Lecture #8 - Receptor Tyrosine Kinases and Phosphatases, and small GTPases

Question 1

Cell proliferation

Answer 1

Cell proliferation/dynamic cell behavior is regulated by RTK and series of phosphorylation events (regulated by signaling pathways)
- Phosphorylation state of proteins is changed during cell proliferation

Cell proliferations - defined by the balance between cell division and cell loss

Question 2

Protein Phosphorylation

Answer 2

Very common post translational modification

Phosphates are mostly added onto proteins BUT can be added onto membrane lipids

Phosphorylation is a small chemical entity BUT can change the property of proteins

Phosphorarylation function - switch mechanism to conduct information transfer (turn processes on/off)

Phosphorylation is done by Tyrosine kinases (receptor and non-receptor) And Ser/Thr kinases or Lipid kinases

Question 3

Phosphatases

Answer 3

Phosphatases dephosphorylate proteins

Have Tyrosine phosphates (PTPs ; receptor and non-receptor) and Ser/Thr Phosphatases
- PTPs - Protein Tyrosine Phosphatase

Question 4

Growth Factors (Ex - EGF + NGF + PDGF + FGF + VEGF)

Answer 4

Growth factors stimulate RTK and Regulate Diverse cellular Functions
- Growth factors are the input (ligand ; primary messenger) for RTK pathways

Signaling pathways start with a primary messenger (ligand ; Ex. Growth factor) –> trigger that initiates signaling pathway activation
- Each growth factor binds to a different receptor and produces a different cellular outcome

Question 5

Output of RTKs

Answer 5

Reponses (output of RTKs) includes Cell growth + proliferation + differentiation + Survival + Migration + Metabolic Response

Outputs depends on and is different for different growth factor (ligand) and cell types

Question 6

Epidermal Growth Factor Signaling

Answer 6

Overall EGF signaling pathway – Growth factor (input) –> growth factor binds to the receptor (RTK) –> Binding of growth factor activates the receptor and is relayed to intracellular signals –> get cell response (output)
- Pathway has feedback regulations (not completely linear flow)

Question 7

Receptor Tyrosine Kinases (RTKs) common features

Answer 7

RTK Common features:
1. Single transmembrane domain(crosses membrane once)
2. Cytosolic side (intracellular) - Tyrosine kinase domain (At C terminus)
3. Extracellular side - Ligand binding domain (N-Terminus)
- More diverse - Each receptor has a different ligand = has a different ligand binding domain

Question 8

Structure of Example RTK

Answer 8

Example - EGF receptor (type of RTK)

Structure:
1. Extracellular side- Ligand binding domain (Domain 1,2,3,4)
2. Transmenrane juxtamembrane (single transmembrane region)
3. Intracellular Side- Tyrosine kinase and regulatory region

Question 9

Conserved structure of a kinase domain

Answer 9

Conserved structure of a kinase domain (mouth and tongue structure)
- Tonge = A loop ; Mouth = ATP and substrate binding site

Inactive kinase – A loop blocks the substate from binding to the substrate binding site in the catalytic pocket

Active Kinase – A loop moves –> substrate can bind to the catalytic domain

Question 10

Overall RTK activation

Answer 10

RTK activation is induced by receptor dimerization leads to transphosphorylation –> Transphosphorylation leads to RTK activation

NOTE - Uses Allosteric activation (protein becomes activate because of direct protein binding (function of local concentration))

Question 11

EGF binding to EGF receptor

Answer 11

No ligand - RTK is inactive (no kinase activity)

EGF ligand binds to ligand binding pocket of EGF receptor (RTK) –> ligand binding domain has a big conformational change that exposes a region that can bind to another EGF receptor –> two receptors (both bound to EGF Ligands and expose the regions for two EGF receptors to bind) homodimerize

THEN EGF receptor dimerization allow the kinase domain of one receptor to interact with the kinase domain on the other receptor –> interaction changes the confirmation of the tyrosine kinase domain (A loop is moved (catalytic open is open) –> NOW the kinase domain is activated through allosteric activation

Question 12

What happens once one kinase domain is activated

Answer 12

After dimerization Kinase domain of 1 EGF receptor has allosteric activation –> Activator EGF receptor kinase domains phosphorylates Tyrosines in the C terminal tails receiver EGF receptor kinase domain –> Phosphorylation of the receiver actives the receiver kinase (actives the second EGF receptor)
- One EGF serves as the enzyme (Activator) –> phosphorylates the receiver EGF receptor (acts as the substrate)
- When C terminal tail is phosphatylated the RTK is filly activated (have activated dimer)

