Ser/Thr Protein Kinases Flashcards
- Describe a phosphorylation reaction (including which amino acids can be phosphorylated) and explain how it can affect a phosphorylated protein.
- List at least two other types of secondary protein modification. (See “Signaling Overview”.)
- Explain the structure of an ATP molecule.
- Explain how protein kinases can be classified and describe examples.
- 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).
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Kinases _____. What class of enzyme performs the opposite function?
The action of kinases (phosphorylation) is opposed by the action of phosphatases (dephosphorylation)
Ser/Thr and Tyr have phosphorylatable ____ groups. Chemically, a phosphorylation reaction catalyzed by a kinase is a:
hydroxyl (-OH); nucleophilic attack of such hydroxyl group onto the γ-phosphate of an ATP molecule
Kinases can be classified based on
1) the amino acid residue they phosphorylate (Ser and Thr; or Tyr)
2) substrate (myosin light chain kinase)
3) activation (insulin receptor, pka, etc)
4) phylogenetic relationship (ie CaMKII and MLCK are Ca2+/calmodulin dependent)
A kinase domain consists of a small and large lobe. ATP binds in the cleft between the lobes. A “closed conformation” of the glycine rich loop in the small lobe forces the γ-Phosphate of the ATP into the right position for phosphorylation (a fast reaction). An “open conformation” of the glycine rich loop then allow exchange of the generated ADP for a new ATP (a slow reaction). Thus, kinase activity is thought to require:
alternating open and closed conformations
Types of protein modification include (5):
- Protein phosphorylation
- Acetylation
- Glycosylation
- Ubiquitinylation
- Proteolytic cleavage
Explain the structure of an ATP molecule.
Adenosine triphosphate (ATP) is a nucleotide made of ribose, a purine base (adenosine) and three phosphate groups. The phosphate bonds of ATP are energy rich and can phosphate groups can be donated to other molecules.
Protein kinases consist of two lobes: one large and one small. Between the lobes is a cleft where the ATP and substrate bind. The substrate associates with the large lobe in the cleft. Protein kinases have highly conserved catalytic cores with a ______ and ______.
glycine rich loop and an activation loop
[The glycine rich loop plays a role in altering the conformation of the enzyme. In the closed conformation, the glycine loop forces the ATP into the right position for the phosphorylation reaction. In the open confirmation, the glycine loop changes its position to allow for the exchange of GDP for GTP. In 70% of kinases, the activation loop must be phosphorylated in order to activate the kinase.]
What is a good drug target vs not with regards to a protein kinase?
The active conformation is highly conserved. Therefore, it is not a useful drug target as drug inhibitors would lack specificity. Inactive conformations, however, have varying conformations and can be specifically inhibited. Inhibition can also be achieved by introducing a “pseudo-substrate” that mimics the actual substrate but blocks kinase activity.
Cyclosporin:
immunosuppressant indicated against graft rejection after transplantation. bind to an “immunophilin” (cyclophilin for cylcosporin) which then inhibits phosphatase activity of Calcineurin.
(in some cases for acute treatment of auto-immune diseases, i.e. psoriasis, Crohn’s disease) Tacrolimus is the second line of defense.
[Calcineurin appears to allow NFAT to localize to the nucleus to transcribe IL-2]
Sirolimus = rapamycin = rapamune:
immunosuppressant; can be used in combination with additional drugs in case of high risk for calcineurin inhibitor associated nephrotoxicity. Binds to FKBP-12 which then inhibits kinase activity of mTOR (mammalian target of rapamycin)
[Calcineurin appears to allow NFAT to localize to the nucleus to transcribe IL-2]
