W19

Question 1

What is the function of PI3 kinase?

Answer 1

Phosphorylates PIs at the C3 position, can form four different PIs, one of which forms PIP3 from PIP2

Question 2

What do class I PI3 kinases do?

Answer 2

Catalyse reaction between PI(4,5)P2 and PI(3,4,5)P3

Question 3

Describe class IA PI3 kinases.

Answer 3

p85 adaptor subunit with SH2 domains recognise and bind two specific pTyr on RTK to become activated. p110 catalytic subunit. Ras binding domain alternative route of activation

Question 4

Describe class 1B PI3 kinases.

Answer 4

Contain p110-gamma catalytic subunit and p101 adaptor subunit which binds beta-gamma subunit of G proteins to become activated

Question 5

Describe PTEN.

Answer 5

PIP3 phosphatase with a phosphorylated hairpin, when dephosphorylated the hairpin unfolds and the C2 domain binds the (negatively charged) plasma membrane via Ca2+. This localises PTEN to the plasma membrane to dephosphorylate PIP3

Question 6

Describe the changes in cell signalling regulation seen in cancer.

Answer 6

Upregulation in EGFR, Ras, and PI3 kinases, downregulation of PTEN

Question 7

Name a PI3 kinase inhibitor.

Answer 7

Wortmannin

Question 8

Describe the activation of Akt.

Answer 8

Akt and PDK1 bind PIP3 in close proximity to each other, PDK1 phosphorylates Akt on T308. mTORC2 phosphorylates Akt on S473. Phosphorylation events activate Akt

Question 9

Describe the general action at insulin receptors in adipocytes and muscle cells.

Answer 9

In the presence of insulin, Glut4 vesicles are promoted to the plasma membrane by Rab GTPases to uptake glucose into the cell. In the absence of insulin, the Rab GAP AS160 stimulates Rab GTPase activity, inactivating the Rab proteins so Glut4 remains in intracellular vesicles

Question 10

Describe in detail the action at insulin receptors in adipocytes and muscle cells when insulin is present.

Answer 10

Insulin receptors dimerise and autophosphorylate each other. The p85 SH2 domains of PI3K bind pTyr and phosphorylate PIP2 into PIP3. Akt binds PIP3 via PH domain and is activated by PDK1 and mTORC2. Akt phosphorylates AS160, which is recognised by 14-3-3 proteins that prevent stimulation of Rab GTPase activity hence Glut4 is promoted to the plasma membrane

Question 11

Describe the action of insulin receptors in liver cells.

Answer 11

Akt is activated and phosphorylates transcription factors (e.g.FoxO1) for gluconeogenic genes. 14-3-3 proteins recognise phosphorylation and inhibit localisation to the genome, reducing transcription of gluconeogenic genes

Question 12

Describe Akt’s role in cell survival - apoptosis.

Answer 12

Apoptosis is balanced by signalling of pro-apoptotic BAD and anti-apoptotic BCL2. Akt phosphorylates BAD, 14-3-3 recognise phosphorylation and inhibit BAD signalling so BCL2 anti-apoptotic signalling predominates

Question 13

Describe Akt’s role in cell survival - proliferation.

Answer 13

GSK3 is active in non-stimulated cells, phosphorylating protein for ubiquitinylation and degradation by the proteasome. A target of GSK3 is the transcription factor c-Myc which stimulates transcription of proliferative genes. When Akt is activated, it phosphorylates GSK3, inhibiting action so c-Myc promotes transcription of proliferative genes

Question 14

Describe Akt’s role in cell survival - cell growth.

Answer 14

mTORC1 integrates signals from activated Akt and signals indicating nutrient availability, phosphorylating S6 kinase which phosphorylates ribosome subunit rS6. This activates ribosomal activity, increasing protein production and promoting anabolism for cell growth

Question 15

Name an mTORC1 inhibitor and its effects.

Answer 15

Rapamycin, inhibits cell growth and cell proliferation as well as being an immunosuppresant

Question 16

Describe the effect of adrenaline at beta1-adrenergic receptors.

Answer 16

Activates Galpha-s subunit which activates adenylyl cyclase to produce cAMP

Question 17

Describe the effect of adrenaline at alpha1-adrenergic receptors.

Answer 17

Activates Galpha-q which activates PLC-beta to produce IP3

Question 18

Describe the effect of cAMP from beta-1 adrenergic receptors.

Answer 18

Activates PKA by binding to regulatory subunits, preventing the pseudo-substrate region from inhibiting the catalytic subunits, catalytic subunits are free to phosphorylate their targets

Question 19

Describe the effects of PKA in skeletal and cardiac muscle.

Answer 19

Phosphorylates phosphorylase kinase, which in turn phosphorylates glycogen phosphorylase which hydrolyses glycogen into glucose-1-phosphate and provides the cell with energy.
PKA also phosphorylates L-type Ca2+ channels, increasing the open conformation to increase intracellular Ca2+ for increased muscle contraction in cardiac muscle.
PKA also phosphorylates CREB transcription factor, allowing it to bind upstream of genes involved in metabolism and myogenesis

Question 20

Describe the effect of IP3 produced by alpha1-adrenergic receptors.

Answer 20

IP3 opens Ca2+ channels on ER, calmodulin then binds Ca2+ and targets calmodulin kinases like MLCK, activating it to phosphorylate myosin, activating it to interact with actin and cause muscle contraction

Question 21

Describe the effect of PKA in smooth muscle.

Answer 21

PKA phosphorylates the calmodulin-binding domain of MLCK, preventing activation so myosin is not phosphorylated (vasodilation)

Question 22

Describe the effect of alpha1-adrenergic receptors on PKC.

Answer 22

IP3 release leaves behind DAG, which binds the C1 domain of PKC. Ca2+ binds the C2 domain of PKC. Together these relieve auto-inhibition by the pseudo-substrate, freeing PKC to have action

Question 23

Describe the effect of phorbol esters on PKC.

Answer 23

Phorbol esters mimic DAG, activating PKC (carcinogenic)