shokat mutation presentation
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PPTTRANSCRIPT
Shokat Mutation
Akanksha Singh & Chloe Chabanon-Hicks
Kinases in Action!
Amplification
The Spark of Kinase Research
The Cori’s (1930)
1st Observation of Phosphorylation Event
Eugene P. Kennedy (1954)
Kinase Transfer of y-Phosphate to Substrate
Edmund and Fisher ( 1959)
The Big Question
Past Methods:
Disrupting enzyme 3 and observe corresponding phenotype.
BUT …
Enzyme 3 is affected by enzymes 1&2 and affects enzyme 4, the result of disrupting the gene coding for enzyme 3 may generate the same phenotype as disrupting the gene that encodes for enzymes 1,2,or 4. THEN, specific kinase function could not be
Verified because kinase disruption was not reversible
If we wanted to study the role of kinase 3…
Kevan Shokat 2000
“I kept asking him, ‘How does this experiment tell you which kinase is involved? There’s some missing tool here. We need a better way of telling which precise kinase is working”.
Exploitation of the Conserved ATP Binding Cassette
The Shokat mutation works by mutating the single active site residue in the ATP binding site of a kinase to a less bulky amino acid like glycine, known as the “gatekeeper residue”
Identifying the Kinase of InterestThis mutation creates a deeper pocket to accept the corresponding artificially created substrate ATP analog
What You Would Expect to Find in the ATP Binding Site…
BUT INSTEAD…
AS SEEN ON ATP Inhibitor Analogs Analogs can not phosphorylate substrates
Identifying Substrates of InterestAn inhibitory molecule is engineered with a bump, interacting with a higher affinity to the modified kinase ATP binding site than ATP, thus preventing kinase activity.
Two Methods: [γ-32P]-labeled ATP analog,
autoradiography
mass spectrometry
Contains a N6-(benzyl) group creating a “binding bump”
ATP analog engineered with phosphorothiolate (PO3S–)
Substrae
PO3S–
Substrae
PO3S–
Substrae
PO3S–
Substrae
PO3S–
Immunoprecipation
Observation of Kinase- Substrate Localization
Antibodies with fluorescent tags
Anti-Breast Tumor Kinase antibody
What the Kinase Sequence Database has to offer…
currently contains 7,128 protein kinases, observed from 948 different organisms.
FLASH CARDThe Shokat mutation works by mutating the single active site residue in the ATP binding site of a kinase to a less bulky amino acid like glycine, known as the “gatekeeper residue”