p90 ribosomal s6 kinases- eclectic members of the human kinome

Signal Transduction 2007, 7, 225 – 239 K. Y. Lee et al. 225

Review Article

p90 Ribosomal S6 kinases- eclectic members ofthe human kinome

Kwok Y. Lee, Paola A. Bignone, Trivadi S. Ganesan*

Cancer Research UK, Molecular Oncology Laboratories, Ovarian Cancer Group. Weatherall Institute ofMolecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK

The p90 ribosomal S6 kinase (RPS6KA1-6; RSK1-6) family represents an important family of con-served serine-threonine kinases among the higher eukaryotes. RSKs are identified by the pre-sence of two non-identical and active kinase domains that is unique amongst the kinome. Thesekinases are involved in a multitude of essential processes in the cell, ranging from apoptosis andtranscriptional regulation and activation of immediate early genes to the maintenance of thecell cycle and growth. They are also important in the regulation of the cell cycle in G2, meiosis Iand II. The mutation and loss of catalytic activity of RSK2 causes Coffin-Lowry syndrome. Thisreview provides a concise and focused perspective on the current understanding of the functionof RSKs and their substrates.

Keywords: RSK / signal transduction /

Received: April 19, 2006; accepted: July 7, 2006

DOI 10.1002/sita.200600091

Introduction

The mitogen activated protein kinase (MAPK) cascadeplays an essential role in conveying signals from recep-tors on the cell surface to the nucleus. The cascade con-sists of three distinct parts: the MAPKs, the MAPK kinases

(MAPKKs, MKKs or MEKs) and the MAPKK kinases(MAPKKK or MEKK). The transfer of signal occurs throughphosphorylation of each MAPK in succession, MAPKKKfiMAPKKfiMAPK. The MAPK is able to phosphorylatemany different targets and therefore able to produce arange of variable cellular outcomes. Evolutionarily con-served, from yeast to mammals, the MAPKs are involvedin a critical number of processes such as cell prolifera-tion, differentiation, cell cycle, growth and apoptosis.The three most well-studied subfamilies of MAPKs are,the extracellular signal-related kinases 1 and 2 (ERK1/2),the p38 MAP kinases and the Jun amino-terminal kinases/stress-activated protein kinases (JNK/SAPK). Whereasmitogenic stimuli activate the ERK pathway, the JNK andp38 proteins are activated by cellular stresses including,UV radiation, oxidative stress and cytotoxic drugs. TheERK1/2 pathway is the best characterised of the MAPKpathways [1]. Like all the MAPKs, ERK1/2 has the ability totranslocate into the nucleus and phosphorylate transcrip-tion factors (TF) and activate the immediate early genes.

Correspondence: Dr. Trivadi S. Ganesan, Cancer Research UK, Mole-cular Oncology Laboratories, Ovarian Cancer Group. Weatherall Instituteof Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX39DS, UK.E-mail: [email protected]: +44 (0)1865 222431

Abbreviations: APC, anaphase promoting complex; CaMK, Ca2+/Calmo-dulin dependent protein kinase; CBP, CREB-binding protein; CLS, Cof-fin-Lowry syndrome; CREB, cAMP response element-binding protein;CSF, cytostatic factor; ER, estrogen receptor; ERK, extracellular signalrelated kinase; FGF, fibroblast growth factor; GSK3, glycogen synthasekinase 3; IGF-1, insulin-like growth factor-1; IjB, inhibitor of NFjB; IKK,IjB phosphorylating kinase; IL-3, interleukin 3; JNK/SAPK, jun amino-terminal kinase/stress-activated protein kinase; MAPK, mitogen-activatedprotein kinase; MEK, MAPK/ERK kinase; MPF, maturation promoting fac-tor; MSK, mitogen and stress-activated protein kinase; mTOR, mamma-lian target of rapamycin; NFkB, nuclear factor jB; NHE-1, Na+/H+ exchan-ger isoform-1; PDK1, 3-phosphoinositide-dependent protein kinase-1;PJS, Peutz-Jeghers syndrome; PKA, protein kinase A; PKC, protein ki-nase C; Plx1, polo-like kinase 1; RPS6KA1-6, RSK1-6, p90 ribosomal S6kinase isoforms 1 to 6; TF, transcription factor; TPA, 12-O-tetradecanoy-phorbol-13-acetate; TSC, tuberous sclerosis complex; wt, wild type

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.signaltrans.com

*Current address: Chairman, Cancer Institute and Institute of MolecularMedicine, Amrita Institute of Medical Sciences and Research Centre, Ela-makkara P.O., Cochin 682026, IndiaE-mail: [email protected]

226 K. Y. Lee et al. Signal Transduction 2007, 7, 225 –239

In addition to controlling gene expression by activatingnuclear TFs, ERK1/2 can also phosphorylate targets in thecytoplasm. An important family of cytoplasmic targetsactivated by the MAPKs are the p90 ribosomal protein S6kinase alphas (RPS6KAs).

Originally called p90rsk or RSK, the protein name wasderived from its molecular weight and the reported abil-ity to phosphorylate the 40S ribosomal subunit S6 inXenopus laevis [2]. In subsequent experiments however, itwas discovered that other kinases (p70S6K) were the majorphysiological kinases [3] of the 40S ribosomal unit S6.Like the MAPKs, the RSK proteins can phosphorylate tar-gets in the cytoplasm and nucleus (Fig. 1), and in thisreview we focus on RSK1-4 (RPS6KA1-3 and 6) and theirinvolvement in cell survival, cell cycle arrest, transcrip-tional regulation and in multiple diseases such as Coffin-Lowry Syndrome and X-linked mental retardation.

The family of RSKs

The RPS6KAs comprise 6 family members, RPS6KA1-6(current HUGO nomenclature) with varying synonyms(Table 1). These names reflect which RPS6KAs were firstdiscovered (HU-1 to 3 corresponds to the first humanRPS6KAs identified) and the varying homology and waysof activation between RPS6KAs. RSK1-4 are largely acti-vated by ERK1 and 2 and are more closely related to eachother than the mitogen-stress activated kinases, MSK1and MSK2, which are quite distinct kinases with differentsubstrate specificities.

