Chapter 26:Insulin Receptor,the PI 3-Kinase Pathway, and the Cytokine Receptors

Question 1

Glucose Homeostasis-The Insulin Effect

Answer 1

• Normal (fasting) blood glucose levels in humans are maintained at ~5.5 mM by the opposing actions of insulin and glucagon
• Increased blood glucose after a meal results in increased secretion of insulin from the β cells of the endocrine pancreas
• Insulin acts to reduce blood glucose by promoting glucose uptake in muscle and adipose tissue and glucose storage in liver

Question 2

Insulin Affects Many Cellular Processes

Answer 2

• Glucose uptake
• Amino acid uptake
• Acetyl Coa –> Fatty Acids
• Glucose –>Glycogen
• Pyruvate –> Glucose (Gluconeogenesis)
• Protein synthesis
• Insulin is anabolic = signals the building of macromolecules, cells, and tissues.

Question 3

The Insulin Receptor Is a “Dimer” of Two Different Proteins Linked by…

Answer 3

• Disulfide bonds
• 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 ‘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 cross phosphorylate each other, just as occurs with other RTKs.

Question 4

Insulin Receptor Is an RTK, So It Signals Like an RTK

Answer 4

IRS (insulin receptor substrate) is 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 5

Adaptor proteins

Answer 5

Link proteins together through protein binding domains such as SH3 domains to facilitate the creation of larger signaling complexes. They do not have any intrinsic enzymatic activity themselves. Ex: Grb2

Question 6

Docking proteins

Answer 6

Can be adaptor proteins but usually this term is limited to those proteins that contain an N-terminal motif or domain for direct membrane association (e.g. a PH domain or myristoylation sequence) and a large number (> 5) of tyrosine phosphorylation sites for effector recruitment. Ex: IRS

Question 7

Scaffold proteins

Answer 7

Interact with multiple members of a signaling pathway and tether them into a signaling complex.

Question 8

The Phoshatidylinositide 3-Kinase (PI3K, PI 3-K) Pathway Is Key to Cellular Growth and Survival

Answer 8

• This pathway is one of the most commonly altered pathways in human tumors.
• This pathway also promotes cell survival and proliferation and chemoresistance.
• Each of these biological outcomes results from interactions of this pathway with other signaling networks.

Question 9

PI 3-kinases Are a Family of Lipid Kinases That Phosphorylate the…

Answer 9

3 position of Phosphoinositols

Question 10

IP₃

Answer 10

Stimulates release of Ca²⁺ from ER

Question 11

PIP₃

Answer 11

Recruits proteins to the membrane

Question 12

PI 3-K Structure and Function

Answer 12

• PI 3-K has two subunits, one of 85 kDa (p85) and one of 110 kDa (p110)
• PI 3-kinase converts the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinostiol-3,4,5-triphosphate (PIP3)

Question 13

PIP3 Serves as a Docking Site for PH Domain-Containing Proteins

Answer 13

Akt (Protein Kinase B, PKB) activation requires its recruitment to the plasma membrane through interaction with PIP3 and subsequent phosphorylation by the protein kinase PDK1 (another PH domain-containing protein)

Question 14

After phosphorylation by PDK1

Answer 14

Activated Akt is released from the plasma membrane to phosphorylate its downstream target proteins on ser/thr. These targets tend to promote cell growth, cell proliferation, and cell survival.

Question 15

PTEN Recognizes PIP3 and Dephosphorylates It

Answer 15

PTEN is a lipid phosphatase and it dephosphorylates PIP3 on the 3’ position to yield PIP2. PTEN thus is a tumor suppressor of this pathway, and it is mutated in many cancers.

Question 16

The Core PI 3-K Signaling Pathway

Answer 16

• Binding of a growth factor or insulin to an RTK activates PI 3-kinase, a lipid kinase.
• PI 3-kinase phosphorylates phosphatidylinositol lipids on the 3 position, most notably converting PIP2 into PIP3.
• PIP3 serves as recognition site for PDK1 (phosphatidylinositol-dependent kinase 1) and other kinases with PH domains such as Akt (protein kinase B).
• PDK1 phosphorylates Akt when it is docked at the membrane, activating Akt
• Akt phosphorylates many proteins that ultimately increase glucose uptake and storage, increase cell growth, increase cell division, override cell cycle checkpoints, and inhibit apoptosis.
• PTEN is a lipid phosphatase and dephosphorylates PIP3, inactivating the pathway. PTEN is thus a tumor suppressor protein.

