Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45

Abstract

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82, 1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signalregulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcomaassociated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK) 2 mitogenactivated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19,20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20 -22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5,8,13,23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/ threonine kinases that lie at the terminus of the ERK pathway (1, 24 -26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1,24,25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activ...

Phosphorylation of Eukaryotic Translation Initiation Factor 4B (EIF4B) by Open Reading Frame 45/p90 Ribosomal S6 Kinase (ORF45/RSK) Signaling Axis Facilitates Protein Translation during Kaposi Sarcoma-associated Herpesvirus (KSHV) Lytic Replication

“…We further demonstrated that depletion of RSK expression or inhibition of RSK activity reduces KSHV lytic gene expression, indicating that RSK signaling is required for optimal KSHV lytic replication (17,33), but the exact functional roles of the persistently active RSK remain to be determined. To characterize the biological consequence of RSK activation by ORF45, we screened some of the known cellular substrates of RSK by Western blot using phosphorylation-specific antibodies (45,46).…”

“…Plasmid pCR3.1-ORF45 full length and its derivatives have been described previously (17,33). Plasmid pRK7-HA-S6K1 (8984) was purchased from Addgene (Cambridge, MA) (40).…”

“…Retrovirus-based Transduction-The knockdown of gene expression by a retrovirus-based siRNA vector including target sequences for RSK1, RSK2, ERKs, and ORF45 has been described previously (17,33). The BCBL1 cells that stably express eIF4B wild type or S422A mutant were established by standard lentivirus vector protocols (43).…”

“…We further revealed the underlying mechanism and demonstrated that ORF45 forms complexes with RSK and ERK, retains active pRSK and pERK in the complexes, and protects them from dephosphorylation, thus promoting sustained activation of these kinases (33). Moreover, we showed that depletion of RSK expression by siRNA or inhibition of RSK activity by specific inhibitors reduces KSHV lytic gene expression, suggesting that the RSK signaling is required for optimal KSHV lytic replication, but its exact roles remain to be determined (17,33). In the work reported here, we showed that ORF45-mediated sustained activation of ERK/RSK signaling contributes to the phosphorylation of eIF4B and plays a critical role in translation regulation during KSHV lytic infection.…”

“…We recently found that the immediate early and tegument protein open reading frame 45 (ORF45) of KSHV interacts with RSK1 and RSK2 and causes sustained activation of RSK and ERK during lytic replication (17). We further revealed the underlying mechanism and demonstrated that ORF45 forms complexes with RSK and ERK, retains active pRSK and pERK in the complexes, and protects them from dephosphorylation, thus promoting sustained activation of these kinases (33). Moreover, we showed that depletion of RSK expression by siRNA or inhibition of RSK activity by specific inhibitors reduces KSHV lytic gene expression, suggesting that the RSK signaling is required for optimal KSHV lytic replication, but its exact roles remain to be determined (17,33).…”