Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Abstract

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 M 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.The cAMP-dependent protein kinase (PKA) 3 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. (4) found that a single mutation conferred specificity for ATP analogs that have a large substituent on the nitrogen at position 6 (N 6 ) on the purine ring of ATP. Two residues that were found within 5 Å of the N 6 position of ATP were based initially on the structures of PKA and CDK2 (cyclindependent kinase 2) since the crystal structure of v-Src was not available at the time (2). In v-Src, these residues are Val 323 and Ile 338 , while the corresponding residues in PKA are Val 104 and Met 120 . Although the Val 323 mutation was found to be not important for conferring specificity in v-Src, mutation of Ile 338 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 t...

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

“…6A). One prominent mitochondrial PKA substrate detected in this screen is the coiled-coil helix protein ChChd3, a peripheral component of the intermembrane space that is essential for maintaining cristae integrity (42,43). This tally's with evidence that SKIP partitions to intermembrane space and matrix subfractions prepared from murine heart mitochondria (Fig.…”

Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation

“…2E) indicate that testis from C␣M120A mutant mice contained reduced amounts of C␣ protein compared to wild-type testis, suggesting that the lower PKA activity observed in C␣M120A mutants is the result of a decrease in the level of the mutant protein. The ATP binding-site mutation that we created lowers the affinity of C␣ for ATP (12), and it is likely that diminished ATP binding interferes with assembly of type I PKA holoenzyme and the consequent stabilization of the C␣ subunit. However, we cannot rule out changes in the level of mutant C␣ mRNA.…”

“…In JEG-3 cells transfected with C␣M120A, the IC 50 for 1NM-PP1 was Ϸ150 nM. The mutant C␣M120A also has been stably expressed in Escherichia coli to provide a tool to identify direct targets of PKA in vitro (12). We have now created a ''knockin'' mouse that can be switched from expressing a wild-type C␣ to the mutant C␣M120A by the action of Cre recombinase.…”

Cross-Talk Between Mitochondrial Fusion and the Hippo Pathway in Controlling Cell Proliferation DuringDrosophilaDevelopment

“…ChChd3 is a mitochondrial inner membrane protein and its mutation affects mitochondrial morphology ChChd3 encodes a highly conserved protein predicted to be a mitochondrial inner membrane protein according to the role of its mammalian homolog (Schauble et al 2007;Darshi et al 2011). It contains a coiled-coil-helix-coiled-coilhelix (CHCH) domain at its C terminus ( Figure 3A).…”