Activator phosphorylating the receiver = transphosphorylation (one kinase phosphorylates the other) vs. CIS phosphorylation – Kinase phosphorylates itself

Question 13

Who phosphorylates who in Transphosphorylation

Answer 13

Transphosphorylation happens from both sides (Kinase domains of BOTH EGF receptors are activated upon dimerization and will phosphorylate the other ; both act as the receiver and the activator)
- Kinase domain of A with phosphorylate kinase domain B and then when kinase domain B is actaivte and kinase domain of B with phosphorylate kinase domain of A

Question 14

Confirmational changes in EGF receptor activation

Answer 14

Conformational changes:
1. EGF ligand binds to the EGF receptorcauses a conformational change in the ligand binding domains (change allows two receptors to dimerize)
2. When receptors dimerize (allosteric activation) –> the kinase domain has a confirmational change –> moves A loop and activates the Kinase domain and allows transphosphorylation to happen

Question 15

EGF Pathway (steps)

Answer 15

Growth factor (ligand) Binds to receptor dimerizes and has transphosphorylation –> activated EGF receptor dimer leads to:
1. JAK/STAT Pathway –> ultimately leads to survival
2. Ras Pathway (RAS –> MAPK) –> ultimately leads to proliferation
3. PI3K –> AKT –> ultimately leads to survival
4. PLD –> PKC –> ultimately leads to proliferation

ALL intracelluar signals downstream of growth factor input (downstream effectors of EGF signlaing) - all lead to an increase in cell number
- Ligand binding can lead to activation of all or a few of the pathways

Question 16

Non-Receptor Tyrosine Kinases

Answer 16

Example - JAK (activated downstream of EGF receptor)

Non-receptor Tyrosine kinases have no transmembrane domain and are in the cytoplasm

Catalytic site of non-receptor tyorsine kinases are similar to the catalytic (Kinase) domain of RTK

Structure of non-receptor Tyrosine kinase:
1. Tyrosine kinase domain
2. NO transmembrane domain (diffuse in cytosol)
3. Can have SH2 or SH3 or PH domain
- Domains enable protein-protein or protein-lipid interaction –> used for Llocalization in cells

Question 17

Structure of JAK

Answer 17

Structure of JAK makes JAK unique

JAK - Has 2 kinase domains –> Tyrosine kinase domain (active) AND a kinase like domain (Pseudokinase domain ; inactive)

Question 18

Steps of JAK activtaion

Answer 18

Start – JAK is pre-asscoiated with C terminus of the RTK ; JAK is not active when RTK is not active

1. Ligand binds to RTK –> RTK dimerization leads to a confirmaional change in JAK –> Confirmation change in JAK leads to the release of a pseudo-kinase domain from the JAK kinase domain –> NOW JAK is activated
2. Activated JAK phosphorylates Tyrosines on the RTK (ex. EGF receptor) –> have phosphotyrosines on RTK
3. RTK phosphotyrosines recruit STAT –> STAT binds to the C terminus of the phosphorylated RTK
4. When STAT is recruited - JAK phosphorylates STAT(get p-STAT)
5. Phosphorylated STAT forms a homodimer –> homodimer moves to the nucelus
6. In the nucelus STAT recognizes and transcribes genes for cell survival (Ex. p21 + BCl-XL + Myc)

Question 19

JAK when RTK is in ligand free state

Answer 19

When receptor is in ligand free state JAK associated with the RTK is not activated because the JAK pseudo kinase domains are facing to the activated kinase domains
- When the pseudokinase is facing the active kinase domains – the active kinase domain is not able to bind to a substrate

THEN the ligand binding and receptor dimerization causing a confirmation change in JAK so that the Psueodkinase domain is not facing the active kinase domain which exposes the active kinase substrate binding site –> confirmation change activates JAK (active kinase can bind to and phosphorylate its substrate ; can phosphorylates tyrosine on the RTK)

Question 20

When ligands (growth factors or Cytokines) bind –> Receptor associated JAK will phosphorylate STAT

Answer 20

Question 21

RAS pathway and EGF signaling

Answer 21

RAS = intracellular signal downstream of EGF signaling (EGF receptor pathway leads to activation of the RAS pathway)

RAS = small GTPase (has intrincice GTPase activity)
- Ras-GTP = active ; Ras-GDP = inactive
- Ras-GTP Vs. Ras-GDP – have different confirmations in switch 1 and switch 2 regions

