Fig 1.
RSK1 undergoes SUMOylation during KSHV lytic replication.
(A) RSK1 is highly SUMOylated in cells. HEK293T cells were co-transfected with indicated plasmids and cell lysates were subjected to IP and IB with indicated antibodies. (B) Endogenous RSK1 is SUMOylated. Endogenous RSK1 was immunoprecipitated with anti-RSK1 or IgG (control) antibodies under denature condition from lysates of BJAB, A549, and HeLa cells and the co-immunoprecipitated proteins were analyzed by IB with indicated antibodies. (C) RSK1 is SUMOylated in vitro. Recombinant RSK1 was incubated with SAE1/UBA2 (E1), Ubc9 (E2), SUMO1, and ATP/Mg in SUMO reaction buffer. The SUMOylation of RSK1 were detected by indicated antibodies. (D) RSK1 SUMOylation is increased during KSHV lytic replication. The SLKRSK1/2 DKO-vector, or SLKRSK1/2 DKO-Flag-RSK1, SLK-iBACRSK1/2 DKO-vector, or SLK-iBACRSK1/2 DKO-Flag-RSK1 cells were treated with doxycycline (2 μg/ml) and sodium butyrate (1 mM) to induce KSHV lytic replication. Cell lysates were harvested at indicated time points and subjected to IP under denature condition and IB with indicated antibodies. (E) The endogenous RSK1 SUMOylation is elevated during KSHV lytic replication. SLK-iBAC cells were treated with doxycycline (2 μg/ml) and sodium butyrate (1 mM) to induce KSHV lytic replication. Cell lysates were harvested at indicated time points and subject to IP with anti-RSK1 or control IgG under denature condition and IB with indicated antibodies.
Fig 2.
Identification of RSK1 SUMOylation sites.
(A) Lys110 and Lys335 of RSK1 are two primary SUMOylation sites in RSK1 N-terminal fragments. HEK293T cells were co-transfected with indicated plasmids and cell lysates were subjected to IP and IB with indicated antibodies. (B) Lys110 and Lys335 are two SUMO sites of RSK1. Similar procedure as in Fig 2A. (C) Lys110 and Lys335, and Lys421 are three major SUMO sites of RSK1. Similar procedure as in Fig 2A.
Fig 3.
RSK1 SUMOylation is required for efficient KSHV lytic replication.
(A) Generation of SLK-iBACRSK1/2 DKO, SLK-iBACRSK1/2 DKO-RSK1 or SLK-iBACRSK1/2 DKO-RSK1K110/335/421R cells by CRISPR-Cas9 and lentivirus-mediated stable expression. (B-C) RSK1 SUMOylation affects viral protein expression at both RNA and protein levels during KSHV lytic replication. Indicated cell lines were treated with doxycycline (2 μg/ml) and sodium butyrate (1 mM) to induce KSHV lytic replication. Cell lysates were collected at indicated time points and subjected to IB with indicated antibodies (B). Indicated SLK-iBAC cell lines were induced with doxycycline and sodium butyrate for 72 h. Total RNA was extracted, reverse-transcribed into cDNA, and used for KSHV whole-genome qPCR array analysis. The ΔCT values for each primer set were calculated and converted to a heatmap using R (C). (D-E) RSK1 SUMOylation is required for efficient viral genomic DNA replication (D) as well as progeny virus production (E) during KSHV lytic replication. The cell lysates or culture medium containing progeny viruses were collected at indicated time point. Total DNA was isolated and viral genomic DNA was quantified by qPCR. (F-G) Generation of iSLK-BAC16RSK1/2 DKO, iSLK-BAC16RSK1/2 DKO-RSK1, iSLK-BAC16RSK1/2 DKO-RSK1K110/335/421R, BCBL1RSK1/2 DKO, BCBL1RSK1/2 DKO-RSK1, BCBL1RSK1/2 DKO-RSK1K110/335/421R cells by CRISPR-Cas9 and lentivirus-mediated stable expression. (H-I) RSK1 SUMOylation affects viral protein expression during KSHV lytic replication. iSLK-BAC16 related cell lines (H) were treated with doxycycline (2 μg/ml) and sodium butyrate (1 mM) and BCBL1 related cell lines (I) were treated with TPA (20 ng/ml) to induce KSHV lytic replication. RNA samples were collected at 72 h post-induction and subjected to RT-qPCR analysis for overall KSHV gene expression. Heatmaps were generated using similar method as Fig 3C.
