Lecture 3: Receptor Tyrosine Kinases

Question 1

What four domains are important to understand for RTK signalling?

Answer 1

SH2- binds to phosphorylated peptide motifs
SHC - binds to phosphorylated peptide motifs
PTB - Different structure but same function as SH2
SH3- proline rich binding motif

Question 2

What relationship does insulin have with phosphorylation?

Answer 2

It provides enough energy for oxidative phosphorylation. It is a tyrosine kinase and therefore phosphorylates tyrosine residues

Question 3

Why might insulin be a risk factor for dementia?

Answer 3

There is a decrease in insulin during aging and its receptor (IR) in the brain

Question 4

Why is alzheimers sometimes referred to as type III diabetes?

Answer 4

Further decrease in insulin, IR, PKB and GSK3 function. Acute insulin administration dramatically improves learning and memory in Alzheimers disease as well as in controls

Question 5

What disease is a higher risk for those with diabetes type II? Why?

Answer 5

In type II diabetes the response to insulin is diminished, and this is defined as insulin resistance. There is a higher risk of Dementia possibly to hyperinsulinemia.

Question 6

What are three important issue when studying insulin in the brain?

Answer 6

1) Blood Brain Barrier
2) In Vitro Studies on primary cultures
3) The signal transduction pathways induced by insulin link synaptic plasticity to neuronal survival

Question 7

Describe the relationship between insulin and glucose

Answer 7

Insulin is a small protein and is synthesised in significant quantities in β-cells in the pancreas. When the β-cell is appropriately stimulated, insulin is secreted from the cell by exocytosis and diffuses into islet capillary blood. Binding of insulin to the insulin receptor regulates the uptake of glucose from the circulation by inducing the translocation of glucose transporters from the cytoplasm towards the plasma membrane. The glucose, taken up by the transporters, is then stored or directly used as fuel.

Question 8

Has insulin been shown to cross the BBB? Describe this pathway

Answer 8

Yes; when glucose enters the pancreas it stimulates insulin production and enters circulation. It then enters the brain through the blood brain barrier.

Question 9

What four pathways are shown on the slides for insulin?

Answer 9

Survival
Synaptic plasticity
Cell death
Disease

Question 10

Describe the activity of EGF receptors

Answer 10

In the absence of ligand, EGFR adopts a compact conformation in which a loop on domain II is buried. Ligand binding promotes a domain rearrangement in which domains I and II rotate and expose the domain II loop. The exposed domain II loop mediates dimerisation of the extracellular regions, which leads to formation of an asymmetric dimer of the kinase regions, activation of the ‘acceptor’ kinase by a ‘donor’ kinase and transphosphorylation of the C-terminal tail region

Question 11

How does this EGFR differ to that of the RTK?

Answer 11

The dimer is already formed for the RTK both in terms of the receptor and kinase region

Question 12

Describe the MAPK and PI3K pathway

Answer 12

(1) MAPK: SHC binds to the phosphorylated tail of the a subunit. Phosphorylated SHC recruits GRB2 via SH2. A conformation change in GRB2 reveals the SH3 domain which proline rich SOS proteins. A conformation change in SOS proteins allow them to activate RAS proteins which phosphorylates Raf which phosphorylates MEK which phosphoylates ERK which travels to the nucleus and induces growth.

(2) PI3K: the a subunit recruits IRS via its PTB domain. PI3K is comprised of p85 and p110. The SH2-domain of the regulatory subunit p85 binds to the phosphorylated IRS. PI3K then phosphorylates PKB and converts PIP2 to PIP3. p85 also activates MKK4 through a CDC42-dependent mechanism leading to activation of JNK.

Question 13

How can this knowledge be utilised when studying signalling pathways?

Answer 13

Use smart inhibitors, for certain pathways try inhibiting downstream targets; e.g by mutating the binding sites of proteins

Question 14

What aspect of autophosphorylation of RTKs is functionally important to consider?

Answer 14

The sequence around it matters; different sequences recruit different proteins.

