Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

PLoS Pathogens

Sneha M. Pinto¹  Renfeng Li¹  Tai-Chung Huang²  Kun Zhang²  Jun Wan²  Raja Sekhar Nirujogi³  Dong-Wen Lv³  Harsha Gowda⁴  Patrick G. Shaw⁴  Srikanth S. Manda⁴  Jiang Qian⁴  Akhilesh Pandey⁵  Gangling Liao⁵  S. Diane Hayward⁵  Xinyan Wu⁶ 

[1] Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America;Institute of Bioinformatics, International Technology Park, Bangalore, India;Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of America;McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America;Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, United States of America;Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America

关键词: Phosphorylation;    Protein kinases;    Viral replication;    DNA damage;    Epstein-Barr virus;    Protein kinase signaling cascade;    Amino acid analysis;    Mitosis;   

DOI  :  10.1371/journal.ppat.1005346

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).

【 授权许可】

CC BY