The RPS6KAs share a unique feature, the presence oftwo non-identical and catalytically active kinase domainsseparated by a linker region. The RPS6KA family is notalone in having dual kinase domains as the four JAKkinases and GCN2 also have two domains, however their


Figure 1. Summary of RSK1-4 interactions. RSKs proteins are activated mainly by MAPK, but RSK3 also receives input from theJNK pathway. The kinases are involved in a multitude of essential processes in the cell, ranging from apoptosis and transcriptionalregulation and activation of immediate early genes to the maintenance of the cell cycle and growth, with their involvement of regula-tion of key players in G2, meiosis I and II.

Signal Transduction 2007, 7, 225 – 239 p90 Ribosomal S6 kinases 227

additional kinase domains are catalytically inactive [4].The NH2-terminal kinase domain of the RPS6KAs shareshomology with the cyclic AMP catalytic domain, whilethe COOH-terminal kinase domain is similar to that ofthe calcium/calmodulin-dependent kinase family(CaMK). The NH2-terminal kinase domain is responsiblefor substrate phosphorylation and the COOH-terminalkinase domain mediates signals from MAPKs and is alsorequired for the complete activation of the NH2-terminalkinase domain. Both domains contain the conserved ATP-binding sites. The total activation of RPS6KAs requiresthe phosphorylation of the COOH-terminal domain byMAPKs and the binding and subsequent phosphorylationof the NH2-terminal domain by PDK1, concomitant withautophosphorylation by the RSK COOH-terminal domain[5]. The current model for RSK phosphorylation is thatERK initially phosphorylates Ser573 (RPS6KA1 accessionnumber NP_002944) in the COOH-terminal activationloop and Ser363 in the linker region. This activates theCOOH-terminal which then, autophosphorylates Ser380,creating a docking site for PDK1. The full activation ofRSK [5] is affected by synergistic action of phosphoryla-

tion of Ser221 and Ser380 (Fig. 2). More recently it hasbeen shown that the muscle A-kinase anchoring protein(mAKAP) provides a platform for assembly and recruit-ment of PDK1 and the ERK/RSK complex and release ofRSK for phosphorylation of its substrates [6].

The closely related RPS6KAs (RSK1-4) share a 72–82%amino acid identity with each other and only 39–42%with MSK1 and 2 (with 61% identity between them).RPS6KA6 (RSK4) was the last member of the RPS6KA familyto be identified and shares the greatest amino acid iden-tity with the first three RPS6KAs (72–75%) and just 39–44% identity to the MSKs.

Although the RSKs1-4 share a close identity, they can-not compensate for the loss of each other as the mutationof RSK2 leads to the disease Coffin-Lowry syndrome [7, 8].

Knockout RSK

RSK2 – / – mice [7] have been found to be 14% shorter and10% lighter than wildtype (wt) mice and have impairedlearning and coordination. The RSK2 mutant mouse


Table 1. Nomenclature of RSKs.

Gene Synonyms Swiss-Prot Designation

RSK1(1p36.11)

HU-1 (LL, GDB),MAPKAPK1A (LL),RPS6KA1(LL, GDB),ribosomal protein S6 kinase, 90kDa, polypeptide 1(GDB, HUGO)

Ribosomal protein S6 kinase alpha 1 [(S6K-alpha 1)(90 kDa ribosomal protein S6)Kinase 1) (p90-RSK 1) (Ribosomal S6 kinase 1) (RSK-1)(pp90RSK1)].

RSK2(Xp22.12)

HU-3 (LL, GDB),ISPK-1 (LL),MAPKAPK1B (LL),RPS6KA3 (LL, GDB),ribosomal protein S6 kinase, 90kDa, polypeptide 3(GDB, HUGO)

Ribosomal protein S6 kinase alpha 3 [(S6K-alpha 3)(90 kDa ribosomal protein S6)kinase 3) (p90-RSK 3) (Ribosomal S6 kinase 2) (RSK-2)(pp90RSK2) (Insulin-stimulated proteinkinase 1) (ISPK-1)].

RSK3(6q27)

HU-2 (LL, GDB),MAPKAPK1C (LL),RPS6KA2 (LL, GDB),ribosomal protein S6 kinase, 90kDa, polypeptide 2(GDB, HUGO)

Ribosomal protein S6 kinase alpha 2 [(S6K-alpha 2)(90 kDa ribosomal protein S6)Kinase 2) (p90-RSK 2) (Ribosomal S6 kinase 3) (RSK-3)(pp90RSK3)].

RSK4(Xq21.1)

RPS6KA6 (LL, GDB)Ribosomal protein S6 kinase, 90kDa, polypeptide 6(GDB, HUGO)

Ribosomal protein S6 kinase alpha 6 [(S6K-alpha 6)(90 kDa ribosomal protein S6 kinase 6) (p90-RSK 6)(Ribosomal S6 kinase 4) (RSK-4) (pp90RSK4)].

MSK1(14q32.11)

RPS6KA5 (LL, GDB)RLPK (LL, GDB)mitogen- and stress-activated protein kinase 1 (LL)ribosomal protein S6 kinase, 90kDa, polypeptide 5(GDB, HUGO)

Ribosomal protein S6 kinase alpha 5 [(Nuclear mito-gen-and stress-activated protein kinase-1) (90 kDa ri-bosomal protein S6 kinase 5) (RSK-like protein ki-nase) (RLSK)].

MSK2(11q13.1)

RPS6KA4 (LL, GDB)RSK-B (LL, GDB)ribosomal protein S6 kinase, 90kDa, polypeptide 4(GDB, HUGO)ribosomal protein kinase B (LL)mitogen- and stress-activated protein kinase 2 (LL)

Ribosomal protein S6 kinase alpha 4 [(Nuclear mito-gen-and stress-activated protein kinase-2) (90 kDa ri-bosomal protein S6 kinase 4) (Ribosomal protein ki-nase B) (RSKB)].

(LL – Locus link, GDB – Genome Database)


could be considered as a candidate model of Coffin-Lowrysyndrome (CLS) wherein RSK2 is mutated and inactive [8].RSK2 – / – cells have an increased and sustained phosphory-lation of ERK and increased activation of glycogensynthase. After insulin-stimulation in RSK2 – / – cells, ERKwas phosphorylated two-fold over wt despite lower levelsof ERK2 in the skeletal muscle.

