Lecture 1 - Protein Kinases Structure and Regulation

Question 1

This lecture focuses on the “AGC” group of protein kinases, which includes many second messenger-dependent kinases such as:
[Give 3]

Answer 1

Cyclic AMP/GMP-dependent kinases (PKA/PKG)

Phosphoinositide-activated kinases (PKB/Akt)

Ca2+/phospholipid-activated kinases (PKCs)

Question 2

PKA and PKG both have the following domains:
[3 Domains]

Additionally, for each domain state whether it is found in the R (regulatory) subunit or the C (catalytic) subunit

Answer 2

1. A dimerisation/docking (DD) domain: Required for dimerisation and subcellular targeting
2. Two tandem domains that bind either cAMP or cGMP (e.g. cAMP-A and cAMP-B)
3. A catalytic kinase domain

R subunit: DD, cAMP-A, cAMP-B

C subunit: Kinase

Question 3

Mechanism of PKA activation by cAMP:

Answer 3

In the absence of cAMP, PKA is inactive:

• Due to binding of kinase domain to pseudosubtrate peptide found in the linker hinge region between the DD domain and cAMP binding domain
• Thus the R subunit inhibits the C subunit via the blockage of the catalytic site, preventing access to other substrates

Binding of cAMP to the R subunit causes active C subunits to dissociate:

• cAMP binding causes a conformational change that prevents pseudosubstrate interaction with C subunit
• Thus, active C subunit is released and able to phosphorylate substrates

The linker peptide contains the pseudosubstrate sequence and binds in the active site cleft, blocking access to substrates.

cAMP initially binds to domain B, which then promotes binding of cAMP to domain A

Binding of cAMP to domain A requires it to first dissociate from the C-lobe of the C subunit.

This causes dissociation of the entire R subunit, enabling substrates to access the active site.

Question 4

How do protein kinases recognise S or T residues that they phosphorylate?

Additionally, is it primary sequence or 3D conformation that is detected by PKA?

Answer 4

There are conserved sequence features/motifs around phosphorylated residues:

• Basic residues (Arg, Lys) at P-2, P-3, P-6
• A hydrophobic residue (e.g. Leu, Ile, Val, Phe, or Met) at P+1

arg-xxx-xxx-arg-arg-xxx-ser-hyd
P-6 P-3 P-2 P P+1

PKA will phosphorylate short peptides containing the above motif, therefore it is primary sequence that is recognised rather than 3D conformation

Question 5

PKA catalytic subunit:

Answer 5

Consists of :

• Small N-terminal Lobe (N-Lobe)
• Larger C-terminal Lobe (C-Lobe)

MgATP binds deep within the cleft between the 2 lobes

Preferred order of substrate binding:

• ATP binds first as a MgATP2- complex
• Then pseudosubstrate peptide binds with its Alanine residue close to the gamma phosphate of ATP

Question 6

Conserved Residues in Kinase Domains and Their Functions:

Answer 6

GxGxxG motif: A loop that closes over the phosphate groups of ATP, Gly has only Hydrogen as a side chain, larger side chains would clash with ATP

Lys72: Binds negatively charged alpha and beta phosphates of ATP

Glu91: -CO2- side chain interacts with side chain of Lys72, stabilising structure of the N-lobe

Asp166: Acts in catalytic mechanism

Asp184: -CO2- interacts with Mg2+ bound to beta and gamma phosphates of ATP

Thr197: Part of the activation loop, lying between the DFG and APE motifs. In most protein kinases, this loop must be phosphorylated for the kinase to be active

Glu208: -CO2- side chain interacts with -NH3+ from Arg280, anchoring the activation loop to the core of the large lobe

Question 7

Asp166 catalytic activity:

Answer 7

The CO2- on the side chain of Asp166 is thought to accept a proton from the side chain hydroxyl of a serine residue, thus acting as a catalytic base.

The oxygen on Ser then acts as a nucleophile to attack the gamma phosphate

In the end:

• Asp166 has gained a proton
• ATP -> ADP
• Peptide has gained a phosphate

Asp residue equivalent to Asp166 is conserved in all protein kinases.

Mutation of this Asp residue -> Total loss of kinase activity

Question 8

Activation of PKA by phosphorylation:

Answer 8

Like other kinases, PKA is only activated when its phosphorylated at Thr197 in the activation loop

The negatively charged phosphate on Thr197 binds to a positively charged pocket formed bu the side chains of 3 basic residues:
-His87, Arg165, Lys189

His87 is in the C-helix, whose conformation is known to be crucial for kinase activity

Arg165 is adjacent to Asp166, which is involved in catalysis