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

Sharmin Schauble,Charles C King,Manjula Darshi,Antonius Koller,Kavita Shah,Susan S Taylor

doi:10.1074/jbc.m609221200

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 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.

Highlights

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
They found that this single mutation opened up a new pocket that was otherwise inaccessible and termed this residue the “gatekeeper.” The best analog, based on the modeling, was found to be nitrogen at position 6 (N6)(phenethyl)ATP and experimentally, N6(phenethyl)-ATP was preferred by the mutant but was a poor substrate for wild type v-Src
Based on its kinetic properties, we demonstrate a clear preference for N6(benzyl)-ATP for protein kinase (PKA)-as1, but not for wild type C-subunit

Summary

Introduction

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. 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 the single mutation [5] They found that this single mutation opened up a new pocket that was otherwise inaccessible and termed this residue the “gatekeeper.” The best analog, based on the modeling, was found to be N6(phenethyl)ATP and experimentally, N6(phenethyl)-ATP was preferred by the mutant but was a poor substrate for wild type v-Src. Substitution of the gatekeeper residue has been applied to find direct substrates of over 30 kinases to date including v-Src [6] and most recently Yersinia PKA (YpkA) [7]. ChChd is endogenously expressed in mitochondria and may play a role in metal binding similar to Cox and Cox19 [10]

Methods

Results

Conclusion