RSK-null Drosophila mutants have also been recentlyconstructed [9]. These mutants showed an enhanced levelof ERK-dependent differentiation that was mediated byblocking the localisation of ERK to the nucleus. Thisinvolved a physical interaction between RSK and ERK butwas independent of kinase activity. This provides furtherevidence that RSK2 participates in a feedback inhibitionloop on ERK as reported previously [10].

Later studies have implicated the transcription factorATF4 in producing the skeletal deformations found inCLS [11]. ATF4 is required for osteoblast differentiationand cell-specific gene expression. RSK2 is able to phos-phorylate and modulate ATF4 function. ATF4 – / – mice dis-play similar bone deformities as RSK2 knockout mice.Therefore, loss of RSK2 appears to indirectly cause the

skeletal phenotype in CLS through its modulation ofATF4.

The involvement of the RSK family in bone develop-ment is only one of the many different abilities thus faridentified in the cell, as the RSKs appear to function invarious important phases of the cell cycle.

Cell cycle regulation – the RSK start and stop

RSK has been implicated in a number of steps during thecell cycle and meiosis, including the G2/M phase transi-tion of meiosis I, the linkage of meiosis I to meiosis II,metaphase arrest in meiosis II and G1 arrest (Fig. 3) [12–20]. In vertebrates, mature oocytes become arrested inmeiosis II at the metaphase stage and meiosis is only com-pleted after fertilisation. In their classic paper over 30years ago, Masui and Markert [21] described how a cyto-plasmic extract from mature Rana pipiens (leopard frog)oocytes could cause two very different phenomena,maturation promoting factor (MPF) and cytostatic factor(CSF) arrest, processes where RSK is involved.


Figure 2. Domain structure in RSKs. The presence of two non-identical and functioning kinase domains is a unique characteristic ofthe RSK family. The NH2 terminal kinase domain (NTKD), essential for substrate phosphorylation, is linked to the COOH-terminalkinase domain (CTKD), that acts as a modulator, through the linker region. Phosphorylation sites in RSKs are schematically drawn inthe activation loops (grey boxes) in NTKD (Ser221) and CTKD (Thr573) and in the linker region (Thr359, Ser363 and Ser380). Theconserved phosphorylation sites of the human RSKs are shown and highlighted in grey.


Oocytes enter meiosis I by increasing the amount ofactive MPF (a complex of cyclinB/Cdc2). RSK appears to beinvolved in G2 to meiosis I transition through the inacti-vation of the inhibitory kinase Myt1, which phosphory-lates and inactivates cyclinB/Cdc2 [18]. Inactivation ofcyclinB/Cdc2 in the cell occurs by phosphorylation ofthreonine and tyrosine residues Thr14 and Tyr15 ofCdc2, which is maintained by the inhibitory kinasesMyt1 and Wee1. Xenopus RSK (similar to human RSK1/2)associates and phosphorylates Myt1 but not Wee1 [18].Overexpression of a constitutively active Xenopus RSK1 inresting oocytes promoted the entry to MI phase in onlyhalf of the oocytes [16], suggesting that the RSK pathwaymay not be solely responsible for the G2 to meiosis I tran-sition. The polo-like kinase (Plx1) also caused MI phaseentry through activation of the phosphatase Cdc25C,responsible for dephosphorylating Cdc2 at Thr14 andTyr15. Plx1 was phosphorylated and constitutively acti-vated in the presence of the MEK1 inhibitor U0126 [13]and Cdc25C activation is completely blocked in Plx1immunodepleted oocyte extracts [22].

RSK2 has been implicated in the initiation of the meio-sis I to meiosis II transition via its effect on cyclinB.Oocytes treated with U0126 are unable to proceed to thenext meiosis cycle and enter S phase, thus implicatingthe MAPK pathway in the meiosis I to meiosis II transi-tion [13]. A prerequisite of entry into meiosis II is theaccumulation of cyclinB, which is degraded in anaphaseI by the anaphase promoting complex (APC), a ubiquitinligase complex composed of 8 core proteins and asso-ciated specificity factors [23]. Anaphase I entry can bedetected in oocytes through a mobility shift of Cdc27,indicating APC activation due to phosphorylation. Afurther shift of Cdc27 is seen as the cells enter metaphaseII which correlates with cyclinB2 stabilisation [24]. How-

ever this second shift is not seen in U0126 treated oocytesand cyclinB protein level is reduced compared to controls[24]. Therefore the rapid degradation of cyclinB can beattributed to MAP kinase activation. Expression of a con-stitutively active RSK could restore both the levels ofcyclinB required for entry into meiosis II and the Cdc27mobility shift [13, 24]. RSK2 was also identified as thedownstream effector of the c-mos proto-oncogene product(MOS), a MAPKKK, in meiosis I to II [13]. ImmunodepletedRSK2 oocyte extracts proceeded to mitosis even in thepresence of MOS compared to catalytically inactive RSK2and mock controls. It therefore appears that the RSKs arenot key players in the G2 to meiosis I transition but havean essential role in the progression from meiosis I tomeiosis II.

Many vertebrate oocytes are arrested at metaphase II byas yet an unknown factor dubbed CSF arrest, which pre-vents the degradation of cyclins. In 1999, two groups inde-pendently confirmed the involvement of RSKs in CSFarrest using Xenopus oocytes [14, 15]. It was found thatboth MOS and constitutively active MEK could cause meta-phase arrest when injected into developing oocytes. How-ever, depletion of MOS or inhibition of MEK stopped thisarrest [25]. To further elucidate this mechanism of CSFarrest, Bhatt and Ferrel [14, 15] produced immunode-pleted RSK2 Xenopus extracts and found that they did notundergo CSF arrest but instead exited mitosis, as seen bythe reformation of nuclei with decondensed chromatin.Oocyte reconstitution with wt RSK2, but not catalyticallyinactive kinase, re-established its ability to undergo meta-phase arrest, thus confirming RSKs requirement for CSFbut not for its maintenance, as RSK2 depleted CSF-arrestedextracts continued to be arrested. Injecting only the NH2-terminal domain of RSK1 into one-half of a two-cell blasto-mere caused cell arrest (morphologically similar to a CSF


