Week 5- Cellular Signalling

Question 1

Cell Signaling: background

Answer 1

• Cells communicate with each other to monitor surrounding conditions and respond to stimuli appropriately
• Cell signaling is a process in which cell work together and coordinate their activities
• Understanding of cell signaling can tie together independent cellular activities, including regulation of cell cycle and cancer development

Question 2

Basic element of cell signaling

Answer 2

• cells communicate through extracellular messenger (hormones)
• receiver cells need transmembrane receptors
• 1st signaling route: cytoplasmic domain of receptor-activated
• 2nd signaling route: cytoplasmic domain of receptor interacts with other signaling proteins (from peripheral membrane to cytoplasmic to nucleus)
• Common outcome: activation of downstream signaling events (kinases and phosphatases)

Question 3

Signal transduction pathways

Answer 3

• Importance of kinases and phosphatases:
  1. main components of signal transduction pathway
  2. kinase phosphorylates its substrate
  3. kinase can be substrate of another kinase
• Effects of phosphorylation:
  1. activate (mostly) or inactivate an enzyme

Question 4

Activation of signal transduction

Answer 4

• Outcome of signal transduction
  1. change in gene expression
  2. alteration of activity of metabolic enzymes

Question 5

G protein-coupled receptors (GPCR)

Answer 5

• named from interaction with G protein
• G protein complex has three subunits
• Ligands bind to outside part of GPCR
• Ligand binding leads to changes in conformation in inside part of GPCR
• Changes in conformation attracts G protein, forming G protein-coupled receptors
• GPCR activates signaling transduction

Question 6

Activation of G protein

Answer 6

• Ligand binding on GPCR triggers G protein binding and replacement of GDP by GTP on G-alpha
• GTP bound G-alpha is active, has decreased affinity for G protein complex leading for its release
• Free GTP bound G-alpha binds to adenylyl cyclase, to generate cAMP from ATP
• cAMP activates other signaling proteins

Question 7

End of activation of G protein

Answer 7

• After interaction with adenylyl cyclase, G-alpha hydrolyzes GTP and becomes GDP-G-alpha
• GDP-G-alpha is inactive, and detaches itself from adenylyl and forms three subunits G protein complex
• Three subunits G protein complex is inactive

Question 8

GPCR and blood glucose level

Answer 8

• Glucose oxidation provides energy in ATP
• Glucose can be stored in glycogen
• Glycogen breakdown to glucose controlled by hormones
• Glucagon, released when blood glucose levels are low, stimulates glycogen phosphorylase to break down glycogen
• Insulin stimulates glycogen synthase to convert glucose in excess to glycogen

Question 9

GPCR and blood glucose level pt 2

Answer 9

• Glucagon binds to GPCR
• GPCR activates heterotrimeric G protein
• Heterotrimeric G protein activated effector- adenylyl cyclase
• Adenylyl cyclase is a two-part transmembrane enzyme
• Active site in cytoplasmic part and convert ATP to cAMP

Question 10

GPCR and blood glucose level pt 3

Answer 10

• cAMP binds to protein kinase leading to its activation
• Activated PKA phosphorylates and activates phosphorylase kinase
• Active phosphorylase kinase phosphorylates and activates glycogen phosphorylase
• Active glycogen phosphorylase removes glucose from glycogen
• Successive removal of glucose from glycogen increases blood glucose level

Question 11

GPCR and PKA

Answer 11

• At the same time, active PKA phosphorylates and inactivates glycogen synthase, thus prevent conversion of glucose to glycogen
• Active PKA also moves to nucleus to phosphorylate and activate transcription factor (CREB)
• CREB activates gene expression of enzymes in glucose metabolism
• cAMP signal transduction is turned off by phosphatases

Question 12

Receptor protein-tyrosine kinases

Answer 12

Protein-tyrosine kinases phosphorylate specific tyrosine residues on target proteins
- These kinases are divided into transmembrane receptor protein-tyrosine kinases and cytoplasmic protein-tyrosine kinases
- Receptor protein-tyrosine kinases are dimers and activated by cross phosphorylation
- Activation of RTKs pass on the message to Ras GTP-binding proteins
- All GTP- binding proteins are active when GTP bound and inactive when GDP bound

Question 13

RTK activated Ras signaling

Answer 13

• Mutant Ras frequently found in cancers
• Ras is GTPase (GTP binding and GTP hydrolysis)
• Ras is attached to plasma membrane
• Molecular switch: active GTP bound and inactive GDP bound
• GTP Ras from action of Exchange proteins
• GDP-Ras from GTP hydrolysis

Question 14

RTK-activated Ras-MAP kinase signaling

Answer 14

1. Growth factor binds to RTK
2. Binding triggers RTK autophosphorylation
3. Autophosphorylation recruits adaptor protein, which recruits signaling protein Sos
4. Sos, a GEF activates Ras by replacing GDP on Ras with GTP

Question 15

RTK-activated Ras-MAP kinase signaling pt 2

Answer 15

4. Activation of Ras by GEF
5. Activated Ras recruits Raf to membrane, followed by its phosphorylation and activation
6. Activated Raf phosphorylates and activates MEK (MAPKK)
7. Activated MEK phosphorylates and activates ERK (MAPK)

Question 16

RTK activated Ras MAP kinase signaling pt 3

Answer 16

7. Activation of MAP kinase (ERK)
8. Activated ERK translocates into nucleus to phosphorylate and activates transcription factors
9. Activated TFs have higher DNA binding affinity and enhance target gene expression
10. End result is cell proliferation