Question 17

Rapamycin

Answer 17

• Inhibits the ser/thr kinase activity of TOR.
• Rapamycin is a bacterial peptide.
• It was originally developed as an antifungal drug, but this was abandoned once it became clear that it had strong immunosuppressant and anti-proliferative actions.
• Mostly used now to prevent rejection in organ transplants, esp. of kidneys

Question 18

Central Role of TOR in Cell Growth

Answer 18

TOR is a ser/thr kinase and it phosphorylates proteins in a number of key pathways. Shown here is activation of glucose uptake and utilization by TOR.

Question 19

The Excessive Production of Glucose in Response to Signaling Through PI 3-K and TOR Is the Basis for Detection of Metastases by PET Scans

Answer 19

Positron Emission Tomography (PET) scan of patient with a malignant sarcoma after infusion of 2-fluoro-deoxyglucose (FDG) before and after therapy with sunitinib (a multi-target tyrosine kinase inhibitor). Excess FDG is excreted in urine and hence kidney (K) and bladder (B) are visualized.

Question 20

TOR Also Stimulates Protein Synthesis

Answer 20

• Signaling of growth factors through RTKs and nutrients both activate TOR. As TOR is a ser/thr kinase, it phosphorylates many proteins to stimulate translation.
• 4E-BP is an inhibitor of eIF4E. When TOR phosphorylates 4E-BP, it no longer inhibits eIF4E, and translation is stimulated.

Question 21

Apoptosis

Answer 21

• ‘Programmed cell death’ - a specific biochemical pathway to remove cells that are not needed or that are damaged, especially DNA damage
• Controlled by a family of proteins that promote (pro-apoptotic) or inhibit (anti-apoptotic) it.

Question 22

BAD

Answer 22

• BAD is a protein that normally functions to encourage apoptosis (pro-apoptotic) by forming a complex with one of the key proteins inhibiting apoptosis known as Bcl2 (BAD is thus an inhibitor of an inhibitor)
• Akt phosphorylates BAD; when it does so, it inactivates it and hence frees up Bcl2, which inhibits apoptosis. Akt thus promotes cell survival indirectly by inhibiting apoptosis.

Question 23

Survival Factors Activate Akt via PI 3-Kinase and Inhibit Bad

Answer 23

• A ligand binds to an RTK or cytokine receptor
• PI 3-kinase is activated
• Akt is activated
  
  • Bad is phosphorylated and inactivatedWhen active, Bad sequesters proteins such as Bcl2 that block apoptosis
  • When Bad is inactive, apoptosis is blocked and cells survive even when they shouldn’t

Question 24

Cytokine Receptors

Answer 24

• Family includes growth hormone and prolactin receptors and the cytokine receptors
  
  • Growth hormone and prolactin are ancient signals for growth.
  • 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. They act locally but are also distributed by the blood stream.
• The receptors transverse the membrane once and do not have intrinsic enzyme activity
• Cytokine receptors associate tightly with tyrosine kinsases such as Src or members of the JAK tyrosine kinase family

Question 25

Cytokine Receptors Functions

Answer 25

• Transverse the membrane once, function as dimers, and are closely linked to intracellular tyrosine kinases
• Other ligands include:
  
  • Prolactin
  • Erythropoietin
  • Leptin
  • Interleukins
  • Interferons

Question 26

Cytokine Receptors II

Answer 26

• Rely on cytoplasmic tyrosine kinases to phosphorylate target proteins, often including the receptors themselves.
  
  • The Src family
  • The JAK family
• These kinases phosphorylate the STAT (signal transducers and activators of transcription) transcription factor family, enabling them to move from the cytoplasm to the nucleus.

Question 27

Activated Cytokine Receptors Activates the Jak-STAT Pathway

Answer 27

Stats activate many genes, including those involved in the immune response, hematopoiesis, Myc, Fos, mammary gland development, milk production, etc.

Question 28

The Jak/STAT Pathway

Answer 28

• 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 one STAT will recognize the phosphotyrosine on another STAT, and they will dimerize.
• Dimerization of STAT leads to its nuclear localization, DNA binding, and regulation of transcription.

Question 29

Specificity in the JAK/STAT Pathway

Answer 29

• >30 cytokines and hormones utilize the JAK/STAT pathway; how is specificity of action achieved?
• In part, due to presence of multiple genes for 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 30

Examples of Cellar Tyrosine Kinase and STAT Combinations

Question 31

Some of the Pathways Triggered by Erythropoietin

Answer 31

Epo induces red blood cell formation

Question 32

The SHP Tyrosine Phosphatases Inactivate JAKs

Answer 32

A Finnish cross-country skier won 3 gold medals in the 1964 Olympics and had 15% more RBCs than normal. Suspected of doping with Epo.

In 1995, Dr. Lodish at MIT showed that this family lacked the part of EpoR that mediates binding of SHP1. Thus, the receptor is not inactivated properly, making it signal as though there were extra Epo around.