Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellularsignaling machineries, including the mitogen-activated protein-kinase pathway,during their infections. We have demonstrated previously that the open readingframe 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90ribosomal 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 themechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45to RSK increases the association of extracellular signal-regulated kinase(ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular massprotein complexes. We further demonstrated that the complexes shielded activepERK and pRSK from dephosphorylation. As a result, the complex-associated RSKand ERK were activated and sustained at high levels. Finally, we provideevidence that this mechanism contributes to the sustained activation of ERKand RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase(ERK)²mitogen-activated protein kinase (MAPK) signaling pathway has been implicatedin diverse cellular physiological processes including proliferation, survival,growth, differentiation, and motility(1-4)and is also exploited by a variety of viruses such as Kaposisarcoma-associated herpesvirus (KSHV), human cytomegalovirus, humanimmunodeficiency virus, respiratory syncytial virus, hepatitis B virus,coxsackie, vaccinia, coronavirus, and influenza virus(5-17).The MAPK kinases relay the extracellular signaling through sequentialphosphorylation to an array of cytoplasmic and nuclear substrates to elicitspecific responses (1,2,18). Phosphorylation of MAPKis reversible. The kinetics of deactivation or duration of signaling dictatesdiverse biological outcomes(19,20). For example, sustainedbut not transient activation of ERK signaling induces the differentiation ofPC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells(20-22).During viral infection, a unique biphasic ERK activation has been observed forsome viruses (an early transient activation triggered by viral binding orentry and a late sustained activation correlated with viral gene expression),but the responsible viral factors and underlying mechanism for the sustainedERK activation remain largely unknown(5,8,13,23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threoninekinases that lie at the terminus of the ERK pathway(1,24-26).In mammals, four isoforms are known, RSK1 to RSK4. Each one has twocatalytically functional kinase domains, the N-terminal kinase domain (NTKD)and C-terminal kinase domain (CTKD) as well as a linker region between thetwo. The NTKD is responsible for phosphorylation of exogenous substrates, andthe CTKD and linker region regulate RSK activation(1,24,25). In quiescent cells ERKbinds to the docking site in the C terminus of RSK(27-29).Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase(MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linkerregion (amino acid numbers refer to human RSK1) and Thr-573 in the CTKDactivation loop. The activated CTKD then phosphorylates Ser-380 in the linkerregion, creating a docking site for 3-phosphoinositide-dependent proteinkinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylatesSer-221 of RSK in the activation loop and activates the NTKD. The activatedNTKD autophosphorylates the serine residue near the ERK docking site, causinga transient dissociation of active ERK from RSK(25,26,28). The stimulation ofquiescent cells by a mitogen such as epidermal growth factor or a phorbolester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usuallyresults in a transient RSK activation that lasts less than 30 min. RSKs havebeen implicated in regulating cell survival, growth, and proliferation.Mutation or aberrant expression of RSK has been implicated in several humandiseases including Coffin-Lowry syndrome and prostate and breast cancers(1,24,25,30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma,primary effusion lymphoma, and a subset of multicentric Castleman disease(33,34). Infection andreactivation of KSHV activate multiple MAPK pathways(6,12,35). Noticeably, the ERK/RSKactivation is sustained late during KSHV primary infection and reactivationfrom latency (5,6,12,23), but the mechanism of thesustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45,an immediate early and also virion tegument protein of KSHV, interacts withRSK1 and RSK2 and strongly stimulates their kinase activities(23). We also demonstratedthat the activation of RSK plays an essential role in KSHV lytic replication(23). In the present study wedetermined the mechanism of ORF45-induced sustained ERK/RSK activation. Wefound that ORF45 increases the association of RSK with ERK and protects themfrom dephosphorylation, causing sustained activation of both ERK and RSK.