Kaposi's sarcoma-associated herpesvirus K-bZIP protein is phosphorylated by cyclin-dependent kinases

The KS locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three altern atively spliced transcripts. The fully spliced transcript encodes K-bZIP, t he KSHV homologue of the EBV immediate-early transcriptional transactivator Z, Here we show that despite the presence of alternatively spliced transcr ipts, the protein from the fully spliced RNA, K-bZIP, is the principal prod uct detectable in KSHV-infected B cells, The protein is detected only in ly tically infected cells and is focalized to the nucleus. We further characte rized K-bZIP by determining its phosphorylation status. Phosphoamino acid a nalysis revealed phosphorylation on serine and threonine, Analysis of the s ites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K- bZIP was phosphorylated on Thr 111 and Ser 167, These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphoryl ated by CDKs, We tested this hypothesis using an in vitro kinase reaction p erformed in whole-cell extracts that resemble in vivo conditions more close ly than standard in vitro kinase reactions. We found that the three CDK-cyc lin complexes we tested phosphorylated K-bZIP but not the control ORF 73 pr otein, which contains four (S/T)PXR sites, Ectopic expression of K-bZIP can not reactivate KSHV from latency, and single and double mutants of K-bZIP i n which alanines replaced the phosphorylated serine and/or threonine also f ailed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the pro tein.