Signaling: Serine-Threonine Kinases and Phosphatases

Question 1

1. Describe a phosphorylation reaction (including which amino acids can be phosphorylated) and explain how it can affect a phosphorylated protein.

Answer 1

• ATP contains energy rich bonds, particularly beta and gamma phosphoanhydride bonds.
• Ser/Thr and Tyr contain OH groups that can be phosphorylated.
• Phosphorylation happens via a nucleophilic attach of the OH group (of an S/T) to the gamma-phosphate of ATP.
• Kinase helps catalyze this reaction by promoting the ideal positioning of the reaction partners (ATP and whatever protein is being phosphorylated).

Question 2

2. List at least two other types of secondary protein modification

Answer 2

Acetylation, glycosylation, ubiquitinylation, and proteolytic cleavage.

Question 3

3. Explain the structure of an ATP molecule.

Answer 3

• Adenine base,
• sugar ring, and
• 3 phosphate groups connected by high-energy phosphoanhydride bonds.

(Beta and gamma are the most high energy.)

Question 4

4. Explain how protein kinases can be classified and describe examples.

Answer 4

Protein kinases can be classified by:

• Their phosphorylated residue (Ser/Thr or Tyr)
• Their substrate protein (MLCK)

-their activating stimulus –> receptor linked (MAPK, EGFR, insulin) versus second messenger (PKA & cAMP, PKC and Ca, CaMKII and Ca) versus cyclins (CDK2)

• Their phylogenic relationships (evolutionarily similar? Ex: MAPs in 2 categories):
  
  • AGC = PKA, PKG and PKC containing
  • CAMK = like CaMKII, MLCK
  • CMGG = CDKs, MAPKs, GSK3, CLK
  • STE = Ste7, 11, 20 (upstream MAP kinases)
  • CK2 = casein kinase 1
  • TK = tyrosine kinases and TLK = Tyr kinase-like

Question 5

5. Describe the structure/function of a protein kinase and principles of their regulation (including requirement for activation loop phosphorylation in some but not all kinases).

Answer 5

• Kinase domain has a small and large lobe.
• ATP binds the cleft between the lobes.
• Closed conformation of gly-rich loop forces gamma-phosphate of ATP into right position for a phosphorylation reaction (fast reaction).
• Open conformation of gly-rich loop allows exchange of ADP–> ATP (slow reaction = rate-limiting step).
• Kinases alternate between conformations.
• Active conformation of kinases = conserved = problem for making specific inhibitors for individual kinases.
• Inactive conformations are not as conserved –> can target this instead.
• ATP binding pocket is distorted in inactive conformation.
• Parts that can be distorted in inactive state: =
• activation loop,
• C-helix,
• gly-rich loop,
• ATP binding pocket.

Open/closed conformation doesn’t mean the same thing as active/inactive.
-Activation loop needs to be phosphorylated for full activity (THIS determines activation/inactivation); can block active site using inhibitory ‘pseudo-substrate’.

• PKA requires phosphorylation by PKA.
• PKB requires phosphorylation by PDK1.
• PKC requires phosphorylation by PDK1.
• CaMKI and IV require phosphorylation by CaMKK.
• CaMKII requires no phosphorylation by anything else –>auto-phosphorylates itself.
• Calcineurin targeted by cyclosporine (or tacrolimus for patients in whom cyclosporine doesn’t work).
• They bind an immunophilin that inhibits calcineurin phosphatase activity.
• mTOR targeted by sirolimus/rapamycin = another immunosuppressant.
• Binds to FKBP-12 which inhibits mTOR kinase activity.
• Ser/Thr kinases can be targeted by fasudil (Rho-kinase inhibitor –> vasodilatory effect)
• Tyr Kinases targeted by Gleevec.
• MAP kinases can be activated by other upstream kinases.
• CaMKII and calcineurin regulate long term potentiation and long term depression of synaptic strength.
• This is an example of Ca as a node in signal transduction.
• Low frequency stimulation = low Ca = calcineurin wins.
• High frequency stimulation = high Ca = CaMKII wins.
• Glutamine released into synaptic cleft, binds NMDAR and AMPAR.
• AMPA opens and allows Na to pass though; NMDA blocked by Mg until enough depolarization happens via AMPA (and then Ca can pass through).
• Ca signaling leads to increased AMPAR = potentiation. -Potentiation vs. depression determined by the type of stimulation.