Exam 4 - IR/PI-3K/Cytokine Receptor

Question 1

What type of receptor is IR?

Answer 1

IR (insulin receptor) is a RTK (receptor tyrosine kinase)

Question 2

What is normal (fasting) blood glucose level? What maintains this?

Answer 2

Normal (fasting) blood glucose levels in humans are maintained at ~5.5 mM by the opposing actions of insulin and glucagon

Question 3

What happens to blood glucose after a meal?

Answer 3

↑ blood glucose after a meal results in:

↑ secretion of insulin from pancreatic β cells

Question 4

What does insulin do to blood glucose levels?

Answer 4

↓ blood glucose levels by promoting glucose uptake in muscle and adipose tissue and glucose storage in liver

Question 5

What happens to excess sugar in the body?

Answer 5

Excess sugar causes insulin release, which causes glucose uptake by tissues and its subsequent storage as fat or glycogen.

Question 6

Insulin is anabolic or catabolic?

Answer 6

Insulin is anabolic = signals the building of macromolecules, cells, and tissues

Question 7

↑ Insulin affects which cellular process we have to know?

Answer 7

↑ glucose uptake
↑ amino acid uptake
↑ acetyl CoA → fatty acids
↑ glucose → glycogen
↑ protein synthesis
↓ pyruvate → glucose (↓ gluconeogenesis)

Question 8

Describe the structure of IR

Answer 8

Insulin receptor (IR) monomer is comprised of an external α-subunit linked by disulfide bonds to the internal β-subunit. Two of these monomers are linked together by disulfide bonds so that IR is already functionally ‘dimeric’ without insulin. When insulin binds, it causes a conformational change in the β– subunits that activates their kinase activity and brings the cytoplasmic domains closer together so they can phosphorylate each other. Thus, the two β–subunits trans- phosphorylate each other, just as occurs with other RTKs.

Question 9

Insulin receptor is an RTK, so it uses adapter proteins like ___________.

Answer 9

IRS (insulin receptor substrate)

Question 10

IRS

Answer 10

IRS (insulin receptor substrate) is an adaptor protein, although it is more commonly called a docking protein that is recruited by its PTB domain to the phosphotyrosines on IR. IRS itself becomes phosphorylated on many tyrosines by the actions of IR. These P-Tyr serve as recognition sites for other signaling proteins as described for other RTKs

Question 11

What type of domain does IRS have?

Answer 11

PTB

Question 12

The docking protein IRS interacts with what other proteins?

Answer 12

Grb2/SOS (Ras-GEF)
PLCγ
PI3-kinase

Question 13

IRS to Grb2

Answer 13

• Adapter protein: SH2 domain of RTK/docking protein to SH3 domains of Ras-GEF (aka SOS)
• Ras-GEF (SOS) exchanges GDP for GTP in Ras and activates it
• Ras phosphorylates Raf, which phosphorylates MEK, which phosphorylates MAPK
• MAPK leads to cell proliferation, gene transcription

Question 14

IRS to PLCγ

Answer 14

Activates inositol phospholipid signaling pathway (diacylglycerol & IP3 allow docking and activation of PKC) leading to cell proliferation and gene transcription

Question 15

IRS to PI3-kinase

Answer 15

Leads to Akt kinase (PKC), then TOR kinase

Leads to: protein translocation, glucose transporter, protein synthesis, cell growth)

Question 16

phoshatidylinositide 3-kinase

Answer 16

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. When activated, PI 3-kinase catalyzes phosphorylation at the 3 position of the inositol ring of Phosphatidylinositol (PI) to generate several phosphoinositides, of which, PI(3,4,5)P3 matters most because it can serve as a docking site for various intracellular signalling proteins that assemble into signaling complexes. Made up of p85 and p110 subunits.

Question 17

What does IP3 do? What does PIP3 do?

Answer 17

IP3 travels to the ER and activates IP3-gated calcium channels, releasing Ca2+ as a second messenger.
PIP3 remains in the membrane and recruits proteins with Pleckstrin homology (PH) domains

Question 18

What protein domain recognises PIP3?

Answer 18

Pleckstrin homology (PH) domain

Question 19

Akt

Answer 19

Akt (Protein Kinase B, PKB) recognizes and binds PIP3 (which was phosphorylated from PIP2 by PI 3-kinase), which brings it in proximity to PDK1 (another PH domain-containing protein, so also bound to a PIP3). PDK1 is a serine threonine protein kinase.
PDK1 phosphorylates Akt, activating and releasing it from the membrane.
Akt can now phosphorylate its downstream target proteins on ser/thr. These targets tend to promote cell growth, cell proliferation, and cell survival.

Question 20

Targets of Akt

Answer 20

Akt is a kinase, so phosphorylates targets:
mTORC (TOR): ↑ cell growth, ↑protein synthesis
MDM2: activates MDM2 which degrades p53 (which arrests cell cycle and promotes apoptosis in damaged cells), promoting growth
BAD: phosphorylation inhibits BAD (a pro-apoptotic protein that inhibits Bcl2, a key anti-apoptotic protein) and prevents apoptosis

Question 21

Negative regulation of PIP3

Answer 21

PIP3 is dephosphorylated at 3’ position by PTEN back to PIP2 (a lipid phosphatase).
PH domain-containing proteins will no longer be recruited to the membrane. PTEN thus is a tumor suppressor of this pathway, and it is mutated in many cancers.

