PSC2002/L23 PK and Protein Phosphatases

Question 1

What can activate protein kinases? (3)

Answer 1

cGMP (PKG)
Increase in Ca2+
PKB & protein tyrosine kinases (only phosphorylate serine residues)

Question 2

What is the key difference between the cGMP and cAMP signalling pathways?

Answer 2

GPCRs not involved in cGMP pathway

Question 3

What are the 2 distinct types of GCs?

Answer 3

Soluble form (sGC) activated by NO
Plasma membrane bound (pGC) activated by peptide agonists

Question 4

Give another abbreviation of PKG.

Answer 4

cGK

Question 5

What is the role of cGMP in the cGMP pathway?

Answer 5

Activates PKG
PKG phosphorylates serine/threonine residues

Question 6

What breaks down cGMP?

Answer 6

cGMP-dependent PDEs

Question 7

How can nitric oxide production be stimulated?

Answer 7

Increasing calcium

Question 8

How permeable are endothelial cells to NO?

Answer 8

Very permeable

Question 9

Where is ANP released from?

Answer 9

Atrial cells

Question 10

How can a singular peptide increase cGMP? (3)

Answer 10

Binds to plasma membrane
Covalent change in guanylyl cyclase
Leads to increase in catalytic subunits which make cGMP

Question 11

Where is NO released from?

Answer 11

Endothelial cells

Question 12

What is the effect of NO in VSM?

Answer 12

Activates sGC/cGMP
Vasodilation and BP decrease

Question 13

What kind of drugs are used to treat angina?

Answer 13

NO generating drugs

Question 14

What kind of drug is Viagra (sildenafil)?

Answer 14

Type 5 cGMP PDE inhibitor

Question 15

What is the effect of viagra (sildenafil)? What is it used to treat? (2)

Answer 15

Rise in cGMP relaxes SM in some tissues
Used to treat erection problems and pulmonary hypertension

Question 16

Describe the effect of heat stable enterotoxin from E. coli in the intestine. (3)

Answer 16

Activates pGC/cGMP
PKG phosphorylates & activates CFTR
Leads to secretory diarrhoea

Question 17

Describe the LPS (endotoxin) from Gram -ve bacteria. (3)

Answer 17

Increases iNOS (inducible NO synthase) expression
Excessive NO production
Can lead to clinical shock due to severe BP drop

Question 18

How many different isoforms and groups of PKC are there?

Answer 18

11 isoforms
3 groups - conventional, novel and atypical

Question 19

What is required for PKC to become active?

Answer 19

Phospholipid binding

Question 20

What keeps PKC inactive? (2)

Answer 20

R domain has a pseudosubstrate motif
Keeps kinase inactive by occupying substrate binding site on C4 domain

Question 21

Describe the activation cycle for cPKC (following a rise in cytosolic Ca2+). (3)

Answer 21

Ca2+ binds to C2 domain
PKC translocates to PM to bind DAG via C1 domain
PS motif disengages from C4 domain, allowing substrates to bind and be phosphorylated

Question 22

How can cPKC be artificially activated? (2)

Answer 22

Phorbol esters (plant alkaloids which are tumorigenic)
Directly bind to PKC

Question 23

What are the 2 main types of Ca2+/Calmodulin-dependent Protein Kinases?

Answer 23

Those with narrow substrate specificities (e.g., phosphorylase kinase only phosphorylates ‘phosphorylase’
Those with broad substrate specificities (e.g., Multifunctional CaM kinase II (CaMKII) which phosphorylates many substrates)

Question 24

Give 3 functions of CaMKII.

Answer 24

1. Regulates N-methyl-D-aspartate (NMDA) receptors by phosphorylating sites on NR2A and NR2B subunits
2. Enhances InsP3 formation by inhibiting inositol phosphorylate 5-phosphatase
3. Central role in frequency decoding of calcium signals and acting as molecular switch in learning and memory
4. Phosphorylates PLB to control SERCA2 pump - works in synergy with PKA to inhibit PLB effect on SERCA

Question 25

What is the function of protein phosphatases (PP)?

Answer 25

Remove phosphate groups from phosphorylated proteins (Ser/Thr & Tyr residues)

Question 26

What are the 4 major classes of Ser/Thr PPs?

Answer 26

1, 2A, 2B, 2C 1, 2A, 2C show broad & overlapping substrate specificity "B (calcineurin- CaN) has more restricted specificity & requires Ca2+/CaM activity

Question 27

How are PPs regulated?

Answer 27

By inhibitory proteins type 1 and 2

Question 28

Give an additional class of PPs.

Answer 28

Tyrosine PPs, in membrane bound and cytosolic forms

Question 29

Give 2 chemical inhibitors of protein phosphatases and what they block.

Answer 29

Okadaic acid (OA) blocks PP1 and PP2A Cyclosporin A very specific for calcineurin (PP2B) used clinically for immunosuppression via effects on T-cells

Question 30

Where does okadaic acid originate from?

Answer 30

Dinoflagellates (algae) produce this toxin Accumulates in marine sponges and shellfish When ingested causes diarrhetic shellfish poisoning (DSP)

Question 31

How does diarrhetic shellfish poisoning (caused by okadaic acid) cause acute diarrhoea?

Answer 31

Increased paracellular permeability More active CFTR, promoting fluid loss from GI tract

Question 32

How does PKA lead to glycolysis? (3)

Answer 32

Switch on PKA through GPCR Phosphorylates PK to A form Glycogen breakdown

Question 33

What is the role of PKA with regards to PP inhibitory proteins?

Answer 33

Activate PP 'inhibitory' proteins Inhibitory proteins bind to and inhibit PPs during glycogen breakdown Rise in [cAMP] favours glycogen breakdown

Question 34

Describe the synergy between cAMP and Ca2+ in skeletal muscle. (2)

Answer 34

Link between depolarisation and opening of Ca2+ stores Ca2+ feeds into PK helping it to work better

Question 35

What are the requirements of PK to work in skeletal muscle?

Answer 35

PKA phosphorylation and Ca2+ binding

Question 36

What are the subunits of PK?

Answer 36

a, B, d, Y PKA phosphorylation of a and B subunits increases calcium sensitivity of PK

Question 37

Which 2 signalling pathways interact to regulate insulin secretion from beta cells?

Answer 37

cAMP signalling and tyrosine kinase signalling pathways