UC San Diego

Protein kinases regulate numerous activities in eukaryotic cells by phosphorylation of substrates. Cyclic-AMP- dependent protein kinase (PKA) is one of the most important and well studied of these protein kinases. In its inactive form, this kinase exists as a tetrameric protein with a regulatory (R) subunit dimer bound to two catalytic (C) subunits. PKA is activated when the second- messenger cAMP binds cooperatively to the regulatory subunits of PKA. Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates. Our lab is particularly interested in PKA substrates at the mitochondria because of the discovery of two Dual A-Kinase Anchoring Proteins (AKAPs). The goal of this dissertation is to engineer the PKA catalytic subunit to accept bulky N6-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (Shah, Liu et al. 1997), and to identify novel substrates at the mitochondria. Methionine 120 was mutated to alanine in the ATP binding pocket of the catalytic subunit. The mutant C-subunit had to be expressed with PDK1 in order to be soluble and phosphorylated. The mutant preferred N6(benzyl)-ATP and N6(phenethyl)-ATP over other analogs, according to results obtained through qualitative and kinetic analyses. Mitochondria isolated from mouse brain were used in an in vitro assay with recombinant M120G C-subunit and radiolabeled N6(benzyl)-ATP in order to find novel direct substrates. Two-dimensional gel electrophoresis was used to separate proteins. From one of the phosphorylated spots, ChChd3 was identified using mass spectrometry and subsequently characterized. ChChd3 has three motifs/ domains of interest: a myristylation motif, where a highly conserved PKA phosphorylation site is located; a DUF737 domain, which has an unknown function; and a CHCH domain, which is characterized by a Cys-X9-Cys motif. The myristylation motif is not sufficient to target the protein to the mitochondria, however, the CHCH domain is necessary for mitochondrial localization. We propose that ChChd3 uses a mitochondrial import mechanism involving Mia40 and Erv1 where the protein is disulfide-bonded and folded once in the inner mitochondrial space. PKA phosphorylation on the N-terminus of ChChd3 may regulate targeting of the protein to membranes