Kaposi's sarcoma-associated herpesvirus K-bZIP protein is phosphorylated by cyclin-dependent kinases.
The K8 locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites 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 phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.
Polson
A G
AG
Departments of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA.
Huang
L
L
Lukac
D M
DM
Blethrow
J D
JD
Morgan
D O
DO
Burlingame
A L
AL
Ganem
D
D
D000595:Amino Acid Sequence; D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors; D019556:COS Cells; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051790:G-Box Binding Factors; D019288:Herpesvirus 8, Human / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D014775:Virus Activation; D017735:Virus Latency
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D020033:Protein Isoforms; D014157:Transcription Factors; D014764:Viral Proteins; D013912:Threonine; D012694:Serine; D018844:Cyclin-Dependent Kinases