Protein Phosphatases Flashcards
Is the protein kinase reaction reversible or irreversible?
Irreversible, protein phosphatases induce a different reaction, hydrolysis
Why is phosphorylation irreversible?
Since it produces ATP and hydrolysis by protein phosphatases does not
What is a phosphorylase?
Causes phosphorylation of a substrate
What is phosphorylase kinase?
Phosphorylates the phosphorylase
What is PP1
Phosphorylase phosphatase - dephosphorylates phosphorylase
How is Src activated?
Dephosphorylation of pTyr508
Phosphorylation of Tyr397
How many kinases are in the kinome?
> 500
How are kinases and phosphatases families different?
Most kinases are similar, phosphatases are different based on their catalytic mechanisms and amino acid sequences
How many phosphatase phynogenic trees are there?
10
What family due tyrosine phosphatases belong to?
CC1
What are dual specificity phosphatases?
They can be serine/threonine phosphatases or tyrosine phosphatases
Which dual specificity phosphatase is different and why?
PTEN
It can be a lipid or protein tyrosine phosphatase
What do phosphatases recognise?
Molecular features instead of consenus sequences around the phosphorylated site
What is the difference between protein tyrosine phosphatase catalytic pockets to serine/threonine phosphatases?
Tyrosine phosphatase catalytic pockets are deeper than serine/threonine phosphatase catalytic pockets
Why do tyrosine phosphatases have deeper catalytic pockets than serine/threonine phosphatases?
Phospho-tyrosine is a big molecule
Describe the active site of PP1
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
What is the mechanism of the phosphate in PP1?
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
What is the inhibitor of PP1?
Inhibitor 1 and 2
What is the inhibitor of PP2A?
Okadaic acid
What is the inhibitor of PP2B?
Trifluoperazine
What is the inhibitor of PP2C?
EDTA
What is the activator of PP2B?
Ca2+-calmodulin
What is the activator of PP2C?
Mg2+
Where is PP2B abundant?
In the brain
What does EDTA do?
Collates magnesium ions and inhibits them
What causes diuretic shellfish poisoning?
Okadaic acid
What does blue-green algae produce?
Microcystin
Where does microcystin bind covalently to of PP1, PP2A and related PPP enzymes?
A cysteine residue in the beta12-13 loop
What happens if you add toxins to paramecium? and why?
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
Name some physiological processes involved in PP1 signalling
Glycogen metabolism Muscle contractility Protein synthesis Ca2+ uptake by SR Cell division cycle
What are the substrates for PP1 for glycogen metabolism?
Glycogen phosphorylase
Glycogen synthase
If PP1 is involved in so many cellular processes, how are these processes regulated so specifically?
The catalytic subunits of PP1 are bound to various location by different targeting or regulatory subunits
How do you prepare microcystin-sepharose for affinity chromatography?
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
What does dephosphorylation of myosin regulatory light chain cause?
Smooth muscle cell relaxation
What happens when PP1C is blocked in the gut by okadaic acid?
Smooth muscle in the gut contacts
What happens when PP1C interacts with regulatory subunit MYPT1?
It makes PP1C better at desphosphorylating myosin light chains than on its own as it provides a bigger platform for interaction with the substrate
What is the two functions of MYPT1?
Brings PP1C to the mysin
Acts as a platform which enhances the phosphatase specificity
What is MYPT1?
A myosin regulatory subunit
How does myosin light chian interact with PP1C?
The arginine and lysine of the mysoin light chain interact with the negatively charged acidic groove
What is a common motif in most regulatory subunits of PP1?
RVxF/W
In response to adrenalin, what does PKA phosphorylate? What effects to these have?
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
What is the function of targeting subunits of PP1?
Targeting or localisation - bring the phosphatase to the substrate
Alter substrate specificity
Regulation of PP1 activity