Question 15

Name the three segments of the B subunit of the IR and name some phosphorylation targets

Answer 15

Juxtamembrane
Tyrosine-Kinase: phosphorylate each other
C terminus: phosphorylate downstream targets e.g IRS, SHC etc

Question 16

What is meant by insulin resistance?

Answer 16

There is no intracellular signalling even with insulin binding

Question 17

Name 6 members of the RAS superfamily

Answer 17

RAS
RHO
RAB
ARF
RAN
RAD

Question 18

What functions does RAS carry out?

Answer 18

Signalling
Cell survival
Cell growth control
Cell migration

Question 19

What functions does RHO carry out?

Answer 19

Signalling
Cytoskeleton organisation
Cell migration

Question 20

What functions does RAB carry out?

Answer 20

Intracellular vesicle targetting

Question 21

What functions does ARF carry out?

Answer 21

Intracellular vesicle formation

Question 22

What functions does RAN carry out?

Answer 22

Nuclear Import
Spindle formation

Question 23

What functions does RAD carry out?

Answer 23

Blood vessel formation

Question 24

What do mutations in RAS often result in?

Answer 24

Cancer

Question 25

What is the relationship between RAS and MAPK?

Answer 25

When SOS binds to RAS it acts as a gunaine exchange factor (GEF), causing GDP to be released and GTP to bind. Activated RAS can then bind to RAF-1 via its RBD which then induces a conformational change causing it to autophosphorylate itself, begininning the RAF-MEK-ERK pathway.

Question 26

What kind of protein kinase is RAS?

Answer 26

It is not a protein kinase, it cannot phosphorylate. It is a small GTP-ase. Some GEFs (RAS-GEFs) activate it so that it can effect downstream effectors via their RBDs while some GAPs (GTPase activating proteins) (RAS-GEFs) inhibit it through hydrolysing GTP to GDP.

Question 27

How could you test for RAS regulation?

Answer 27

RAS binding assay: The assay works on the principle that Ras only binds to its downstream kinase, Raf-1, when in its active-GTP bound state. In this state, Ras binds to a domain of Raf-1 referred to as the Ras Binding Domain (RBD). Ras is unable to bind to Raf-1-RBD in its inactive/GTP bound state.

A recombinant Raf-1-RBD is provided for the capture of activated Ras from your sample. Raf-1-RBD binds to the wells of a glutathione-coated plate via a GST/Glutathione interaction, thus capturing the active Ras and allowing the inactive/GDP-bound Ras to be washed away. The captured active Ras is detected and measured quantitatively through the addition of an anti-Ras antibody.

An HRP conjugated secondary antibody is then added for the detection. Following addition of the chemiluminescent substrate, signals can be measured using a luminometer or with a CCD camera or a gel assay. Alternativelt can use the radioactive GTPyS.

Question 28

How can the efficiency and specificity of the Raf-Mek-Erk pathway be improved?

Answer 28

Via the scaffolding protein KSR: has binding sites for all three of the kinases in the Raf/MEK/Erk cascade. By binding all three in appropriate orientations, the scaffold makes interactions among the proteins rapid and efficient.

Question 29

How does negative feedback occur within this Raf-Mek-Erk pathway?

Answer 29

When Erk has been activated, it phosphorylates the binding site for Raf, forcing a conformational change that displaces Raf and thereby prevents the phosphorylation of MEK. The result of this feedback regulation is that MEK phosphorylation is temporary.

Question 30

How is PI3K activated and what does it activate?

Answer 30

One of the proteins that binds to the intracellular tail of RTK molecules is the plasma-membrane-bound enzyme phosphoinositide 3-kinase (PI 3-kinase). This kinase principally phosphorylates inositol phospholipids rather than proteins, and both RTKs and GPCRs can activate it. It plays a central part in promoting cell survival and growth.

Question 31

What is meant by the name phosphoinositide?

Answer 31

Phosphatidylinositol (PI) is unique among membrane lipids because it can undergo reversible phosphorylation at multiple sites on its inositol head group to generate a variety of phosphorylated PI lipids called phosphoinositides.

Question 32

What typically happens when PI3K is activated?