Figure 3. RSKs involvement in the cell cycle. RSKhas been shown to be involved in three steps dur-ing meiosis, G2 to meiosis I (MI), MI to meiosis II(MII) and the metaphase arrest at MII, also knownas CSF. During meiosis the oocyte is arrested atG2 just before the start of meiosis I. RSK inhibitsMyt1, which leads to progression of the cell cycle.At the MI transition, RSK is required for the accu-mulation of cyclinB. RSK is also requires for CSFactivity, most likely thought the activation of Bub1and its downstream effect on the APC.


arrested cell) after 2–3 divisions yet the other half contin-ued to divide. MOS, MAPK and endogenous RSK were notactivated in these cells indicating that RSK alone was ableto induce arrest. RSK can therefore mediate metaphasearrest in developing oocytes by an as yet unknown action.The prevailing theory for the mechanism of RSK-mediatedCSF arrest is that RSK induces arrest in the cell throughinhibition of the APC [16]. The RSKs can phosphorylatetwo known APC inhibitors, Bub1 and Emi1. There is how-ever experimental evidence now that in mouse oocytesthe injection of RSK does not mediate CSF arrest [26]. Inaddition, the authors show that RSK1/2/3 – / – mouseoocytes undergo a normal CSF arrest. This suggests thatthere are species differences in the role of RSK and CSFarrest. Mori et al. suggested the role that RSK plays in CSFmay have decreased in higher organisms, as it is an essen-tial component for G1 arrest in starfish oocytes but not formetaphase II-CSF in mouse oocytes [27].

Bub1 (budding uninhibited in benzimadazole) is partof the evolutionarily conserved spindle assembly check-point, which can induce metaphase arrest when the spin-dle is disrupted [28] or when Bub1 is lost [29]. RSK1 canphosphorylate xBub1, the Xenopus homologue of theyeast serine/threonine kinase. Bub1 along with other pro-tein members of the spindle assembly checkpoint, loca-lise to kinetochores in prophase and metaphase of thecell cycle as do RSK and MAPK [16]. Although otherkinases could phosphorylate Bub1, only RSK was foundto be phosphorylated concomitantly and able to activateBub1 in a histone H3 assay. Phosphorylation of Bub1 wassustained in the presence of U1026, when constitutivelyactive RSK was added. Bub1 therefore appears to be down-stream of the MAPK cascade and activated by RSK. Inhibi-tion of the APC complex through activated Bub1, causesthe inhibition of Cdc20 [30], a specificity factor for APCwhich is required for activity in mitotic and meiotic cellcycles. Emi1 has also been implicated in CSF arrest as itcan sequester Cdc20. In a recent report, it was establishedthat RSK2 and Emi1 directly interact and induce meta-phase arrest. A truncated Emi1 mutant, which could notbe phosphorylated by RSK2, loses its ability to impairmouse oocyte maturation [31].

In addition to RSKs' effect on meiosis, it may also pro-mote cell cycle progression in mitosis via inhibition ofp27Kip1 [32]. p27Kip1 is a member of the Cip/Kip cyclin-dependent kinase inhibitor family and blocks progres-sion of G1 to S phase in the cell cycle [32]; p27Kip1 is asso-ciated with cyclinD-Cdk4/6 complexes in proliferatingcells. In arrested cells however, p27Kip1 is complexed withcyclinE or Cdk2, which become sequestered and lead toinhibition of the cell cycle [33]. RSK1 and RSK2 can physi-cally associate with and phosphorylate Thr198 of p27Kip1,

which stimulates the binding to 14-3-3 and subsequentcytoplasmic localisation of p27Kip1 [20]. Therefore RSK1and RSK2 can sequester p27Kip1 from the nucleus and indoing so, may promote S phase entry. Much of the earlywork involving RSKs focused on RSK1-3 (as they wereidentified and cloned initially) but new work on RSK4has uncovered some surprising results.

The inhibitory effect of RSK

The last member of the RSK family to be discovered wasRSK4 (RPS6KA6). Implicated in X-linked mental retarda-tion, two recent publications highlight the diverse func-tions of the RSK family of kinases [34]. Berns et al. identi-fied RSK4 as a component of the p53 pathway that med-iates proliferation arrest [34]. Their experimentalapproach used primary human fibroblast cells that ecto-pically expressed a temperature sensitive large T antigenwhich binds p53 and Rb at 328C allowing cells to prolifer-ate. However at 398C the large T antigen is inactivatedand the cells arrest. A retroviral RNA library (knockdownof 7914 genes) was used to identify genes that inhibit pro-liferation arrest. Six genes were identified: RPS6KA6(RSK4), HTATIP (histone acetyl transferase TIP60), HDAC4(histone deacetylase 4), KIAA0828 (a putative S-adenosyl-L-homocysteine hydrolase, SAH3), CCT2 (T-complex protein1, b-subunit) and TP53 itself. The proliferation of cellsafter reduction of RSK4 expression using siRNA stronglyimplicates this gene in p53 mediated cell cycle arrest.Further evidence has shown that the knockdown of RSK4also confers resistance to p19ARF-dependent proliferationarrest (as mediated by p53). In addition, the p53-depen-dent G1 cell cycle arrest due to ionising radiation is alsoabolished when using RSK4 siRNA. Finally, levels ofp21cip1 (a cdk inhibitor downstream of p53) are greatlyreduced at both protein and RNA levels when p53 orRSK4 is knocked down. However another target gene ofp53, BAX, remained unaffected. Therefore, RSK4 and theother p53 modulators appear to only target some effec-tors of p53. While p53 was one of the siRNAs picked up bythis method of screening, p21Cip was not even thoughexpression of this siRNA could also produce the sameresults as the other modulators above. This shows thatthe screening did not detect all the genes involved in p53dependent proliferation arrest, and more genes may yetbe found.

In another screening system, Myers et al. identifiedRSK4 as an inhibitor of the FGF-RAS-ERK pathway [35].The mouse homologue of RSK4 was able to disrupt Xbra-chyury (Xbra) expression, a T-box transcription factorand molecular marker of mesoderm. Although highly



related, the other members of the RSK family (RSK1-3)were unable to disrupt Xbra protein expression. Howeverunlike RSK1, RSK4 could not induce maturation ofembryos [12]. The unique 96aa amino terminal of mouseRSK4 was required but not sufficient for inhibition as theNH2-terminal kinase domain is also needed to disruptXbra expression. After treatment with a FGF inhibitor(SU5402), ERK expression was reduced, typically near theembryonic/extraembryonic junction. In the presence ofSU5402 RSK4 expression expanded into areas previouslyoccupied by ERK, suggesting that RSK4 expressionrestricts ERK activity in the developing mouse embryo.

