Protein Kinases Flashcards

1
Q

How many protein kinases are in the human genome?

A

> 500

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2
Q

What does the AGC group of kinases induce?

A

Many classical second messenger-dependent kinases

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3
Q

Give examples of kinases which AGC kinases induce

A

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

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4
Q

Give examples of kinases which CaMK group induce

A

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

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5
Q

How was PKG probably made?

A

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

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6
Q

What makes up the R subunit of PKA and PKG?

A

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

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7
Q

What is the dimerisation/ docking domain for?

A

The dimerisation and subcellular targetting

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8
Q

How does cAMP bind to PKA?

A

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

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9
Q

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

A

They are fused in PKG and are seperate in PKA

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10
Q

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

A

Through positive co-operativity

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11
Q

What is positive co-operativity?

A

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

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12
Q

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

A

An R2C2 complex and is inactive

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13
Q

How does cAMP impact the R2C2 structure?

A

When it binds, it causes active C subunits to dissociate

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14
Q

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

A

Proteolysis

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15
Q

What does the linker region form when it undergoes proteolysis?

A

A flexible hinge

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16
Q

What two isoforms do R subunit occur as?

A

RI and RII

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17
Q

Describe the RI hinge region

A

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

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18
Q

Describe the RII hinge region

A

It has a phosphorylation site

19
Q

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

A

The pseudosubstrate domain

20
Q

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

A

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
21
Q

What does PKA catalytic subunit contain?

A

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

22
Q

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

A

Substrate analogues

23
Q

Name and describe a PKA substrate analogue

A

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

24
Q

How does PKI protein inhibit PKA?

A

binds like a substrate and blocks the binding domain

25
Where does MgATP bind to PKA?
Deep within the cleft between the 2 lobes
26
What does ATP binding to PKA induce?
Closure of the N- and C-lobes by flexing the hinge region between them
27
Where does the pseudosubstrate bind?
Partly to the groove on the C-lobe and partly to the cleft between N- and C-lobes
28
What is the kinase domain sequence?
G50xGxxG--K72--E91--RD166LKxxN--D184FG--T197--APE208--R280--
29
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
30
Describe the K72 motif
Has a -NH3+ group on its side chain | Binds to the negatively charged alpha and beta phosphates of ATP
31
Describe the E91 motif
Has a -CO2- on its side chain | interacts with K72 side chain to stabilise the structure of the N-lobe
32
Describe the D166 motif
acts in the catalytic mechanism
33
Describe the D184 motif
Has a -CO2- which interacts with Mg2+ bound to beta and gamma phosphates of ATP
34
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
35
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
36
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
37
What does the hydrophobic isoleucine at P+1 interact with?
P+1 pocket
38
What is the P+1 pocket
A hydrophobic pocket on the large lobe formed by Leu198, Cys199, Pro202 and Leu205
39
What is the pseudosubstrate sequence of the RI regulatory subunit?
RRGAI
40
At what position is PKA phosphorylated to become activated?
Thr197 in the activation loop
41
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
42
What 3 basic residues form the positively charged pocket?
His87 Arg165 Lys189
43
Where is His87?
In the C-helix
44
Where is Arg165?
Adjacent to Asp166