Question 22

What does binding of insulin or growth factor to an RTK do?

Answer 22

1) Activates PI 3-kinase, a lipid kinase.
2) PI 3-kinase phosphorylates phosphatidylinositol lipids on the 3 position, most notably converting PIP2 to PIP3.
3) PIP3 serves as recognition site for PH domain-containing kinases such as PDK1 (phosphatidylinositol-dependent kinase 1) and Akt (protein kinase B).
PDK1 phosphorylates and activates Akt when it is docked at the membrane
4) Activated Akt phosphorylates many proteins, including TOR, MDM2, and BAD. The ultimate consequence is an increase glucose uptake and utilization, increase cell growth, increase cell division, overriding of cell cycle checkpoints, and inhibition apoptosis.

Question 23

PDK1

Answer 23

PDK1 phosphorylates and activates Akt when it is docked at the membrane, both are brought into proximity by binding to PIP3s in the membrane via PH domains

Question 24

How does Rapamycin work?

Answer 24

It inhibits the ser/thr kinase activity of TOR

Question 25

TOR

Answer 25

TOR is a ser/thr kinase and it phosphorylates proteins in a number of key pathways, coordinating cell growth with the environmental conditions.
Ex. ↑ glucose uptake, ↑ glycolysis which will lead to ↑ lipid synthesis

Question 26

How is TOR and PI 3-K involved in detecting cancer?

Answer 26

Both increase glucose uptake significantly (100x normal cells) in constitutively active forms found in cancer, so screening with labelled glucose can find tumours.

Question 27

How does TOR increase protein synthesis?

Answer 27

• Affects transcription regulatory proteins
• Inhibits 4E-BP, which itself inhibits eIF4E, a transcription activator
• Activates S6K which phosphorylates and activates S6, increases ribosome translation of mRNAs encoding ribosomal components

Question 28

BAD

Answer 28

BAD is a pro-apoptotic protein that promotes apoptosis by forming a complex with Bcl2, one of the key anti-apoptotic proteins, and inhibits it. Akt phosphorylates BAD, which allows Bcl2 to inhibit apoptosis. Akt indirectly promotes cell survival through BAD and Bcl2.

Question 29

cytokine

Answer 29

– Cytokines are small proteins, peptides, or glycoproteins that can be made by virtually any cell. Thus, they differ from the true ‘hormones,’ which by definition come only from endocrine glands.
– Cytokines are very analogous to hormones in their affinities for receptors.
– Cytokines act locally and hormones act systemically, but both can enter the blood stream

Question 30

Do cytokine receptors have intrinsic enzyme activity?

Answer 30

No, they associate tightly with cytoplasmic tyrosine kinases such as Src or members of the JAK family

Question 31

Cytokine receptor

Answer 31

Cytokine receptors transverse the membrane once, function as dimers, and are closely linked to intracellular tyrosine kinases (Src or JAK)

Question 32

Cytokine receptor ligand examples

Answer 32

– Cytokines (duh)
– Prolactin
– Erythropoietin
– Leptin
– Interleukins
– Interferons

Question 33

What do activated cytokine receptors activate?

Answer 33

The JAK-STAT pathway

Question 34

Describe the JAK-STAT pathway

Answer 34

The Jak/STAT Pathway
• In their inactive state, STATs are found in the cytosolic compartment as monomers.
• The STAT proteins are recruited via their SH2 domains to the phosphorylated cytokine receptor
• Once there, a JAK will phosphorylate the STAT, causing it to come off the receptor. The SH2 domain of one STAT will recognize the phosphotyrosine on another STAT, and they will dimerize.
• Dimerisation of STAT leads to its nuclear localisation, DNA binding, and regulation of transcription.

Question 35

> 30 cytokines and hormones utilise the JAK/STAT pathway; how is specificity of action achieved?

Answer 35

In part, specificity comes from multiple genes encoding both JAK kinases (4) and STATs (7)
• These JAK kinases interact with different cytokine receptors and phosphorylate distinct STATs that recognize different genes
• Not all cells have all of the isoforms of the kinases or the STATs

Question 36

What pathways can Cytokine R activate?

Answer 36

All RTK pathways (Src, PLC/PKC, Grb2/MAPK,PI3K/Akt) plus JAK/STAT

Question 37

What does Epo induce?

Answer 37

RBC (red blood cell) formation

Induces JAK2 which leads to STAT5, Grb2/MAPK, PLCγ, PI-3K/Akt

Question 38

STATs

Answer 38

STATs are latent cytosolic transcription factors that are recruited to phosphorylated cytokine receptors via their SH2 domains and become tyrosine phosphorylated by JAKs, which triggers their dimerization, nuclear localization, and binding to target genes

Question 39

How is PTEN involved in cancer?

Answer 39

PTEN is a tumor suppressor protein that opposes the Akt pathway (↑ cell growth, ↑ anabolism, hijacked by cancer) by dephosphorylating the membrane PIP3. PTEN is often mutated in cancers.