Similarly to RSK4, RSK2 has also been suggested todown regulate the Ras-ERK pathway through SOS in anegative feedback loop [10]. SOS functions as an activatorof Ras by catalysing GDP release on Ras. Phosphorylationof SOS on Ser/Thr residues in the COOH-terminus nearthe Grb2 binding site, has been linked to the dissociationof Grb2 (an adaptor) from SOS or dissociation of the Grb2-SOS complex from the receptor leading to a possiblenegative feedback on Ras [36].

The potential of RSK4 to be involved in p53 prolifera-tion arrest adds to the published reports that RSK1-3 hasanti-apoptotic properties via its interaction with BAD.

Apoptosis – the good, the BAD and the RSKs

Apoptosis or programmed cell death is a key physiologi-cal process in multicellular animals. RSK1-3 have all beenshown to phosphorylate BAD on Ser112 and Ser155thereby protecting against BAD-mediated cell death(Fig. 4) [37–40].

BAD is active as an unphosphorylated molecule; in itsphosphorylated inactive form (phosphorylation sitesSer112, Ser136 and Ser155), BAD is bound to 14-3-3 pro-teins and sequestered in the cytosol [41]. Growth factorstimulation of cells induces a number of kinase pathwaysthat can phosphorylate BAD. AKT has been shown to pro-mote cell survival by phosphorylating BAD at Ser136 in

cells stimulated with interleukin 3 (IL-3) and insulin-likegrowth factor 1 (IGF-1) via activation of phosphatidylino-sitol 3-kinase (PI3K). IL-3 can also cause protein kinase A(PKA) stimulation and subsequent BAD phosphorylationat Ser112 [41]. Phorbol ester-induced activation ofHEK293 cells stimulates the PKC pathway, which in turnactivates RSK1-3 and phosphorylates BAD at Ser112 [40].This is inhibited by a PKC inhibitor (GF109203X) but nota MAPK (PD98059) or p38MAPK (SB203580) inhibitors [37,40]. BAD Ser136 can also be phosphorylated by RSKs butto a lesser degree than by AKT [38, 39]. It appears thatdifferent kinases have different affinities to particularphosphorylation sites of BAD. Overexpression of domi-nant negative RSK2 reduces BAD-mediated cell death tothat of control cells [37, 40] and only BAD with wt Ser112could be rescued from apoptosis [40], implicating Ser112as an important site in BAD-mediated phosphorylation.However, in different cell lines there appears to be dis-tinct requirements for BAD phosphorylation at Ser112and Ser136 sites in suppressing apoptosis [39, 41]. BADtherefore seems to be a convergence point for the integra-tion of different kinase pathways to suppress apoptosisand increase cell survival.

The effect of RSKs on BAD phosphorylation has alsobeen studied in human melanoma cells [42]. Melanomasand melanocytes both have high levels of phosphoryla-tion of BAD at Ser75 (corresponding to murine Ser112),but only melanocytes and 1/7 melanoma cell lines hadphosphorylated Ser99 and Ser118 (equivalent to mouseSer136 and Ser155) [42]. MEK inhibitors are able to induceBAD-mediated apoptosis in melanoma cell lines correlat-ing with BAD Ser75 dephosphorylation. This effect wasreversed by overexpression of constitutively active RSK1.Therefore, in melanoma cells BAD inactivation occursthrough a survival signal mediated by RSK, not found innormal melanocytes. Apoptosis mediated by the evolutio-narily conserved BCL-2 (B-cell CLL/lymphoma 2) proteinfamily requires the oligomerisation of BAK and BAX. Thisprocess is moderated by the binding of an activator mole-cule, BID. The antiapoptotic members of the BCL-2 family


Figure 4. Multiple mechanisms of inactivation ofBAD by RSK. BAD-mediated apoptosis can be haltedby RSK by two different ways. Phosphorylation ofBAD at Ser112 creates a docking site for the 14-3-3protein that can be sequester BAD away in the cyto-plasm. Alternatively, phosphorylation at Ser155 byRSK blocks the binding of BAD to Bcl-XL, which inturn allows Bcl-XL to inhibit apoptosis by binding theproapoptotic proteins BAK and BAX.


(Bcl-XL and BCL-2) sequester BID and prevent the BAK/BAXinteraction [43]; BAD can also bind to BCL-2 family mem-bers and inhibit the sequestration of the activating BIDprotein.

RSK1-3 can also inhibit apoptosis through phosphory-lation of BAD at Ser155 which prevents its binding to theBcl-XL antiapoptotic protein [42]. Expression of the BADmutant S155A induced apoptosis similar to that of wtBAD. RSK could rescue apoptosis in wt BAD [44], and thiscapacity decreased with the number of alanine muta-tions introduced into BAD (S155A A S112A/S136A A

S112A/S136A/S155A). The S155E BAD, that mimicks con-stitutive phosphorylation, suppressed apoptosis nearlytwice as effectively as wt BAD.

Another way in which RSK could prevent apoptosis isthrough the phosphorylation of DAPK (death-associatedprotein kinase). Phosphorylation of Ser289 in DAPK mayocclude the binding of the kinase to calmodulin, main-taining its autoinhibitory conformation [45].

RSK can also phosphorylate GSK3a and b, which mightprevent apoptosis in neutrophils. GSK3s are kinases thatphosphorylate and inactivate glycogen synthase. Ectopicexpression of GSK3a induces attenuation of apoptosis insome cultured cells [46]. Inhibition of GSK3a is mediatedby phosphorylation of Ser21 (Ser9 on GSKb) by RSK andAKT [47]. GSK3 is inhibited by RSK phosphorylation inearly Xenopus embryogenesis and also in NIH3T3 cells [48,49]. GSK3a inhibition has also been studied in neutro-phils where apoptosis can be delayed by the use of che-moattractants that activate the AKT or ERK pathway.RSK2 was found to phosphorylate and inhibit GSK3a

after stimulation with the chemoattractant fMLP [47].Cell survival is additionally increased in cultured cells

