • NMDA = substrate for PKA
  • PKA combines with AKAP and substrate - localised
  • phosphotase and PKA co-localised too, fine control and ion channel
  • PP1 inhibitors also located close to PP1 for fine tuning
47
Q

explain the function og mAKAP cardio myocytes.

A
  • PDE 4D3 = substrate for PKA
  • once C-sub is released, it phosphorylates PDE 4D3
  • upon phosphorylation by PKA, PDE 4D3 is active
  • this enhances the degradation of cAMP (by PDE 4D3)
    • then C can re-bind R
  • act as -ve feedback mechanism that prevents over-stimulation of PKA
48
Q

Describe the function of Yotiao.

A
  • Target protein substrates: NMDAR, PKA, PP1
  • function: co-localise PP1 to reg the phosphorylation status and in turn the Ca2+ activity of NMDAR
  • Yotiao targets both PKA and PP1 to NMDAR
  • active PP1 + inact PKA = small amount of Ca2+ allowed in
  • active PKA phosphorylates NMDAR
  • phosphorylation enhances the opening of channels
    • Glu binding elicits a much higher influx if phosphor
    • when PP1 active - return to basal state
49
Q

How are phosphotases classified?

A
  • 1 PP for many kinases

classified by:

  • dephosphorylate α or β of phosphorylase kinase
  • inhibition by groupd of endogenous PP inhibitors
    • inhibitor-1/inhibitor-2
  • inhibition by okadaic acid
  • activation by Ca2+ - CaM
  • dependence on Mg2+
50
Q

what are the differences between PP1, PP2 and PP3?

A

PP1

  • preference for β, no dependence on Mg2+ or CaM
  • regulated by inhibitor-1 + DARP-32

PP2

  • very potent inhibition by okadaic acid

PP3

  • dependent on Ca2+ - CaM
51
Q

describe the regulation of PP1 activity governed by glycogen-binding-subunit (G).

A
  • G-sub regulates location and activity of PP1
  • G-sub targets PP1 to glycogen where PP1 can readily de-phosphorylate phosphorylase a
    • i.e. co-localise phosphorylase a and PP1
52
Q

describe the relaxation caused by PP1 in smooth muscle cells.

A
  • PP1 targeted to myosine by myosine-targeting subunit
  • de-phosphorylation of the light chain –> relaxation
  • phospho-light chain allow ATP hydrolysis to drive contraction
53
Q

what are 2 regulatory/targeting subunits that target PP1 and NMDAR that are not AKAP?

A

yotiao and spinophilin

54
Q

Many signalling proteins contain SH2 domain, describe it in terms of PLC-gamma

A
  • PLC-gamma has SH2 domain
  • binds to autophosphorylated Tyr on EGF-R
  • leads to activation of PLC by phosphorylation
55
Q

what techniques are commonly used to investigate the neuronal death mechanism?

A
  1. assays for viability of cultured neuronal cells
  2. S35-Methionine labelling of cellular proteins to study rate of biosynthesis of a specific protein and its stability
  3. gene transfer to exp recombinant proteins or KO of exp of an endogenous protein in specific region of rat brains
56
Q

what types of assays (4) can be used to assess neuron death i.e. cell viability?

A
  1. MTT assay
  2. Lactate dehydrogenase leakage assay
  3. Calcein-AM conversion to calcein live cells
  4. Ethidium homodimer-1 assay for dead cells
57
Q

describe a MTT assay to assess cell viability.

A
  • MTT cleaved into coloured formazan product by NADH oxidase in mitochondria
  • MTT (yellow) –> formazan (violet)

redox reaction

58
Q

describe the lactate dehydrogenase leakage assay to assess

A
  1. LDH converts NAD–>NADH (at the same time as pyruvat –>lactate)
  2. NADH helps produce 1-methoxy PMS
  3. 1-methoxy PMS then converts WST-8 (colourless) –> WST-8 formazan (orange)
  • measure metabolic enzyme (LDH)
  • measure relative LDH release over control = indication of death
59
Q

describe calcein-AM conversion to clacein live cells as a mechanism for measuring cell viability.

A
  • green fluorescent viability dye calcein AM
    • live cells contain esterases capable of converting non-fluorescent calcein-AM –> fluorescent calcein
  • calcein-AM = membrane permeable
  • green = live
60
Q

how does ethidium homodimer-1 assay for cell viability work?

A

EthD-1

  • red fluorescent dye penetrates damaged cell membrane
  • upon binding RNA EthD-1 emits fluorescence with great intensity
  • cannot enter live cells
  • red=dead
61
Q

discuss S35-Methionine labelling as tool for styuding rate of biosynthesis

A
  • radioactive S35 methionine is cell permeating
  • to measure protein synth:
    • pulse phase - incorp of S35-Met in protein during biosynth
    • analyse amount of S35-Met by PAGE and autoradiography
    • chase phase: add XS Met (non-radioact) to prevent further labelling of proteins
    • monitor decay of S35-labelled proteins
62
Q

describe gene transfer to exp recombinant proteins or KD exp of protein in specific region of rat brains.

