Abstract
RSK1, a downstream kinase of the MAPK pathway, has been shown to regulate multiple cellular processes and is essential for lytic replication of a variety of viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. We demonstrate that RSK1 undergoes robust SUMOylation during KSHV lytic replication at lysine residues K110, K335, and K421. SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Compared to wild-type RSK1, the overall phosphorylation level of RxRxxS*/T* motif is significantly declined in RSK1K110/335/421R expressing cells. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B. Sequence analysis showed that eIF4B has one SUMO-interacting motif (SIM) between the amino acid position 166 and 170 (166IRVDV170), which mediates the association between eIF4B and RSK1 through SUMO-SIM interaction. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication.
Highlights
SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B since small ubiquitinlike modifiers (SUMOs)-SUMO-interacting motif (SIM) interaction mediates the association between eIF4B and RSK1. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication
We previously demonstrated that the ORF45, a tegument as well as immediate-early protein of KSHV, interacts with and persistently activates cellular p90 ribosomal S6 kinases (RSKs), which is critical for KSHV lytic replication and progeny virus production [3,4]
RSK1 SUMOylation is enhanced during KSHV lytic replication
Summary
Antibodies and chemicalsAnti-RxRxxS /T (#10001S), RSK1 (#9333S), RSK2 (#5528S), p-eIF4B (#3591S), eIF4B (#3592S) antibodies were ordered from Cell Signaling Technology. HRP anti-HA (#901519), HRP anti-Flag (#637311) antibodies were purchased from BioLegend. PRSK1 (Thr359/Ser363) (#AP0539), pRSK1 (Ser380) (#AP1147), RSK3 (#A16305), ERK1/2 (#A10613), and SUMO1 (#A19121) were purchased from ABclonal. Anti-RTA (ORF50) monoclonal mouse antibody was given by Dr Ke Lan (Wuhan University, China) [34]. Anti-ORF65 monoclonal mouse antibody was given by Dr Shou-Jiang Gao (University of Pittsburgh, USA) [35]. SUMOylation of RSK1 regulates KSHV lytic replication antibodies against ORF45 and ORF52 and polyclonal antibodies against K3, K8, and ORF55 were described previously [36,37,38]. EZview Red ANTI-FLAG M2 Affinity Gel (#F2426) and sodium butyrate (#B5887) were ordered from Sigma, Doxycycline (#S4163) was ordered from Selleck, Glutathione Sepharose 4B (#17-0756-01) were ordered from GE Healthcare.
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