via RSKs interaction with the transcription factor NFjB.This activates survival genes in the nucleus such as themitochondrial membrane stabilising proteins Bcl-XL andBlf-1, the caspase inhibitors cIAP1/cIAP2 and XIAP (inhibi-tor of apoptosis proteins) [50]. The action of NFjB is inhib-ited by IjB (Inhibitor NFjB) which binds to the nuclearlocalisation signal of NFjB thereby preventing transloca-tion to the nucleus. The prototypic and best studied ofthe IjBs is IjBa. The inhibitory action of IjB is disruptedby phosphorylation of two critical residues, Ser32 andSer36, which initiates the ubiquitination and subsequentdegradation of IjBa. NFjB can then translocate to thenucleus and activate transcription. In baby rat kidneycells, RSK1 physically associates with IjBa and phosphor-ylates it at Ser32 [51]. A dominant negative RSK1 inhib-ited phosphorylation of IjBa and its subsequent degrada-tion in response to TPA. However, ubiquitination of IjBa

requires phosphorylation of Ser32 and Ser36. It is nowknown that the IKK (IjB kinase) complex regulates IjBa

ubiquitination and phosphorylates IjBa at Ser32 andSer36 sites [52]. Analysis of p53 null cells showed thatRSK1 associates with the IKK-2 subunit of the IKK com-plex and is required for NFjB activation, upon treatmentwith anthracycline doxorubicin (an inducer of DNA dou-ble stranded breaks) [53]. Silencing expression of RSK1 (byRNAi) abolished IKK phosphorylation. This could explainprevious data showing RSK1 and IjBa interaction, as itsuggests that RSK1 indirectly leads to IjBa phosphoryla-tion by inducing IKK activity and thereby promoting cellsurvival.

In summary, RSK can mediate a variety of signals toprevent apoptosis including the inhibition of GSK3a andBAD, to the activation of pro-survival signals such asNFjB. Yet, this is still only a small amount of the numberof potential interactions that this eclectic family ofkinases have been reported to have.

RSK substrates and transcriptional regulation

When RSK is activated it can translocate into the nucleus,in association with ERK, and activate immediate-earlygene expression and transcription factors. There is betterunderstanding of the spatial and temporal regulation ofphosphorylation of RSKs. Active ERK localises to the dock-ing domain which is present in the C-terminus of RSK (D-domain). The CD domain of ERK mediates this interac-tion. This interaction seems to be quite independent ofthe DEF domain interactions of ERK which is responsiblefor induction of immediate early growth response [54].

Originally RSK2 was identified as a CREB kinase, but itis now thought that MSK1 and MSK2 in the p38 pathwayare the major CREB kinases [55–57]. Indeed, embryonicstem cells lacking MSK1 and fibroblast cells from MSK1and MSK2 knockout mice showed a significant reductionin CREB phosphorylation, even though RSK2 was acti-vated normally compared to wt cells [55, 56]. MSK1 andto a lesser extent MSK2 could be activated by ERK1/2 orSAPK2/p38 in primary fibroblasts.

The phosphorylation of histone H3 has also beenreported as a major activity for RSK2 as CLS cells showeddiminished phosphorylation of H3 [58]. However, usingMSK1 and 2 double knockout cells, it has been found thatMSK1 and 2 are the kinases most likely to phosphorylateH3 in vivo. Further experiments have shown that CLSfibroblast cells are able to phosphorylate H3 even in theabsence of detectable RSK2 [59], contrary to the originalreport.

RSK2 can also interact with CBP (CREB-binding protein)a coactivator of CREB [60, 61]. CBP co-ordinates transcrip-tion with chromatin remodelling by acetylating his-



tones; opening the chromatin and making it more acces-sible. CBP can also activate transcription directly via itsinteraction with TFIIB and act as a “bridge” betweenCREB and the transcriptional machinery. The majority oftranscription factors studied interact with CBP, includ-ing c-Fos, c-Jun, E1A, E2F, NFjB, p53 and CREB. In quies-cent cells, CBP and RSK2 were found to be complexed butbecome dissociated once RSK2 was activated. RSK1, RSK3and MSK1 can also associate with CBP. The use of amutant RSK2 showed that Ser227, the site of PDK1 phos-phorylation, is a critical residue which mediates CBP dis-sociation when phosphorylated [60]. The association ofRSK2 and CBP disrupts the histone acetyltransferaseactivity of CBP, as the RSK binding site is adjacent to acysteine-histidine-rich zinc finger motif (C/H3). There-fore, activation of RSK2 causes dissociation of the RSK/CBP complex, the increase in CREB-mediated (and alsoCBP) transcription, but also the release of CBP to acetylatehistones in the chromatin. The interaction between CBPand RSK occurs in the same region as the CBP-E1A interac-tion, which represses transcriptional activation, limitingits ability to induce transcription. This contrasts with aprevious report showing that CBP and an unidentifiedRSK isoform interact and form a complex in response toRas activation [61]. In this study, the RSK/CBP complexalso repressed transcription of genes mediated by CREB.The differences observed between the two reports may bedue to differences in the cell lines used (PC12 comparedto NIH3T3, COS and HEK293) or in the specificity of anti-bodies used to identify the different RSK isoforms.

The RSK family has also been implicated in the phos-phorylation of one of the immediate-early genes, c-Fos,part of the AP-1 transcription factor dimeric complex[62]. Stimulation of cells with PDGF activates the c-Fospromoter via the Ras-Raf-MAPK-RSK pathway. Overexpres-sion of c-Fos causes osteosarcoma in mice. In a fibroblastcell line derived from CLS patients and RSK2 knockoutmice, c-Fos expression was severely impaired [62]. In thesecell lines RSK1 appeared at wt levels and there was amajor reduction in c-Fos phosphorylation after IGF-1 sti-mulation [63]. In addition there was normal CREB phos-phorylation and transcriptional activation, indicatingagain the dispensability of RSK2 in CREB phosphoryla-tion.