Fig 4.
RSK1 SUMOylation does not alter its activation or kinase activity.
(A) SUMOylation site mutations of RSK1 do not change ERK-RSK-ORF45 complex formation. HEK293T cells were co-transfected with indicated plasmids and cell lysates were subjected to IP and IB with indicated antibodies. (B) SUMOylation site mutations do not affect RSK1 phoshporylation by KSHV ORF45. SLKRSK1/2 DKO-vector, SLKRSK1/2 DKO-RSK1 and SLKRSK1/2 DKO-RSK1K110/335/421R cells were infected with lentiviruses containing empty vector, ORF45WT or ORF45F66A as indicated. The cells were serum-starved for 24h before sample collection and cell lysates were analyzed by IB with indicated antibodies at 48 h post-transfection. (C) SUMOylation site mutations do not affect RSK1 kinase activity in vitro (Methods section). RSK1 kinase complexes with or without ORF45 were purified from HEK293A cells transfected with indicated plasmid. GST-S6 peptide was purified from E.coli. RSK1 (5 μl) were mixed with GST-S6 (2.5 μg) in kinase assay buffer at 30 °C for 30 min. The reaction was stopped by adding 2 x SDS-PAGE loading buffer, followed by IB analysis with indicated antibodies.
Fig 5.
RSK1 SUMOylation affects the phosphorylation of its downstream substrates.
(A) SUMOylation of RSK1 contributes to the overall phosphorylation of its substrates during KSHV lytic replication. SLKRSK1/2 DKO-vector, SLKRSK1/2 DKO-RSK1, SLKRSK1/2 DKO-RSK1K110/335/421R, SLK-iBACRSK1/2 DKO-vector, SLK-iBACRSK1/2 DKO-RSK1, or SLK-iBACRSK1/2 DKO-RSK1K110/335/421R cells were induced with doxycycline (2 μg/ml) and sodium butyrate (1 mM) and cell lysates were subjected to IB with indicated antibodies. (B) SUMOylation of RSK1 affects the overall phosphorylation of RSK1 substrates in the presence of KSHV ORF45. SLK-vector, SLKRSK1/2 DKO-vector, SLKRSK1/2 DKO-RSK1 and SLKRSK1/2 DKO-RSK1K110/335/421R cells were infected with lentiviruses containing empty vector, ORF45WT or ORF45F66A as indicated. The cells were serum-starved for 24 h before sample collection and cell lysates were analyzed by IB with indicated antibodies at 48 h post-transfection. (C) SUMOylation site mutations affect eIF4B phosphorylation by RSK1 in vitro. RSK1 kinase complexes with or without ORF45 were purified from HEK293A cells transfected with indicated plasmid. HA-eIF4B was purified from transfected HEK293T cells by affinity purification. RSK1 (5 μl) were mixed with eIF4B (0.5 μg) in kinase assay buffer at 30 °C for 30 min. The reaction was stopped by adding 2 x SDS-PAGE loading buffer, followed by IB analysis with indicated antibodies.
Fig 6.
SUMO-SIM interaction contributes to RSK1-eIF4B association.
(A) SUMOylation of RSK1 is required for efficient interaction between RSK1 and eIF4B. HEK293T cells were co-transfected with indicated plasmids and cell lysates were subjected to IP and IB with indicated antibodies at 48 h post-transfection. (B) Alignment of SIMs among eIF4B, PIAS1, PIAS2, PIAS3, and TTRAP. (C) eIF4B SIM binds to endogenous SUMO1 and RSK1. GST vector control and GST-tagged eIF4B SIM or its mutant were purified from E.coli and subjected to GST pull-down assay with HEK293T cell lysates. The co-precipitated proteins were analyzed by IB with indicated antibodies. (D) SIM mutation within eIF4B reduced the RSK1-eIF4B association. HEK293T cells were co-transfected with indicated plasmids and cell lysates were subjected to IP and IB with indicated antibodies at 48 h post-transfection.