Question 22

Ras GTPase cycle

Answer 22

1. Start have Ras-GDP (inactive)
2. GEF helps Ras convert from GDP to GTP –> Ras released GDP
3. GTP binds to Ras (NOW Ras is activated)
4. Ras-GTP binds to downstream effectors (ex. MAPKKK)
5. Intrinsic RAS GTPase activity and GAP lead Ras to hydrolyze GTP –> get Ras GDP (inactivated)
   
   • Ras turns off because Ras has intrinsic GTPase activity

Question 23

How does GEF and GAP work

Answer 23

GEF is NOT an enzyme
- GEF binds to Ras-GDP –> binding makes Ras-GDP unstable –> Forces Ras to release GDP

GAP is NOT an enzyme
- GAP binds to Ras-GTP and helps promoter Ras GTPase activity
- Ras can hydrolyze GTP –> GDP BUT its intrinsic GTPase activity is very slow –> Solution is GAP which meakes Ras GTPase activity faster

Question 24

Ras a molecular switch

Answer 24

Want precisie on/off control of signlaing pathways in cells

Because of the Ras GTPase cycle Ras can function as a molecular switch that tightly regulates signaling pathway
- Ras-GDP = pathway is on ; Ras-GTP = pathway is off (Ras can be a switch for pathway to be on or off)

Question 25

How does RTK phosphorylaton lead to Ras activation?

Answer 25

Once have transphosphorylation of RTKs (EGF receptor) have phosphorylated Tyrosine residues on the RTK --> Ras becomes activated using RAS GEF For a while they didn't know how phosphorylated RTK leads to activated Ras GEF - Before RTK activation (at rest ; without ligand) - Ras-GDP is tethered to the plasma membrane through cystein residues + RTK is at the plasma membrane Vs. Ras GEF is in the cytoplasm --> did not know how Ras GEF goes to the plasma mebrane in order to acaive Ras-GDP when RTK is phosphorylated/activated

Question 26

How is Ras GEF is recruited to the plama membrane to activate Ras once ligand binds to RTK

Answer 26

Use phage display --> found GRB2 (adapter protein that recruits Ras-GEF to the plasma membrane to acivate Ras once Ligand binds to RTK) GRB2 has 2 SH3 domains and 1 SH2 domains --> Domains allows for protein-protein interaction - SH2 binds to phosphorylated Tyrosines on RTK - 2 SH3 domains binds to RasGEF (RasGEF = SOS) END - Once RTK is activated GRB2 brings Ras GEF to the membrane --> Ras GEF is brough close enough to can interact with Ras-GDP to make Ras-GTP -> Ras-GTP actiavtes downstream signals

Question 27

Ras regulation of pathways

Answer 27

Ras regulates diverse signaling pathways Image – see Ras at the membrane --> eveyrthing downstream is activated by Ras (Ras does many things) Major downstream Ras effector that Ras activates = Map Kinase signaling pathway (Ras --> Raf --> Mek --> ERK) - Map Kinase = Ser/Thr kinase

Question 28

EGF signaling and PI3K

Answer 28

Activated/phosphorylated EGF leads to PI3K activation --> AKT --> Survival  PI3K translates information from protein level to lipid level - PI3K rxn - PIP2 (substrate)--> PIP3 - PI3K kinase domain phosphorylates the 3rd position of the inositol ring of PIP2 to make PIP3 PIP3 is recognized by AKT and activates AKT at the membrane --> AKT (Ser/Thr Kinase) phosphorylates downstream machinery - Example - AKT phosphorylates mTOR (Ser/Thr kinase)

Question 28

PI3K structure

Answer 28

PI3K has dimer complex of catalytic and regulatory subunits Tandem SH2 domains of PI3K regulatory subunit recognizes the phosphorylation on the tyrosine of the activated RTK --> regulatory SU of PI3K binds to the phosphorylated RTK --> binding to the RTK allows the catyltic subunit of PI3K to bind to PIP2 substrate

Question 29

Result of AKT and mTOR activation

Answer 29

AKT and mTOR will promote cell survival and growth by suppressing apoptosis and autophagy and producing cell materials

