Identification of Endogenous Substrates for a Protein Kinase from Sulfolobus solfataricus P2 by Screening a Genomic Expression Library

Abstract

Protein phosphorylation and dephosphorylation events are one way cells actuate molecular responses. We are tracing the origins of protein phosphorylation events through the study of the extremophilic archaeon Sulfolobus solfataricus P2. A novel method for identifying potential substrates for protein kinases was developed to facilitate these studies. A genomic library was constructed using a λ-phage vector placing gene expression under lac promoter control. Membrane-bound recombinantly-expressed proteins were incubated with purified kinase, PK4 (ORF sso3182) and [γ]-32P-ATP. Genomic DNA phage inserts expressing 32P-labeled proteins were sequenced, the proteins identified by searching the S. solfataricus genome, and the corresponding ORFs were cloned. Their protein products were expressed, purified, and assayed with PK4 to confirm identity. The protein product of the ORF sso0563, the catalytic A-type ATPase subunit A, was phosphorylated in vitro by PK4. Additional subunits of the ATPase (AtpB, AtpG, AtpE) were also overexpressed and ATPase activity was reconstituted in vitro. Current studies focus on the effects of phosphorylation of the ATPase subunits and the ability of the ATPase complex to hydrolyze ATP. Ultimately studies will be performed in vivo to determine interactions between PK4, ATPase, and phosphorylation effects. This work funded by a grant from the National Science Foundation.