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An ATP-analog sensitive mutant to identify direct substracte of cAMP-dependent protein kinase

Sharmin Schauble

Appointment Period: 2002-2004, Grant Years: [18,19]

Sharmin SchaubleFinding substrates of cyclic-AMP dependent protein kinase (PKA) at the mitochondria will help define mechanisms for the onset of cancer. PKA is implicated in breast and prostate cancers, but is also found to help protect against cancer by stimulating apoptosis. Misregulation of programmed cell death, or apoptosis, is one of the steps leading to all types of cancer. PKA is localized to the mitochondria through A-kinase anchoring proteins and it is known to stimulate and inhibit apoptosis. The goal of my research was to identify novel, undiscovered substrates of PKA in the mitochondria. I have mutated the catalytic subunit of PKA to allow it to accept N6-substituted ATP analogs, based on work done with v-Src. Mutating methionine-120 to glycine in the catalytic subunit of PKA opens up a hydrophobic pocket in the ATP-binding pocket so that the kinase preferentially accepts N6-(benzyl)ATP. By engineering the kinase to allow it to accept ATP analogs, the mutant can transfer a radiolabeled gamma-phosphate of the ATP analog to its substrate. These phosphorylated substrates can then be separated by one- or two-dimensional gel electrophoresis, and detected by autoradiography. We are interested in possible substrates of PKA in the mitochondria, because PKA is localized to the mitochondria through A-Kinase Anchoring Proteins (AKAPs). Using this modified catalytic subunit of PKA that accepts ATP analogs, I have identified possible novel substrates. This was accomplished using assays consisting of radiolabeled ATP analogs, mouse brain mitochondria, and purified engineered catalytic subunit. Labeled proteins were removed from two-dimensional gels and subjected to identification through mass spectrometry. One of the novel substrates is a completely uncharacterized protein which may be involved in oxidative phosphorylation, and is abundant in the mitochondria. By elucidating the pathways within the cells we will be able to define the specific role that PKA plays in either enhancing or preventing the onset of cancer.

Schauble S, King CC, Darshi M, Koller A, Shah K, Taylor SS. Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit. J Biol Chem. (2007) 282:14952-9. PMID: 17242405.