A
  • introduce specific proteins to specific region of brain while keeping rat alive
  • gene transfer using adrenoviral and lentiviral :
  1. viral vector contains gene encoding protein
  2. use sterotexic inhection to intro virus into regions of choice
  3. after recovery, behavioural effects monitored
  4. brain sections which stained for presence of recombinant protein/absence are obtained
  • expose skull and use guide
63
Q

what are the 5 steps in cell signalling research?

A
  1. hypothesis
  2. choice of system/model
  3. interventions
  4. observations
  5. results and conclusions
64
Q

what are the differences between primary cell lines and established cell lines?

A

primary cell lines: cutlures from it consist of linages of cells originally present in the primary culture

establised cell lines: lines that originate with humans

  • HEK293 cells
  • readily cultures and suitable for general studies
  • easily cultured
65
Q

what does
DAPI show?

A

cells not astrocytes

66
Q

how are primary cells produced?

what are the advantages/disadvantages?

A
  1. take tissue
  2. dissociate cells by proteases
  3. put in proper environment in 1o culture dishes –> 1o cells

advantages: similarities to cells from original tissue
disadvantages: access to tisse source (ethics), mixed cell populations (must purify), cell numbers limited, cells difficult to manipulate

67
Q

describe a basic experiment with HEK293 cells and transfecting them with GOSPEL.

A
  1. HEK293 cells culture (grown easily)
  2. transfect cells with plasmid to allow expression of HA epitope-tagged GOSPEL
  3. fix and stain cells with anti-HA antibody
  4. stain with labelled 2o antibody, co-stain with DAPI
68
Q

what are the 4 types of interventions can be used to manipulate protein expression in cells?

A
  1. Challenging the cells with growth factors, stresses or removing GF’s
  2. increasing levels of a protein -whether mutant forms of protein are useful
    1. consider whether it should be “epitope tagged”
    2. lipofection or viral vectors
  3. decreasing levels of a protein - how will loss of exp affect cell?
    1. suitable RNAi/siRNA approach
  4. use of cell-permeable versions of chemical inhibitors or activators of known targets in cells
69
Q

what does epitope labelling achieve?

A

epitope = part of antigen that is recognised by immune system

gives capacity for real live imaging

70
Q

what is chronic myeloid leukaemia?

A

biphasic disease - stem cell disorder

  1. chronic phase: process of differentiation they begin to acquire philadelphia chromosome
    1. specific region of chromosome 22 called Bcr joined to sequences upstream of exon ABL on chromosome 9
  2. blast phase: additional mutations required - very aggressive as they proliferate quickly
71
Q

what are the critically critical residues of Brc-Abl and their function?

A

Brc-Abl = constitutively active PK, which activate a bumber of signalling pathwyas

catalytically critical residues:

  • Gly-rich loop (anchors ATP)
  • Lys (coordinate a/b subunits of ATP)
  • catalytic loop: base (Asp)
  • activation loop: contains Ser/Thr/Tyr

function of activation loop

  • P-Thr197 in activation loop, makes interaction with side chain Arg-165
  • positions Arg-166 properly
72
Q

what are the mechanisms for inactivation of PK’s?

A
  • PKA inhibited by PKI and R
  • C-Src kinase is inactivated by phosphorylation
  • Insulin receptor kinase is activated by insulin binding
  • Abl is kept inactive by activation loop, phosphorylation of Tyr389 = active
73
Q

what structures are conserved in PK’s?

A

Lys salt-bridged with ahelix-C such that Lys is aligned properly for ATP-binding

catalytic loop is properly aligned for phospho-transfer

active site

74
Q

describe the development of Glivec.

A
  • add 3 pyridyl group to increase activity
  • amide group: against TK’s
  • methyl group: abolishes PKC activity
  • piperazine group increases solubility and bioavailability
75
Q

describe the action of Glivec and how it binds

A
  • binds selectively to Bcr-Abl, blocks downstream effects
  • also inhibits C-kit and PDGFR (platelet-derived GFR)
  • P-Tyr393-c-Abl amd Bcr-abl AS = identical
  • glives binds inactive conformation (therefore specific) and shifts equalibrium
  • binds to ATP BS for inactive Abl
    • Phe382 occludes BS in active conformation
76
Q

what is a Bcr-Abl dependent mechanism of Glivec resistance?

A
  • point mutations in kinase domain
  • over-expression of bcr-abl gene (give more glivec)
  • gene amplification
77
Q

what are some brc-abl independent mechanism for Glivec resistance?