Question 30

2nd messenger

Answer 30

Examples - PIP3 + DAG + IP3 2nd messenger - Small molecule that is at low concentrations when the signaling pathway is off and increases in concentration when pathway is on - Increase in second messenger concentration causes a change in cell behavior - After activation the concetration of the 2nd messenger will decrease Increase/decrease of 2nd messenger allows 2nd messengers to act like a switch - pathway is on when 2nd messenger is there and off when the 2nd messenger is gone - Similar to Ras (switch ; on/off regulation)

Question 31

Is Ras a 2nd messenger

Answer 31

NO because Ras is not a small molecule so it is not a second messenger

Question 32

EGF signaling and PLC

Answer 32

PLC is a downstream effector of EGF receptor Catalytic pocket of PLC is occupied by its own SH2 domains (creates steric hinderance) --> when RTK is phosphorylated 1 SH2 domain of PLC binds to the phosphorylated RTK which allows PLC to go to the plasma membrane --> when PLC binds to phosphorylated RTK the RTK phosphorylates the PLC After RTK phosphorylates PLC the 2nd SH2 domain (one not bound to RTK) of PLC will bind to the phosphorylated domain --> SH2 domains moves out of the catalytic pocket --> NOW PLC is activated and at the plamsa membrane and can cleave PIP2 to DAG and IP3

Question 33

PLC

Answer 33

Phospholipase C (PLC) has 10 domains (more modular/has more domains than most intracellular effectors) PLC rxn (substarte is lipid) – PIP2 (substrate) --> IP3 + DAG - IP3 and DAG are both second messengers (DAG activates PKC ; IP3 increase intracellular Ca to activate PKC) - PKC is activated by DAG (directly) and IP3 (indirectly) --> PKC (Ser/Thr kinase) phosphorylates effectors --> leads to cell proliferation

Question 34

Effect of phosphorylated RTK on PLC

Answer 34

Phosphorylated tyrosine’s RTK changes the location of PLC and changes the confirmation of PLC --> allows PLC to be activated Activated PLC makes 2nd messengers that activates further downstream things that leads to cell proliferation - PLC needs the right domains to recognize the target protein (Ex. Need SH2 to recognize phosphorylated tyrosine residues)

Question 35

Types of proteins consisting of EGFR signal pathways

Answer 35

Question 36

Phosphotyrosine binding domains

Answer 36

Phosphotyrosine binding domains – SH2 and PTB - Proteins with SH2 and PTB use the domains to bind to phosphotyrosine on target protein Proteins with SH2 and PTB domains includes: 1. GRb2 – adapter protein 2. P85 – Regulatory SU of PI3K has tandem SH2 domains 3. PLC – Has tandem SH2 domains Domains plays important roles in signaling pathways for information transfer

Question 37

Phosphatidylinositol-binding domain

Answer 37

Phosphatidylinositol-binding domain = PH domain (Plekstrin Homology) PH domain – domain of proteins that allows protein to bind to a lipid - Allow cells to utilize lipids to regulate signaling pathways Proteins with PH domain: 1. PLC - binds to membrane lipids (ex. binds to PIP2) 2. AKT/PkB - PH domain binds to PIP3

Question 38

Answer 38

Answer – GTP (always abdnent = not a second messenger ; because small moelcule needs to change concetration to be a second messenger) HIS notes: - Second messengers are informational, small molecules such as Ca2+, DG, IP3, and PIP3 - Their concentration is usually kept low in resting cells, rises upon stimulation, and then gets rapidly removed - cGMP, that is made from GTP, is a second messenger, but GTP itself is not a second messenger - GTP is abundant in cells (~0.5mM), and supports activity of small GTPases such as Ras

Question 39

Answer 39

Answer - D EGF receptor has a kinase domain = it is an enzyme; Ras is a small GTpase = it is an enzyme His Notes: - An enzyme is a substance that catalyzes a chemical reaction to its substrate - Small GTPases such as Ras have an inherent catalytic activity (GTPase) - Gbr2 is an adaptor protein that binds to both RTK and Ras GEF - GEF and GAP are proteins that bind to small GTPases, but retain no catalytic activity

Question 40

Tyrosine Phosphatases

Answer 40

Tyrosine Phosphatases includes receptor (has transmembrane domain) and non-receptor types Structure of Receptors: 1. Cytosolic side – Tyrosine phosphatase domain (In C-terminus) - Can have one or two phosphatase domains - Domain is the same for all tyrosine phosphatase) 2. Extracellar side – Ligand binding domain (N-Terminus) - Variable - Different structures to match different ligands 3. One transmemenrane domain Example – Receptor Tyrosine phosphatase (PTP)

