Protein Kinases

Question 1

How many protein kinases are in the human genome?

Answer 1

> 500

Question 2

What does the AGC group of kinases induce?

Answer 2

Many classical second messenger-dependent kinases

Question 3

Give examples of kinases which AGC kinases induce

Answer 3

cyclic AMP/GMP dependent (PKA/PKG)
Phosphoinositide-activated (PKB/AKT)
Ca2+/ phospholipid-activated (PKC)

Question 4

Give examples of kinases which CaMK group induce

Answer 4

Ca2+/ calmodulin-dependent (CaMKs)
AMP-activated (AMPK)
AMPK-related kinases (ARKs)

Question 5

How was PKG probably made?

Answer 5

By a fusion of genes encoding the seperate R and C subunits of PKA-like ancestors

Question 6

What makes up the R subunit of PKA and PKG?

Answer 6

A dimerisation/ docking (DD) domain and two tandem domains which bind cAMP or cGMP, respectively

Question 7

What is the dimerisation/ docking domain for?

Answer 7

The dimerisation and subcellular targetting

Question 8

How does cAMP bind to PKA?

Answer 8

It binds to the C-terminus first and then the N-terminus but activation only occurs at the N-terminus

Question 9

What is the difference between the R subunit and C subunit of PKA and PKG?

Answer 9

They are fused in PKG and are seperate in PKA

Question 10

How do cAMP and cGMP bind to PKA and PKG, respectively?

Answer 10

Through positive co-operativity

Question 11

What is positive co-operativity?

Answer 11

Where when one binds it makes it easier for the other to bind

Question 12

In the absence of cAMP, what does PKA exist as?

Answer 12

An R2C2 complex and is inactive

Question 13

How does cAMP impact the R2C2 structure?

Answer 13

When it binds, it causes active C subunits to dissociate

Question 14

What is the linker region between cAMP-binding domain and DD sensitive to?

Answer 14

Proteolysis

Question 15

What does the linker region form when it undergoes proteolysis?

Answer 15

A flexible hinge

Question 16

What two isoforms do R subunit occur as?

Answer 16

RI and RII

Question 17

Describe the RI hinge region

Answer 17

It contains a pseudosubstrate sequence which resembles a substrate but lacks a phosphorylatable serine/threonine residue

Question 18

Describe the RII hinge region

Answer 18

It has a phosphorylation site

Question 19

Where does the kinase bind to PKA in the inactive conformation of RI

Answer 19

The pseudosubstrate domain

Question 20

Why does PKA not phosphorylate every serine or threonine in a protein?

Answer 20

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

Question 21

What does PKA catalytic subunit contain?

Answer 21

A small N-terminal lobe and a larger C-terminal lobe

Question 22

What are used to stop kinases phosphorylating their substrates all the time?

Answer 22

Substrate analogues

Question 23

Name and describe a PKA substrate analogue

Answer 23

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

Question 24

How does PKI protein inhibit PKA?

Answer 24

binds like a substrate and blocks the binding domain

Question 25

Where does MgATP bind to PKA?

Answer 25

Deep within the cleft between the 2 lobes

Question 26

What does ATP binding to PKA induce?

Answer 26

Closure of the N- and C-lobes by flexing the hinge region between them

Question 27

Where does the pseudosubstrate bind?

Answer 27

Partly to the groove on the C-lobe and partly to the cleft between N- and C-lobes

Question 28

What is the kinase domain sequence?

Answer 28

G50xGxxG–K72–E91–RD166LKxxN–D184FG–T197–APE208–R280–

Question 29

Describe the GxGxxG motif

Answer 29

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

Question 30

Describe the K72 motif

Answer 30

Has a -NH3+ group on its side chain

Binds to the negatively charged alpha and beta phosphates of ATP

Question 31

Describe the E91 motif

Answer 31

Has a -CO2- on its side chain

interacts with K72 side chain to stabilise the structure of the N-lobe

Question 32

Describe the D166 motif

Answer 32

acts in the catalytic mechanism

Question 33

Describe the D184 motif

Answer 33

Has a -CO2- which interacts with Mg2+ bound to beta and gamma phosphates of ATP

Question 34

Describe the T197 motif

Answer 34

part of the activation loop
lies between the DFG and APE motif
in most protein kinases, has to be phosphorylated to be activated

Question 35

Describe the E208 motif

Answer 35

the -CO2- side chain interacts with the -NH3+ side chain of R280
Anchors the activation loop to the core of the large lobe

Question 36

What do the positively charged nitrogens of P-6, P-3 and P-2 of PKI bind to?

Answer 36

Negatively charged oxygens on glutamate side chains (Glu-203, -170 and -230) in the large lobe

Question 37

What does the hydrophobic isoleucine at P+1 interact with?

Answer 37

P+1 pocket

Question 38

What is the P+1 pocket

Answer 38

A hydrophobic pocket on the large lobe formed by Leu198, Cys199, Pro202 and Leu205

Question 39

What is the pseudosubstrate sequence of the RI regulatory subunit?

Answer 39

RRGAI

Question 40

At what position is PKA phosphorylated to become activated?

Answer 40

Thr197 in the activation loop

Question 41

How does PKA phosphorylation induce PKA activation

Answer 41

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

Question 42

What 3 basic residues form the positively charged pocket?

Answer 42

His87
Arg165
Lys189

Question 43

Where is His87?

Answer 43

In the C-helix

Question 44

Where is Arg165?

Answer 44

Adjacent to Asp166