A
  • over exp of Scr kinases
  • α glycoprotein, decreases levels, binds glivec
  • over exp of P-glycoprotein (pumps drug out)
78
Q

describe the Thr315 mutation in Glivec treatment and how is it treated?

A
  • glivec binds Hb pocket and αhelix C and activation loop
  • mutation Thr315 –> Ile
  • cannot h-bond
  • steric clash between glivec and Ile
  • limits acces to AS
  • DRUG = dasatinib
    • less selective
    • effective in most glivec-resistant drugs
79
Q

what is acute myeloid leukaemia and how is it diagnosed?

A

AML - maligancy in stem cells. >20% of cells in bone marrow = blast cells, proliferate at expense of other cells

sample taken from spongy bone in bone marrow and diagnosis is based on the flow cytometry results

80
Q

why do people with AML get urinary stones?

A

when blast cells breakdown they release uric acid

81
Q

what is FLT3? Explain its role in AML.

A

FLT3

  • type III TM receptor TK
  • normally exp in bone marrow: critical for proliferation regulation and differentiation
  • present in all stem cells
  • expressed in 70% of leukamic cells of AML patients
  • stem cells lacking FLT3 decrease ability to reconstitute lympohid precursors
82
Q

what is the structure of FLT3?

A
  • EC portion: immunoglobulin-like loops
  • IC portion:
    • tandem of duplication of domains
    • 2 kinase domains
    • juxtamembrane domain
83
Q

what are the 2 types of mutations that occur on FLT3 proteins?

A

FLT3-ITD (internal tandem duplication)

  • insertions at the JM region at JMZ
  • induces olidolconal myeloproliferative disease

FLT3-TKD (TK domain mutations)

  • insertions near the catalytic loop
  • induces oligoclonal lymphoid disorder
84
Q

how is FLT3 auto-inhibited?

A

JMB wedges between the N and C loops, preventing them coming together and activating

85
Q

what is the mechanism of action of FLT3?

A

ligand binds which induces dimerisation

they trans-phosphorylate each other at Tyr589 and Tyr591

JMB cannot insert into auto-inhibitory domain

downstream signalling can occur e.g. GF binding

86
Q

what occurs in the FLT3-ITD mutation?

A
  • makes FLT3 auto-inhibition leaky - constitutively active
  • result = uncontrolled hematopoiesis
  • FLT3-ITD can phosphorylate STAT5 to increase cell proliferation and survival
87
Q

describe EGFR in the treatment of cancer

A
  • EGFR variant expressed in a number of cancer cells
  • variant = constitutively active (lig cannot bind or dimerise)
  • inhibit:
    • EGFR tyr kinase inhibitors
    • block receptor
    • inhibit ligand
    • kill receptor (link to immune system)
  • drugs: Erlontinib + Gefitinic (TKI), Cetuximab (MAb), H447 (MAb linked to antiCD64)
  • SU11248 = non specific TKI, anti-angiogenic, many s.e.
88
Q

describe the activation/inactivation of IRK

A

inactive

  • activation loop Tyr located at position where substrate Tyr should be BUT not phosphorylated as ATP cannot bind
  • catalytic loop with Asp for catalysis

active, autophosphorylated IRK

  • activation loop out of AS
  • ATP comes in
  • undergoes cis-phosphorylation - locked inactive form
89
Q

describe inactive/active forms of Abl

A
  • Phe382 - occludes ATP BS
  • Tyr383 - occludes substrate BS at same location as where subtrate Tyr would bind but is not phosphorylated because ATP cannot bind
90
Q

specificity is a major challenge in using PK inhbitors, why? how do you get around this?

A
  • all active conformations of PKs are very similar, inactive conformations are different
  • cannot inhibit ATP-BS (even though there are differences) becauses peptide inhibitors are not effective as not permeable
  • target inactive conformation and stabilise
  • avoids s.e.
  • in equalibrium with active/inactive just shifted towards active
91
Q

why is Abl constitively active in CML?

A
  • “cap region” encoded on exon1 (which is taken off) is inhibitory –> consitutively active
  • cap region allosterically inhibits c-Abl
    • myristoyl group with cap
    • myristoyl group binds kinase domain to inactivate it
    • GNF2=Abl allosteric inhibitor, mimics myristoyl
92
Q

what does Glivec work well?

A

just a single biochemical mutation

gain-of-function mutation of Abl, so specific inhibitor will work

specificity of Glivec towards Abl and 2 other kinases

93
Q

How is GNF2 implicated in the treatment for CML?

A
  • high selectivity for BCR-ABL
  • GNF2 binds myristoyl-binding pocket in major lobe of kinase domain BCR-ABL
  • myristoyl-regulates and targets BCR-ABL to membrane near N-terminal but this group is chopped off (so no inhibition) but binding pocket still there
  • So GNF2 tries to restore physiological inhibition
  • GNF2 can inhibit T35II mutant

Cell Signalling and Neurochemistry (3 decks)

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