Protein Phosphatases

Question 1

Is the protein kinase reaction reversible or irreversible?

Answer 1

Irreversible, protein phosphatases induce a different reaction, hydrolysis

Question 2

Why is phosphorylation irreversible?

Answer 2

Since it produces ATP and hydrolysis by protein phosphatases does not

Question 3

What is a phosphorylase?

Answer 3

Causes phosphorylation of a substrate

Question 4

What is phosphorylase kinase?

Answer 4

Phosphorylates the phosphorylase

Question 5

What is PP1

Answer 5

Phosphorylase phosphatase - dephosphorylates phosphorylase

Question 6

How is Src activated?

Answer 6

Dephosphorylation of pTyr508

Phosphorylation of Tyr397

Question 7

How many kinases are in the kinome?

Answer 7

> 500

Question 8

How are kinases and phosphatases families different?

Answer 8

Most kinases are similar, phosphatases are different based on their catalytic mechanisms and amino acid sequences

Question 9

How many phosphatase phynogenic trees are there?

Answer 9

10

Question 10

What family due tyrosine phosphatases belong to?

Answer 10

CC1

Question 11

What are dual specificity phosphatases?

Answer 11

They can be serine/threonine phosphatases or tyrosine phosphatases

Question 12

Which dual specificity phosphatase is different and why?

Answer 12

PTEN

It can be a lipid or protein tyrosine phosphatase

Question 13

What do phosphatases recognise?

Answer 13

Molecular features instead of consenus sequences around the phosphorylated site

Question 14

What is the difference between protein tyrosine phosphatase catalytic pockets to serine/threonine phosphatases?

Answer 14

Tyrosine phosphatase catalytic pockets are deeper than serine/threonine phosphatase catalytic pockets

Question 15

Why do tyrosine phosphatases have deeper catalytic pockets than serine/threonine phosphatases?

Answer 15

Phospho-tyrosine is a big molecule

Question 16

Describe the active site of PP1

Answer 16

Contains a bimetal centre 
Bridged by a water molecule, which is displaced by the binding of phosphate 
Has 3 grooves:
- an acidic groove 
- a C-terminal groove 
- a hydrophobic groove

Question 17

What is the mechanism of the phosphate in PP1?

Answer 17

The phosphate undergoes nucleophilic attack by a hydroxyl ion
Histidine125 provides a proton to the alcohol group
Metal ions are important in coordinating this mechanism

Question 18

What is the inhibitor of PP1?

Answer 18

Inhibitor 1 and 2

Question 19

What is the inhibitor of PP2A?

Answer 19

Okadaic acid

Question 20

What is the inhibitor of PP2B?

Answer 20

Trifluoperazine

Question 21

What is the inhibitor of PP2C?

Answer 21

EDTA

Question 22

What is the activator of PP2B?

Answer 22

Ca2+-calmodulin

Question 23

What is the activator of PP2C?

Answer 23

Mg2+

Question 24

Where is PP2B abundant?

Answer 24

In the brain

Question 25

What does EDTA do?

Answer 25

Collates magnesium ions and inhibits them

Question 26

What causes diuretic shellfish poisoning?

Answer 26

Okadaic acid

Question 27

What does blue-green algae produce?

Answer 27

Microcystin

Question 28

Where does microcystin bind covalently to of PP1, PP2A and related PPP enzymes?

Answer 28

A cysteine residue in the beta12-13 loop

Question 29

What happens if you add toxins to paramecium? and why?

Answer 29

They swim backwards A calcium pump regulates the directionality of the flagella, if this is blocked in the phosphorylated state then it jams the flagella in terms of directionality

Question 30

Name some physiological processes involved in PP1 signalling

Answer 30

``` Glycogen metabolism Muscle contractility Protein synthesis Ca2+ uptake by SR Cell division cycle ```

Question 31

What are the substrates for PP1 for glycogen metabolism?

Answer 31

Glycogen phosphorylase | Glycogen synthase

Question 32

If PP1 is involved in so many cellular processes, how are these processes regulated so specifically?

Answer 32

The catalytic subunits of PP1 are bound to various location by different targeting or regulatory subunits

Question 33

How do you prepare microcystin-sepharose for affinity chromatography?

Answer 33

The O in microcystin makes the carbon slightly electropositive The S in sepharose can give a negative charge which attacks the carbon Forms a covalent bond between sulphur and carbon

Question 34

What does dephosphorylation of myosin regulatory light chain cause?

Answer 34

Smooth muscle cell relaxation

Question 35

What happens when PP1C is blocked in the gut by okadaic acid?

Answer 35

Smooth muscle in the gut contacts

Question 36

What happens when PP1C interacts with regulatory subunit MYPT1?

Answer 36

It makes PP1C better at desphosphorylating myosin light chains than on its own as it provides a bigger platform for interaction with the substrate

Question 37

What is the two functions of MYPT1?

Answer 37

Brings PP1C to the mysin | Acts as a platform which enhances the phosphatase specificity

Question 38

What is MYPT1?

Answer 38

A myosin regulatory subunit

Question 39

How does myosin light chian interact with PP1C?

Answer 39

The arginine and lysine of the mysoin light chain interact with the negatively charged acidic groove

Question 40

What is a common motif in most regulatory subunits of PP1?

Answer 40

RVxF/W

Question 41

In response to adrenalin, what does PKA phosphorylate? What effects to these have?

Answer 41

``` Glycogen synthase (inhibits it) - reduce glycogen synthesis Phosphorylate phosphorylase kinase (activates) - increase glycogen breakdown Site 1 and 2 of glycogen-binding subunit - PP1c cannot bind to glycogen-binding subunit - translocates to the cytoplasm Inhibitor 1 (activates) - inhibitor 1 inhibits PP1c in the cytoplasm ```

Question 42

What is the function of targeting subunits of PP1?

Answer 42

Targeting or localisation - bring the phosphatase to the substrate Alter substrate specificity Regulation of PP1 activity