TY - JOUR
T1 - Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling
AU - Li, Renfeng
AU - Liao, Gangling
AU - Nirujogi, Raja Sekhar
AU - Pinto, Sneha M.
AU - Shaw, Patrick G.
AU - Huang, Tai Chung
AU - Wan, Jun
AU - Qian, Jiang
AU - Gowda, Harsha
AU - Wu, Xinyan
AU - Lv, Dong Wen
AU - Zhang, Kun
AU - Manda, Srikanth S.
AU - Pandey, Akhilesh
AU - Hayward, S. Diane
N1 - Funding Information:
This work was supported by NIH K99AI104828/R00AI104828 to RL, NIH R21AI101377 to SDH, NIH R01EY024580 and P30EY001765 to JQ and NIH U54GM103520, U24CA160036 and HHSN268201000032C to AP (http://grants.nih.gov/ grants/oer.htm). The authors acknowledge the joint participation by the Adrienne Helis Malvin Medical Research Foundation and the Diana Helis Henry Medical Research Foundation in conjunction with The Johns Hopkins Hospital and the Johns Hopkins University School of Medicine and the Foundation’s Parkinson’s disease Programs. RL received support from the VCU Philips Institute for Oral Health Research and the VCU NCI Designated Massey Cancer Center (NIH P30 CA016059). RSN is a recipient of Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Publisher Copyright:
© 2015 Li et al.
PY - 2015
Y1 - 2015
N2 - 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).
AB - 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).
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U2 - 10.1371/journal.ppat.1005346
DO - 10.1371/journal.ppat.1005346
M3 - Article
C2 - 26714015
AN - SCOPUS:84953246099
SN - 1553-7366
VL - 11
JO - PLoS Pathogens
JF - PLoS Pathogens
IS - 12
M1 - e1005346
ER -