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

Abstract

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.

Conserved Herpesvirus Protein Kinases Target SAMHD1 to Facilitate Virus Replication

“…Although CHPKs from beta-and gamma-herpesviruses are structurally similar to the cellular cyclin-dependent kinases 1/2 (CDK1 and CDK2) (Kuny et al, 2010;Romaker et al, 2006), recent studies from our lab and others suggest that these CHPKs have a broader substrate recognition than the cellular ortholog CDKs (Li et al, 2011(Li et al, , 2015Oberstein et al, 2015;Umañ a et al, 2018;Zhu et al, 2009). The mimicry of CDK activity by CHPKs results in the phosphorylation of cell-cycle-related proteins to create a pseudo-S-phase environment suitable for efficient viral DNA replication (Chen et al, 2010;Hume et al, 2008;Iwahori et al, 2009Iwahori et al, , 2015Kawaguchi and Kato, 2003;Kudoh et al, 2006;Kuny et al, 2010;Lee et al, 2007).…”

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis

“…Systems-level approaches have provided important insights into the molecular functions of CHPKs. For example, yeast 2-hybrid screens revealed several binary binding partners of CHPKs 27,28 and phosphoproteome profiling was successfully used to assess CHPK substrate specificity 7,12,29,30 . However, comprehensive and comparative information about CHPK interaction partners at the proteome level is lacking.…”

Mass Spectrometry-Based Discovery of <i>in vitro</i> Kinome Substrates

“…e introduction of a chemical tag using γ-thiophosphate derivatives of the ATP analog was also utilized to enrich substrate proteins of a target analog-sensitive mutant. [48][49][50] ATP analog-sensitive kinase-based approaches are very powerful tools for reducing the e ects of other endogenous kinases and accurately identifying positive kinase substrates in both cases; however, this strategy is applicable only if analog-sensitive kinases are available. Furthermore, wildtype kinases may also utilize the ATP analog as a phosphate source to some extent.…”