Answer 32

When activated, PI 3-kinase catalyzes phosphorylation at the 3 position of the inositol ring to generate several phosphoinositides. The production of PI(3,4,5)P3 matters most because it can serve as a docking site for various intracellular signaling proteins, which assemble into signaling complexes that relay the signal into the cell from the cytosolic face of the plasma membrane

Question 33

How does this PI3K-PI route differ to that of GPCRs and RTKs described earlier?

Answer 33

PI(4,5)P2 is cleaved by PLCβ (in the case of GPCRs) or PLCγ (in the case of RTKs) to generate soluble IP3 and membrane-bound diacyglycerol.

By contrast, PI(3,4,5)P3 is not cleaved by either PLC. It is made from PI(4,5)P2 and then remains in the plasma membrane until specific phosphoinositide phosphatases dephosphorylate it.

Question 34

What can dephosphylate the 3 position on the inositil ring?

Answer 34

Prominent among these is the PTEN phosphatase, which dephosphorylates the 3 position of the inositol ring. Mutations in PTEN are found in many cancers: by prolonging signaling by PI 3-kinase, they promote uncontrolled cell growth.

Question 35

To which class of PI3K do RTK and GPCRs belong?

Answer 35

There are various types of PI 3-kinases. Those activated by RTKs and GPCRs belong to class I. These are heterodimers composed of a common catalytic subunit and different regulatory subunits.

RTKs activate class Ia PI 3-kinases, in which the regulatory subunit is an adaptor protein that binds to two phosphotyrosines on activated RTKs through its two SH2 domains.

GPCRs activate class Ib PI 3-kinases, which have a regulatory subunit that binds to the βγ complex of an activated trimeric G protein (Gq) when GPCRs are activated by their extracellular ligand. The direct binding of activated Ras can also activate the common class I catalytic subunit.

Question 36

How do intracellular signalling proteins bind to PIP3?

Answer 36

Intracellular signaling proteins bind to PIP3 produced by activated PI 3-kinase via a specific interaction domain, such as a pleckstrin homology (PH) domain, first identified in the platelet protein pleckstrin. PH domains function mainly as protein–protein interaction domains, and it is only a small subset of them that bind to PIP3

Question 37

What is the relevance of this alternative protein binding function?

Answer 37

At least some of these also recognise a specific membrane-bound protein as well as the PI(3,4,5)P3, which greatly increases the specificity of the binding and helps to explain why the signalling proteins with PI(3,4,5) P3-binding PH domains do not all dock at all PI(3,4,5)P3 sites.

Question 38

Name a PH domain containing protein important for this pathway

Answer 38

One especially important PH-domain-containing protein is the serine/threo- nine protein kinase Akt. The PI-3-kinase–Akt signaling pathway is the major path- way activated by the hormone insulin.

Question 39

Name a signal (not insulin) and receptor which induces the PI3k pathway

Answer 39

Members of the insulin- like growth factor (IGF) family of signal proteins, for example, stimulate many types of animal cells to survive and grow. They bind to specific RTKs, which activate PI 3-kinase to produce PI(3,4,5)P3.

Question 40

What is the consequence of producing PIP3?

Answer 40

The PI(3,4,5)P3 recruits two protein kinases to the plasma membrane via their PH domains—Akt (also called protein kinase B, or PKB) and phosphoinositide-dependent protein kinase 1 (PDK1), and this leads to the activation of Akt. Once activated, Akt phosphorylates various target proteins at the plasma membrane, as well as in the cytosol and nucleus. The effect on most of the known targets is to inactivate them; but the targets are such that these actions of Akt all conspire to enhance cell survival and growth.

Question 41

What large protein kinase does this PI3K mediated cell growth depend on?

Answer 41

The control of cell growth by the PI-3-kinase–Akt pathway depends in part on a large protein kinase called TOR

Question 42

How was TOR named?

Answer 42

Named as the target of rapamycin, a bacterial toxin that inactivates the kinase and is used clinically as both an immunosuppressant and anticancer drug. TOR was originally identified in yeasts in genetic screens for rapamycin resistance; in mammalian cells, it is called mTOR

Question 43

In what two ways can TOR exist in the cell?