RSKs are able to bind and phosphorylate the two mam-malian estrogen receptors (ERa and b), the ligand-acti-vated transcription factors that can activate or represstranscription. The ERs can interact with several proteinsand influence the cell cycle (MAD2, cyclinD1); bind totranscription factors (TFIIH); mediate chromatin modifi-cations to repress (N-CoR, SMRT, MTA-1) and activate tran-scription (p300, CBP, SRC-1, TIF2) [64]. ERa and b have

both a hormone-independent (associated with the AF-1domain) and hormone-dependent transcriptional activa-tion function (AF-2 domain). Phosphorylation of Ser118and Ser167 in ERs appears to be crucial for their tran-scriptional activity; the mutant S118A showed decreasedtranscription activity [65, 66]. RSK1 can physically associ-ate with ER, phosphorylates Ser167 specifically in an invitro kinase assay and phosphorylates Ser167 in vivo inCOS-1 cells compared to a kinase dead mutant and vectorcontrols. The ER mutant S167A and the kinase dead RSK1mutant both suppress ER regulated transcription. ERtranscription was also allosterically regulated by RSK2,with binding at the hormone binding domain (326–394in ERa specifically) producing a conformational switchin ERa that enhances AF-2 activation. This exposes thephosphorylation site Ser167 that when phosphorylatedincreases transcription by activating the AF-1 domain[67].

More RSK functions

The Na+/H+ exchanger isoform-1 (NHE-1) is a ubiquitousion exchanger that is important for cell growth since itregulates intracellular H+. Growth factors activate NHE-1rapidly and the increase in intracellular pH is a universalresponse to mitogen stimulation. After serum stimula-tion and two-dimensional tryptic phosphopeptide map-ping, only one peptide (containing Ser703 of NHE-1) hasincreased phosphorylation which is blocked by PD98059[68] and when mutated abolishes the increased affinityfor H+ after serum-stimulation. RSK2 was found to beresponsible for the phosphorylation of Ser703. A domi-nant negative RSK2 mutant inhibited the ability of NHE-1to undergo phosphorylation of Ser703 after growth fac-tor addition compared to wt. Phosphorylation of Ser703by RSK mediates the binding of 14-3-3 to NHE-1 [69]. Thisbinding is lost if the MAPK pathway is inhibited asdemonstrated by the addition of PD98059. The 14-3-3/NHE-1 complex prevents the dephosphorylation ofSer703 (as shown by addition of PP1). Therefore, phos-phorylation of NHE-1 by RSK is thought to promote stabi-lisation of the NHE-1 conformational change by provid-ing a binding site for 14-3-3 binding.

RSK2 can further phosphorylate LKB1, a Ser/Thr kinaseand product of a tumour suppressor gene that whenmutated causes the autosomal dominant inherited disor-der Peutz-Jeghers syndrome (PJS). Individuals with PJSdevelop benign gastrointestinal polyps (hamartomas)and develop other tumours in lung, breast, ovary, testis,pancreas, stomach and intestine [70]. Ectopic expressionof LKB1 can suppress cell growth in cancer cell lines lack-



ing endogenous LKB1 protein [71]; the inhibition of cellproliferation is dependent on the phosphorylation statusof Ser431 as shown by the use of catalytically inactivemutant LKB1 proteins (S431A) which inhibited cellgrowth [72]. RSK2 phosphorylation of LKB1 at Ser431 mayplay an important role in the LKB1 effect as a tumour sup-pressor gene, although it is still unknown how signifi-cant a role RSK2 plays in this phosphorylation.

TSC1 and TSC2 are tumour suppressors associated withthe tuberous sclerosis complex (TSC), a disorder charac-terised by the development of hamartomas and tumour-like growths in a variety of organs. TSC1 and TSC2 form aheterodimeric complex that inhibits mTOR, a proteinrequired to mediate cell proliferation and growth via p70ribosomal S6 kinase 1 (S6K1) and eukaryotic initiationfactor 4E-binding protein1 (4E-BP1) [73]. RSK1 phosphory-lates TSC2 (also known as tuberin) in vitro and in vivo atSer1798 (Fig. 5). RSK phosphorylation appears to causeupregulation of S6K1 activity, inhibiting the tumour sup-pressor function of the tuberin/harmatin complex. In asimilar way, the phosphorylation of DAPK by RSK alsoinhibits its tumour suppressor activity [45]. The TSC1/2complex acts as a convergence point for AKT and MEK sig-nalling as stimulation of AKT can also stimulate TSC2,yet is independent of RSK signalling. More recently, it hasbeen shown that inactive RSK1 associates with the eukar-yotic initiation factor elF3. Cell stimulation promotes

mTOR binding to the elF3 complex and phosphorylationof RSK1 at its hydrophobic motif. Phosphorylation resultsin the release of RSK1, and the phosphorylation of elF4Band its targets [74, 75]. Phosphorylation of eIF4B by S6Kand RSK at site Ser422 [76], represents a point where bothpathways come together, with different kinetics andgrowth-factor dependencies, that results in the promo-tion of protein synthesis.

RSK1 appears to also play a role in the differentiationof PC12 cells. The ectopic expression of a constitutivelyactive RSK1 (but not constitutively active RSK2) inducesthe outgrowth of neurites in this cell line [77]. Thisfurther reinforces the notion that although highly homo-logous to each other, the different RSKs have profoundlydistinct substrate specificities. Interestingly, it has beenshown recently that RSKs can bind PDZ domain proteinsvia its C-terminus. RSK2 binds Shank-3, a PDZ domainprotein, and its phosphorylation is important for excita-tory synaptic transmission in neurons [78].

The role of RSK in cancer has been further illuminatedby the use of the first RSK inhibitor, SL0101 [79]. Thiscompound is able to inhibit RSKs and not the relatedkinases, p70S6K, PKA or MSK1. Furthermore, in the breastcancer cell line MCF-7, SL0101 inhibits cell proliferationby arresting cells in G1 while the ,normal’ breast cell lineMCF-10A remains unaffected. This suggests that MCF-7cells have become reliant on the RSK pathway much likethe abnormal melanoma cell lines. It would be interest-ing to determine if the cells also undergo BAD-mediatedapoptosis.

The importance of the RSK family in diseases is high-lighted by the loss of activity of RSK2 in Coffin-Lowry syn-drome.

