Identification of Chchd3 as a Novel Substrate of Pka Using an Analog-sensitive Catalytic Subunit

Address Correspondence to: Susan S. Taylor, Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093-0654, (858) 534-3677, FAX (858) 534-8193, E-mail: [email protected] 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 Nsubstituted ATP analogs, using a chemical genetics approach initially pioneered with vSrc (1). Methionine 120 was mutated to glycine in the ATP binding pocket of the catalytic subunit. To express the stable mutant C-subunit in E. coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr197 and Ser338. Based on its kinetic properties, the engineered C-subunit preferred N(benzyl)ATP and N(phenethyl)-ATP over other ATP analogs, but still retained a 30 μM Km 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. PKA is expressed in all mammalian cells and plays a major role in processes such as metabolic control, memory, growth, development, and apoptosis. Thus, it has many substrates which usually are highly tissue specific. One such example is the bifunctional enzyme phosphofructokinase-2 (PFK2), which upon phosphorylation by PKA is converted to its phosphatase active mode in liver, but is stabilized in its kinase mode in heart, and is not a PKA substrate in skeletal muscle (1). Although PKA has been widely studied in most cells, the direct substrates of PKA are difficult to distinguish from downstream substrates that are phosphorylated as a result of the PKA activation. A possible way to determine whether identified substrates are modified by the kinase directly or indirectly via an intermediary kinase is to engineer the ATP binding pocket of the kinase in question to accept a bulky ATP analog that cannot be used by the wild type kinase. This strategy was developed initially for a tyrosine kinase, v-Src, (2) and has subsequently been used for other protein kinases (3). Shah et al., found that a single mutation conferred specificity for ATP analogs that have a large substituent on the nitrogen at position 6 (N) on the purine ring of ATP (4). Two residues that were found within 5 Å of the N position of ATP were based initially on the structures of PKA and CDK2 (cyclin-dependent kinase 2) since the crystal structure of v-Src was not available at the time (2). In v-Src, these residues are Val323 and Ile338, while the corresponding residues in PKA are Val104 and Met120. Although the Val323 mutation was found to be not important for conferring specificity in v-Src, mutation of Ile338 to Ala did alter the specificity of v-Src and allowed the enzyme to use an orthogonal bulky ATP analog. Once the crystal structure of hematopoietic cell kinase (Hck), a Src family tyrosine kinase was available, molecular modeling was employed to explore more fully the binding pocket of Hck to find the best analog for http://www.jbc.org/cgi/doi/10.1074/jbc.M609221200 The latest version is at JBC Papers in Press. Published on January 22, 2007 as Manuscript M609221200