Question 41

Affect of ligand binding on PTP activity

Answer 41

Ligand free state - PTP is a monomer (PTP is active) - PTP can dephosphorylate a substrate Ligand bound state – PTP dimerizes --> PTP is inactive (C terminus phosphatase domain is inactivated) - Similar dimileration as RTKs

Question 42

Affect of PTP dephosphorylation

Answer 42

Active PTP decreases the amount of phosphorylated tyrosine (supressed phosphorylated tyrosines levels) because PTP removes the phosphate When the phosphates is inactive (ligand bound) there is an increase in phosphorylation events (because there is no phosphtase to take the phosphate off)

Question 43

Outcomes of ligand binding to RTKs and PTP

Answer 43

The outcomes of ligand binding to RTKs and PTP is the same --> both lead to increase in phosphorylation of Tyrosine residues Ligand binding to RTK -> actaives RTK --> increase phosphorylation Ligand binding to PTP --> Inactives PTP --> not removing phosphtaes --> Increase phosphoryplation

Question 44

Physiological role of PTP

Answer 44

Pleitrophin (Neurite outgrowth promoting factor ; ligand) binds to PTP receptor --> PTP dimerizes --> phosphatase domain is inactaived --> have increase in Tyrosine phohsphorylation (increase phosphorylation of Beta catenin + Beta adducin + Rho GAP) - Beta catenin + Beta adducin + Rho GAP are the subsrates of PTP = inactived PTP then incerase phosphorylation of the substartes Nueronal outgrowth needs morphological changes at the nueronal edges (use adducin and Rho GAP because controls cytoskelatol regulation) AND neuronal outgrowth needs adhesions to stay at the right location (need beta catenin for adhesion)

Question 45

What dephosphorylates EGF receptor (RTK) - what turns off signaling pathways

Answer 45

Each activation reaction needs to have a counter reaction to turn off a pathway BUT don’t know what dephosphorylates the EGF receptor to turn off signaling (just know there must be a molecules that turns off phosphorylation of the EGF receptor) Idea – Protein phosphatases (receptor and non-receptor) may play a role in dephosphorylating EGF receptor to turn off EGF receptor (Ex. SHP1 or PTP-1B or LAR or SAP1) - Issue – don't know if they are physiologically relevant

Question 46

EGF Vs. NGF

Answer 46

EGF and NGF are both RTKs - Ligand binding domains of EGF and NGF are different because NGF and EGF bind to different molecules BUT the intracellular kinase domain is very similar Even though they have similar kinase domains NGF and EGF receptors have different outcomes: 1. EGF – Causes Cell proliferation 2. NGF – Causes Differentiation (cell number stays the same)

Question 47

Proliferations (EGF) Vs. Differentiation (NGF) - Experiment

Answer 47

In cell culture (PC12 cells) --> give cells EGF --> cell proliferates (no change in morphology = no differentiation) In cell culture (PC12 cells) --> give cells NGF --> cell differentiation (morphology of the cells change) SHOWs the cells have different outcomes even through EGF and NGF receptors have similar kinase domains (small differences in kinase domains lead to different outcomes)

Question 48

How does EGF and NGF lead to different outcomes

Answer 48

When add NGF to cell - Get sustains inctease in Ras-ERK levels (sustained increase in activity of Ras/Map kinase/ERK downstream effector machinery) When add EGF to cell - Get transient increase in Ras-ERK levels (transient increase in activity of Ras/Map kinase/ERK downstream effector machinery) END - different outs in NGF and EGF is due to receptors using different feedback regulation

Question 49

How does feedback regulation affect the outcome of EGF and NGF

Answer 49

NGF = sustained increase in Ras-ERK because have a positive feedback actaivtion of downstream machinery (Erk1/2 Positivley feedbacks to Raf1) --> Ras-ERK activity lasts longer EGF = Transient increase in Ras-ERK because have a negative feedback activation of downstream machinery (ERK negatively feeds back to supress Raf-1) --> Less Ras-ERK activity - Have suppressive signal once pathway is activated = get a transient activation Different activation of Ras pathway/different feedback regulation = leads to different outcome (proliferation vs. Differentiation)

Question 50

Does regulation only happen where a upstream molecules activates a downstream molecule

Answer 50

NO - there is regulation where a downstream molecule acts on an upstream molecules --> feedback changes the strength of signaling and the timescale of signlaing Pathways don't only include an upstream molecules activating a downstream molecule END - signaling pathways can have complex regulation