Answer 43

mTOR complex 1: contains the protein raptor; this complex is sensitive to rapamycin, and it stimulates cell growth—both by promoting ribosome production and protein synthesis and by inhibiting protein degradation. Complex 1 also promotes both cell growth and cell survival by stimulating nutrient uptake and metabolism.

mTOR complex 2: contains the protein rictor and is insensitive to rapamycin; it helps to activate Akt, and it regulates the actin cytoskeleton via Rho family GTPases.

Question 44

How is mTOR in complex 1 relevant to the PI3K-AKT pathway?

Answer 44

The mTOR in complex 1 integrates inputs from various sources, including extracellular signal proteins referred to as growth factors and nutrients such as amino acids, both of which help activate mTOR and promote cell growth. The growth factors activate mTOR mainly via the PI3K–Akt pathway.

Akt activates mTOR in complex 1 indirectly by phosphorylating, and thereby inhibiting, a GAP called Tsc2. Tsc2 acts on a monomeric Ras-related GTPase called Rheb. Rheb in its active form (Rheb-GTP) activates mTOR in complex 1. The net result is that Akt activates mTOR and thereby promotes cell growth

Question 45

Hollistically describe a pathway in which PI3K-AKT inhibits apoptosis

Answer 45

An extracellular survival signal activates an RTK,
which recruits and activates PI 3-kinase. The PI 3-kinase produces PIP3, which serves as a docking site for two serine/threonine kinases with PH domains—Akt and the phosphoinositide-dependent kinase PDK1—and brings them into proximity at the plasma membrane.

The Akt is phosphorylated on a serine by a third kinase (usually mTOR in complex 2), which alters the conformation of the Akt so that it can be phosphorylated on a threonine by PDK1, which activates the Akt. The activated Akt now undergoes a conformational change in the PH domain and dissociates from the plasma membrane and phosphorylates various target proteins, including the Bad protein.

When unphosphorylated, Bad holds one or more apoptosis-inhibitory proteins in an inactive state. Once phosphorylated, Bad releases the inhibitory proteins, which now can block apoptosis and thereby promote cell survival. The phosphorylated Bad binds to a ubiquitous cytosolic protein called 14-3-3, which keeps Bad out of action.

Question 46

How does MTORC2 affect MTORC1?

Answer 46

MTORC1 also phosphorylates AKT (which inhibits TSC2 (in complex with TSC1), which acts as a GAP for RHEB, which activates MTORC1).

Question 47

What effect does MTORC1 have on this signalling? (2)

Answer 47

MTORC1 activates S6K1. S6K can inhibit insulin/PI3K signaling through inhibition of IRS1.

mTORC1 also activates GRB10 which inhibits both MAPK and PI3K pathways through effects on growth factor receptors such as the IR.

Question 48

What are RAPTOR and RICTOR to their respective complexes?

Answer 48

scaffold proteins regulating the assembly and substrate binding of MTORC1 (raptor) and MTORC2 (rictor)

Question 49

Give a general MTOR inhibitor and a specific inhibitor of one complex

Answer 49

deptor: inhibitor

pras40/ rapamycin: MTORC1 inhibitor

Question 50

Give roles for each complex

Answer 50

MTORC1:
Macromolecule biosynthesis
Lipid synthesis
Autophagy (autophagosome formation)
Cell cycle progression
Growth
(energy) Metabolism

MTORC2:
Cell survival (via Akt- foxo)
Cytoskelatal organisation
Metabolism

Question 51

How can the MAPK pathway affect the PI3K pathway?

Answer 51

The MAPK pathway can inhibit PI3K and downstream signaling through inhibition of either EGFR or IRS1. ERK can activate mTORC1 via inhibition of the TSC complex, while S6K can inhibit mTORC2 through direct phosphorylation of RICTOR by S6K. The TSC complex while it inhibits mTORC1 can activate mTORC2

Question 52

Given that insulin may be a risk factor for dementia, how may we study this?