Losing the RSK – diseases associated withRSK

Two members of the RSK family are implicated in dis-eases, RSK2 in Coffin-Lowry syndrome (CLS) and RSK4 in X-linked mental retardation. Both RSK genes are found onthe X-chromosome. CLS is characterised by mental retar-dation, short stature and craniofacial and skeletalabnormalities. In a minority of cases, hearing and cardiacproblems have also been noted. Females that are affectedby the syndrome may manifest less severe forms of thedisease, which provided clues towards its sex-linkage.Missense and nonsense mutations on or around phos-phorylation sites, ATP-binding sites, or the ERK dockingsite, as well as intragenic deletions in the RSK2 gene canbe responsible for CLS [8]. So far there is no correlationbetween the different mutations and severity of pheno-


Figure 5. RSK and its effects on the tumour suppressor mTOR.RSK is able to directly phosphorylate tuberin. This phosphoryla-tion of the tumour suppressor upregulates p70S6K, which subse-quently enhances the transcription of mRNAs and downregu-lates the inhibitor of translation initiation, 4E-BP1.


type. However, mutations may be linked to the alteredmental status [80]. Skeletal deformations are sufficientlyevaded in CLS when RSK2 is only 20% active (perhapsindicating that this amount of activity is sufficient forATF4 activation) [11]. Nevertheless this level of kinaseactivity can still lead to mild mental impairment.Although there is no direct knowledge yet as to howRSK2 causes the CLS phenotype, likely candidates are RSKsubstrates in the brain. In situ hybridisation of the adultmouse brain showed that although there was expressionof all RSK1-3 in all regions tested, there were differencesin levels of expression [81]. In the developing mousebrain, RSK1 is more strongly expressed in proliferatingcells, perhaps indicating a role in development, RSK3expression is highest in the central and peripheral ner-vous system and RSK2 is relatively weak during embryo-nic development. However, in the adult mouse brainRSK2 is expressed strongly in cells with high synapticactivity such as the pyramidal cells of the hippocampusand Purkinje cells of the cerebellum. This suggests a pos-sible link between CLS patients with mental retardationand RSK2 function.

The X chromosome is frequently implicated inimpaired mental function, which can be part of a morecomplex syndrome (MRXS) with or without specific clini-cal features (MRX or X-linked non-specific mental retarda-tion). Xq21 deletions can cause mental retardation and X-

linked deafness type 3 (DFN3) and choroideremia (CHM),a degeneration of the choroid, retinal epithelium andthe retina that causes blindness. The two genes DFN3 andCHM have been identified and cloned, with the gene thatcauses mental retardation mapping between these twogenes. The most likely candidate for this MRX locus isRSK4 [82]. Although no mutations of RSK4 have yet beenfound in 200 patients, RSK4 is still a putative gene linkedto MRX in this region, as there is a lack of other genes inthis area.

Conclusions and perspective

The RPS6KA gene family is involved in multiple activitieswithin cells including major cell cycle steps, apoptosisand transcriptional regulation, with more functions stillbeing discovered (Fig. 1 and Table 2). RSK can phosphory-late BAD which antagonises its ability to promote apopto-sis while RSK interactions with CREB and CBP/p300 pro-motes survival mechanisms, therefore, providing bothanti-apoptotic and pro-survival signals. However, theredoes appear to be additional roles for the different RSKisoforms. RSK1 is also implicated in cell cycle progressionin meiosis, has anti-apoptotic activity through phosphor-ylation of GSK3, BAD and IKK and also promotes tran-scription via activation of CREB, CBP/p300 and c-Fos. The


Table 2. RSKs potential interactions.

Protein Substrate Proposed Effect

RSK1 Myt1BADCREB, ERCBPIKKp27Kip1

GSK3a/b

Decreases inhibition of MPFIncreases cell survivalActivates transcriptional activityModulates transcriptionDegradation of IjBaInhibition of S phase entryInhibition of apoptosis

RSK2 Bub1BADERa, c-FosCBPSOSATF4p27Kip1

NHE1LKB1GSK3aShank-3

Stabilises cyclinB levelIncreases cell survivalActivates transcriptional activityModulates transcriptionNegative feedback loop of MAPKOsteoblast expressionInhibition of S phase entryStabilises conformational changeInhibition of cell proliferationInhibition of apoptosisSynaptic transmission

RSK3 BADCREBCBP

Increases cell survivalActivates transcriptional activityModulates transcription

RSK4 Xbrap53

Disrupts normal mesoderm formationp53 pathway


generalisation of RSK functions can only be taken so faras RSK1 is also implicated in CSF arrest via its phosphory-lation of Bub1. The newly discovered RSK4 appears tohave a more inhibitory role, as it is involved in the p53pathway as well as acting as an inhibitor for ERK itself.RSK2 appears to be the most pleiotropic of all RSKs, shar-ing many of RSK1 substrates including, p27Kip1, GSK3,ERa and NHE1. The only unique function of RSK2 is itsability to suppress the Ras/Raf pathway by phosphorylat-ing SOS. There are no unique functions of RSK3 known todate.

There is still considerable uncertainty as to whichRPS6KA family member performs which particular func-tion. The cell cycle research on MPF and CSF has beenmost extensively carried out using Xenopus oocytes and itis still unknown whether this can be extrapolated toother organisms. In the case of histone H3 and CREBphosphorylation, it now appears that the more physiolo-gically relevant kinases are not RSKs but the relatedRPS6KA members, MSK1 and 2, a situation reminiscentof the initial discovery of the RPS6KA family in Xenopusand p70S6K. It is clear that although there are a number ofoverlapping functions between the different RSKs, thereis not complete compensation, as in the case of Coffin-Lowry syndrome, where RSK2 is lost but both RSK1 andRSK3 are at normal levels.

The discovery of RSKs has led over the last 20 years tothe identification of many different functions. It is alsoapparent that they have probably separate and non-over-lapping functions. These are probably specialised, asRSKs are limited to metazoans and mammalian cells.Although RSK1 and 2 broadly function as positive effec-tors of growth and differentiation, RSK4 has an inhibi-tory role. The precise functions of RSK3 are still poorlyunderstood relative to the other isoforms. To furtherdelineate RSK function in the future, it will be necessaryto use RNAi and other knockout strategies in cell lines,stem cells and mice to determine the exact effect of a par-ticular RSK as well as use more specific inhibitors to theRSKs. Therefore further investigations of this importantkinase family will be necessary to elucidate the exact spe-cificity of each individual member in relation to its func-tion.

We apologise to the many authors whose work could not becited due to space limitations. We would like to thank CancerResearch UK, The Association for International CancerResearch and Wellbeing for support. We would also like tothank Stephan Feller for critical reading of the manuscriptand all the members of the Ovarian Cancer Group for their gen-erous and unique assistance.

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p90 ribosomal s6 kinases- eclectic members of the human kinome

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