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1: J Biol Chem. 2007 May 18;282(20):14952-9. Epub 2007 Jan 22.
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Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit.

Schauble S, King CC, Darshi M, Koller A, Shah K, Taylor SS.

Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0654, USA.

Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.

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PMID: 17242405 [PubMed - indexed for MEDLINE]