Protein Kinases Flashcards
How many protein kinases are in the human genome?
> 500
What does the AGC group of kinases induce?
Many classical second messenger-dependent kinases
Give examples of kinases which AGC kinases induce
cyclic AMP/GMP dependent (PKA/PKG)
Phosphoinositide-activated (PKB/AKT)
Ca2+/ phospholipid-activated (PKC)
Give examples of kinases which CaMK group induce
Ca2+/ calmodulin-dependent (CaMKs)
AMP-activated (AMPK)
AMPK-related kinases (ARKs)
How was PKG probably made?
By a fusion of genes encoding the seperate R and C subunits of PKA-like ancestors
What makes up the R subunit of PKA and PKG?
A dimerisation/ docking (DD) domain and two tandem domains which bind cAMP or cGMP, respectively
What is the dimerisation/ docking domain for?
The dimerisation and subcellular targetting
How does cAMP bind to PKA?
It binds to the C-terminus first and then the N-terminus but activation only occurs at the N-terminus
What is the difference between the R subunit and C subunit of PKA and PKG?
They are fused in PKG and are seperate in PKA
How do cAMP and cGMP bind to PKA and PKG, respectively?
Through positive co-operativity
What is positive co-operativity?
Where when one binds it makes it easier for the other to bind
In the absence of cAMP, what does PKA exist as?
An R2C2 complex and is inactive
How does cAMP impact the R2C2 structure?
When it binds, it causes active C subunits to dissociate
What is the linker region between cAMP-binding domain and DD sensitive to?
Proteolysis
What does the linker region form when it undergoes proteolysis?
A flexible hinge
What two isoforms do R subunit occur as?
RI and RII
Describe the RI hinge region
It contains a pseudosubstrate sequence which resembles a substrate but lacks a phosphorylatable serine/threonine residue
Describe the RII hinge region
It has a phosphorylation site
Where does the kinase bind to PKA in the inactive conformation of RI
The pseudosubstrate domain
Why does PKA not phosphorylate every serine or threonine in a protein?
They have to have conserved sequence features or motif around the phosphorylated residue
- Basic residues, Arg or Lys, at position P-2, P-3 and sometimes P-6, to the phosphorylated site
- Hydrophobic residue, e.g. Leu, Ile, Val, Phe or Met, at P+1
What does PKA catalytic subunit contain?
A small N-terminal lobe and a larger C-terminal lobe
What are used to stop kinases phosphorylating their substrates all the time?
Substrate analogues
Name and describe a PKA substrate analogue
Protein kinase inhibitor
Has the sequence motif Arg-x-x-Arg-Arg-x-Ala-Ile
it is not phosphorylated since there is an Ala instead of a Ser
How does PKI protein inhibit PKA?
binds like a substrate and blocks the binding domain
Where does MgATP bind to PKA?
Deep within the cleft between the 2 lobes
What does ATP binding to PKA induce?
Closure of the N- and C-lobes by flexing the hinge region between them
Where does the pseudosubstrate bind?
Partly to the groove on the C-lobe and partly to the cleft between N- and C-lobes
What is the kinase domain sequence?
G50xGxxG–K72–E91–RD166LKxxN–D184FG–T197–APE208–R280–
Describe the GxGxxG motif
It is a loop which closes over the phosphate group of ATP
It interacts with the adenine moiety of ATP
Glycine has only a H side chain which can restrict the ATP binding site
Describe the K72 motif
Has a -NH3+ group on its side chain
Binds to the negatively charged alpha and beta phosphates of ATP
Describe the E91 motif
Has a -CO2- on its side chain
interacts with K72 side chain to stabilise the structure of the N-lobe
Describe the D166 motif
acts in the catalytic mechanism
Describe the D184 motif
Has a -CO2- which interacts with Mg2+ bound to beta and gamma phosphates of ATP
Describe the T197 motif
part of the activation loop
lies between the DFG and APE motif
in most protein kinases, has to be phosphorylated to be activated
Describe the E208 motif
the -CO2- side chain interacts with the -NH3+ side chain of R280
Anchors the activation loop to the core of the large lobe
What do the positively charged nitrogens of P-6, P-3 and P-2 of PKI bind to?
Negatively charged oxygens on glutamate side chains (Glu-203, -170 and -230) in the large lobe
What does the hydrophobic isoleucine at P+1 interact with?
P+1 pocket
What is the P+1 pocket
A hydrophobic pocket on the large lobe formed by Leu198, Cys199, Pro202 and Leu205
What is the pseudosubstrate sequence of the RI regulatory subunit?
RRGAI
At what position is PKA phosphorylated to become activated?
Thr197 in the activation loop
How does PKA phosphorylation induce PKA activation
The negatively charged phosphate on Thr197 binds in the positively charged pocket formed by the side chains of 3 basic residues
Causes a conformational change
What 3 basic residues form the positively charged pocket?
His87
Arg165
Lys189
Where is His87?
In the C-helix
Where is Arg165?
Adjacent to Asp166
