Direct Substrate Identification with an Analog Sensitive (AS) Viral Cyclin-Dependent Kinase (v-Cdk)

Viral cyclin-dependent
kinases (v-Cdks) functionally emulate their
cellular Cdk counterparts. Such viral mimicry is an established phenomenon
that we extend here through chemical genetics. Kinases contain gatekeeper
residues that limit the size of molecules that can be accommodated
within the enzyme active site. Mutating gatekeeper residues to smaller
amino acids allows larger molecules access to the active site. Such
mutants can utilize bio-orthoganol ATPs for phosphate transfer and
are inhibited by compounds ineffective against the wild type protein,
and thus are referred to as analog-sensitive (AS) kinases. We identified
the gatekeeper residues of the v-Cdks encoded by Epstein–Barr
virus (EBV) and human cytomegalovirus (HCMV) and mutated them to generate
AS kinases. The AS-v-Cdks are functional and utilize different ATP
derivatives with a specificity closely matching their cellular ortholog,
AS-Cdk2. The AS derivative of the EBV v-Cdk was used to transfer a
thiolated phosphate group to targeted proteins which were then purified
through covalent capture and identified by mass spectrometry. Pathway
analysis of these newly identified direct substrates of the EBV v-Cdk
extends the potential influence of this kinase into all stages of
gene expression (transcription, splicing, mRNA export, and translation).
Our work demonstrates the biochemical similarity of the cellular and
viral Cdks, as well as the utility of AS v-Cdks for substrate identification
to increase our understanding of both viral infections and Cdk biology.