Answer 52

Consider its effects on the brain. We know that insulin activates both the RAS-RAF-MEK-ERK and PI3K-PKB-survival pathway. We could consider what roles they have in learning and memory.

Question 53

What might be a good model for studying the roles of these in learning and memory?

Answer 53

Long term depression and potentiation

Question 54

Describe how LTP and LTD can be induced and measured

Answer 54

Long-term potentiation (LTP) and long-term depression (LTD) can be induced by applying an electrical stimulus by placing an electrode placed in the Schaffer collateral-commissural (SCC) pathway and recording from the CA1 subfield. (The CA1 pyramidal cell layer receives input from the entorhinal cortex through the dentate gyrus and the CA3 pyramidal layers and the SCC; the subiculum carries hippocampal efferents.)

LTP: Initially, low-frequency stimulation (LFS) is applied to the Schaffer collaterals to establish a stable baseline, after which LTP is induced by high-frequency stimulation(HFS), followed by LFS. Successful induction of LTP can be assumed when the post-HFS EPSP peak amplitude exceeds that seen before HFS and is maintained for at least 60 min.

LTD: After initial baseline recording, low-frequency stimulation is applied to the SCC; successfully induced LTD can be assumed when the post-LFS EPSP peak amplitude is smaller than that observed before LFS.

Question 55

Roughly describe the molecular biology behind LTP and LDP

Answer 55

In normal firing the NMDA receptor in the post synapse is activated by glutamate binding but only after depolarisation removes inhibitory Mg2+. Once the Mg2+ is removed, Ca2+ can enter the cell. Calcium entry through postsynaptic NMDA receptors can initiate the two different forms of synaptic plasticity.

LTP: The stimulation causes a calcium- and CaMKII-dependent cellular cascade, which results in the insertion of more AMPA receptors into the postsynaptic membrane. The next time glutamate is released from the presynaptic cell, it will bind to both NMDA and the newly-inserted AMPA receptors, thus depolarizing the membrane more efficiently.

LTD: LTD occurs when few glutamate molecules bind to NMDA receptors at a synapse (due to a low firing rate of the presynaptic neuron). The calcium that does flow through NMDA receptors initiates a different calcineurin and protein phosphatase 1-dependent cascade, which results in the endocytosis of AMPA receptors. This makes the postsynaptic neuron less responsive to glutamate released from the presynaptic neuron.

Question 56

How did Lars and the crew study insulin on memory?

Answer 56

Cultured hippocampal slices, applied insulin and did immuno blotting for AKT and phosphorylated AKT. They also did electrophysiological recordings following insulin application to measure LTD and LTP. Additionally they measured this with the presence of an NMDAR and PI3K antagonist.

Question 57

What were the results of Lars’ study on insulin and memory?

Answer 57

They found that insulin phosphorylated PKB in the slices, indicating it activated the PI3K-PKB pathway

The also found that insulin induces LTD & LTP in hippocampal slices in an NMDAR & PI3K dependent manner

Therefore insulin influences synaptic plasticity in a PI3K dependent manner

Question 58

Does synaptic plasticity affect the PI3K pathway?

Answer 58

PI3-kinase and its downstream effectors Akt and p70S6K are activated in LTP.

Question 59

To reiterate what were the points for insulin being a risk factor for dementia?

Answer 59

1) Aging : decrease in insulin and its receptor (IR) in the brain

2) Alzheimer’s disease: further decrease in insulin, IR, PKB and GSK3 function
Acute insulin administration dramatically improves
learning and memory in Alzheimers disease as well as in controls

3) Diabetes Type II: Hyperinsulinemia, higher risk of Dementia => insulin resistance?

Question 60

What are some treatments for insulin defects in the brain?

Answer 60

1) Aging and Alzheimers disease: Short term insulin treatments (insulin-IR-PI3K route)

Learning and memory games/ spatial memory (NMDAR- PI3K route)

Activate pathways that convert onto PI3K e.g. BDNF, Leptin)

2) Diabetes: Activate pathways that convert onto PI3K e.g. BDNF and Leptin