Question 51

Posisble feedback mechanisms

Answer 51

1. PKA mediated positive feedback 2. Rapid Ras combined with slow Rap activation 3. Rapid internalization of EGFR

Question 52

RTKs/PTPs and cancer

Answer 52

RTKs and PTPs and their downstream effectors (PI3K and RAS) are often mutated in cancer Ras + Pi3K + MAPK + JAK + PLC + EGFR + PKC = all oncogenes EGFR gene is amplified and mutated in human brain tumors --> leads to constitutive activation of the growth pathway (more cell proliferated correlated to EGF receptor amplification)

Question 53

Causes of EGF related cancers

Answer 53

1. Upregulated expression of EGF receptor and or increased EGF production and release - Have more quantity of EGF receptors - Need EFG to bind to RTK (EGF dependent) BUT have more EGF receptors - Cancer drug – Erbitux (ligand binding/receptor dimerization inhibitor -- RTK can't activate) 2. Mutation or truncation of EGF receptor that causes the receptor to always be turned on even in the absence of ligand (EGF) - EGF independent activation of EGF receptor (receptor is on even when EGF is not there) - Cancer drug – Iressa and Tarceva (Receptor kinase inhibitor)

Question 54

Cosimic

Answer 54

Cosimic – catalogue of somatic mutations in cancer Use – search to see if gene of interest is mutated or truncated in cancer patients AND can see the frequency the gene is mutated in cancer patients

Question 55

Cosimic results (looking at EGF receptor)

Answer 55

Cosmic shows there are some cancer mutations in the ligand binding domain BUT there is a higher frequencey of cancer mutaions in the tyrosine kinase domain (in C terminus) Most mutations occur in the Tyrosine kinase residues Chart: - Each bar = Amino Acid residue in EGF receptor - Height of bar = number of reports of mutaion in cancer pateints at that amino acid (Higher bar = more reports of mutations in cancer pateints)

Question 56

Frequent mutation in RTK

Answer 56

Frequent mutation in RTK = L85R L85R - Leu in the A loop of the RTK kinase domain is mutated to an Arginine --> Because of mutation A loop is always in the open state (even iif the EGF receptor is not actaivted ; no ligand bound)

Question 57

Activation in L85R mutated RTK

Answer 57

Images: A and B – EGF receptor before and after activation - Once ligand binds/dimerization - have a Kinase domain that activates the receiver kinase domain of another EGF receptor --> once activator activates the receiver kinase domain A loop sticks out so the catalytic pocket is open C – shows the confirmation when kinase domain has the mutation - A loop is out regardless of dimerization (EGF receptor kinase is active without EGF ligand/dimerization)

Question 58

Drugs to treat mutant RTK

Answer 58

Erlotinib and Gefitinib are drugs to treat cancers by inhibiting tyrosine kinase activity of EGFR (RTK kinase domain inhibitors) Bar chart - Mutated RTK has more kinase activity (kinase is always active) than WT

Question 59

Survival Curve with Geftinib treatment

Answer 59

Survival Curve with Geftinib treatment - SHOWS Mutant cells are senstaive to Gefitinib treatment - Geftinib – RTK inhibitor Cancer mimicing cells with mutant EGF receptor die with the Gefitinib treatment (L858R line) WT (WT EGF receptor) are not senstive to treatment until higher doses (have higher cell viability) 100 nm is a good dose window --> WT cells are not killed (have a higher viability) and the Mutant cells are killed

Question 60

How do Erlotibinib and Gefitinib work

Answer 60

Erlotibinib and Gefitinib inhibit kinase activity by binding ATP binding site How Gefitinib works - Gefitinib is a competitive inhibitor for the ATP binding site (inhbits kinase activity)

Question 61

Crystal structure of Gefitinib binding to RTK

Answer 61

Crystal structure – shows mutant EGF without ligand has the same structure of an activate EGF with ligand - Shows binding pocket where ATP binds is the same pocket where Gefitinib binds (Gefitinib binds instead of ATP) Gefitinib has a higher affinity for mutant RTK than WT RTK --> Gefitinib will preferntially bind to mutated RTK and won’t block WT RTK activity - Shows why Gefitinib only kills mutated cancer cells that express the mutated EGF receptor and not WT cells

Question 62

Answer 62

Don’t need ligand to bind to EGF it is activated independetley of ligand binding

Question 63

FDA approved small molecule inhibtors and monoclonal Antibodies against RTKs for cancer therapy

Answer 63