Lecture 4 - Regulation of protein activity (protein kinases)

Question 1

Proteins and their functions

Answer 1

A protein’s function is determined by its shape

A protein shape is determined by the interactions between its amino acids

Change the properties of its amino acids and thus the protein’s shape
This can modify the protein’s function

Alter an amino acid in the amino acid sequence of a protein then …
Change in the final structure and therefore function - this could turn an enzyme on or off, allow an interaction with another molecule, change protein’s location
Final shape is determined by amino acid sequence

Question 2

Post-translational modification

Answer 2

THe regulated addition of a small molecule
Modifications that happen after the protein is translated
Most often the addition of a phosphate
Adds a large negative charge
Will cause the folded protein to adjust its shape
Phosphate bonds to serine, threonine or tyrosine amino acid residues

Question 3

Protein phosphorylation and its significance

Answer 3

Protein phosphorylation is carried out by protein kinases
Transfer of a phosphate from ATP to the target protein
Protein kinases add phosphates
Protein phosphates remove them

Protein kinases are categorised after their amino acid target…
Serine/threonine kinases
Tyrosine kinases
Adds phosphates to the first word

…and often named by what activates them
Protein kinase A dependent protein kinases
Protein kinase C dependent protein kinases
Cyclin-dependent protein kinases
Receptor tyrosine kinases

Note: protein kinases must be regulated

There are many different types of protein kinases - grouped into families based on theyr sequences, some are ‘promiscuous’ meaning that they have many protein “targets” and others are highly specialised to a single protein 
Calcium-calmodulin-dependent protein kinases (CAMK) 
Tyrosine kinases (receptor or cytoplasmic) 

Protein kinases are regulated by many receptor types.
They provide a mechanism for signal transduction
We have already seen the role of protein tyrosine kinases in cytokine receptor actions

Question 4

Many different GPCR

Answer 4

Respond to hormones, neurotransmitters, paracrine signals, odours and light
> 1000 types in human
Receptor gives specificity
G protein transducer the signal (into the cell and changes the behaviour of protein kinase)
Effector (generates intracellular signals - alpha and beta-gamma subunits may provide different signals) (goes on to affect the protein kinases)

Question 5

Many GPCRs signal via protein kinases …

Answer 5

Protein kinase A (PKA) 
PKA exists as an inactive complex with its regulatory subunits 
cAMP binds to the regulatory subunits 
Causes them to dissociate 
Releasing the active kinase

May act via phospholipase C to activate
Protein kinase C (PKC)
Ca2+/calmodulin-dependent protein kinase (CaMK)
PKC activation
Events depicted as realistic molecular structures
Inactive PKC located at cell membrane
Intereacts with lipid produced following receptor activations
PKC complex disassociates and releases active (catalytic) subunits of PKC

Question 6

GPCRs have multiple different effectors

Answer 6

Adenylyl cyclase - forms cAMP

Phospholipase C - releases intracellular Ca2+

PI3-kinase
These signals may activate protein kinases
Kinases regulate protein activity
In cytoplasm - enzymes
In the nucleus - transcription factors
Puts phosphates on proteins and there are two groups that it will do = enzymes which will change enzyme activity or on to transcription factors which will head into the nucleus and change transcription

Question 7

how is the activity of an enzyme maybe regulated by protein kinases

Answer 7

Protein kinases may increase the expression of a protein via transcription factors - changes the amount of a particular protein in a cell
Protein kinases may also change the activity of existing protein -increase the activity of an enzyme
Illustrate the latter with reference to the enzyme tyrosine hydroxylase

Question 8

how is the activity of an enzyme maybe regulated by protein kinases - example with enzyme tyrosine hydroxylase

Answer 8

Tyrosine hydroxylase (TH) 
Catecholamines have multiple functions 
Dopamine, noradrenaline and adrenaline 
All made by the same pathway 
First and rate-limiting step is carried out by TH - Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis
Dopamine provides a negative feedback 

Process
Dopamine synthesised in nerve terminals (when dopamine is made it feedback and turns TH off so that we do not get anymore made
Now the dopamine is released in response to nerve stimulation
Need more dopamine since the original amount has been used
Increase TH activity via phosphorylation
Phosphorylation reduces the negative feedback by dopamine (becomes resistant to the negative feedback therefore more dopamine can be formed)

TH can be phosphorylated at 4 different sites (Ser8, Ser19, Ser31, Ser40)
Different kinases act at each site
Different sites have different effects on activity

The order of phosphorylation matters
Ser-19 allows other sites to be phosphorylated