Biochem. J. (2012) 441, 553–569 (Printed in Great Britain)
553
doi:10.1042/BJ20110289
REVIEW ARTICLE
Regulation and function of the RSK family of protein kinases
Yves ROMEO†, Xiaocui ZHANG† and Philippe P. ROUX†‡1
†Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, P.O. Box 6128, Montreal, QC, Canada, H3C 3J7, and ‡Department of Pathology and Cell Biology,
Faculty of Medicine, Université de Montréal, P.O. Box 6128, Montreal, QC, Canada, H3C 3J7
The RSK (90 kDa ribosomal S6 kinase) family comprises a group
of highly related serine/threonine kinases that regulate diverse
cellular processes, including cell growth, proliferation, survival
and motility. This family includes four vertebrate isoforms
(RSK1, RSK2, RSK3 and RSK4), and single family member
orthologues are also present in Drosophila and Caenorhabditis
elegans. The RSK isoforms are downstream effectors of
the Ras/ERK (extracellular-signal-regulated kinase) signalling
pathway. Significant advances in the field of RSK signalling have
occurred in the past few years, including several new functions
ascribed to the RSK isoforms, the discovery of novel protein
substrates and the implication of different RSK isoforms in
cancer. Collectively, these new findings increase the diversity of
biological functions regulated by RSK, and highlight potential
new directions of research. In the present paper, we review the
structure, expression and activation mechanisms of the RSK
isoforms, and discuss their physiological roles on the basis of
established substrates and recent discoveries.
INTRODUCTION
are generally thought to be more responsive to cellular stresses [8].
The past few years have witnessed a wealth of new information
on the biological roles of the RSKs, and in the present review we
discuss these insights and also highlight novel biological pathways
that are modulated by these kinases.
The Ras/MAPK (mitogen-activated protein kinase) pathway
(Figure 1) plays a central role in transducing extracellular
signals to intracellular target proteins involved in cell growth
and proliferation (reviewed in [1–3]). Inappropriate regulation
of this pathway leads to a variety of disorders and diseases,
including many types of cancers [4]. In this pathway, the GTPase
Ras activates Raf isoforms (A-, B- and C-Raf), which are
serine/threonine kinases frequently mutated in human cancers
[5,6]. In turn, activated Raf phosphorylates and activates MEK
[MAPK/ERK (extracellular-signal-regulated kinase) kinase]1/2,
which are dual-specificity kinases capable of phosphorylating
and activating the MAPKs ERK1/2. Once activated, ERK1/2
phosphorylate several substrates, including members of the RSK
(90 kDa ribosomal S6 kinase) family [7,8]. This family consists
of four human isoforms (RSK1–4) and two structurally related
homologues, termed MSK1 [mitogen- and stress-activated kinase
1; also known as RLPK (RSK-like protein kinase)] and MSK2
[also known as RSK-B (RSK-like B]. Despite being highly related
to the RSKs, the MSKs have very different biological functions
and have been reviewed extensively [9,10]. Whereas the RSKs are
directly activated by ERK1/2 in response to various stimuli, the
MSKs are activated by both the ERK1/2 and the p38 pathways, and
Key words: cell signalling, mitogen-activated protein kinase
(MAPK), phosphorylation, proliferation, protein kinase, 90 kDa
ribosomal S6 kinase (RSK).
DISCOVERY
The regulated phosphorylation of rpS6 (ribosomal protein S6)
has attracted much attention since its discovery in 1974 [11].
This is due to the temporal correlation of rpS6 phosphorylation
with the initiation of protein synthesis and the suggestion that
its phosphorylation facilitates polysome assembly [12]. Several
laboratories deployed great efforts to identify the protein kinases
responsible for rpS6 phosphorylation [13,14], and in 1985, the
groups of Erikson and Maller purified an intracellular kinase
activity, termed ribosomal S6K (S6 kinase), that phosphorylated
rpS6 from unfertilized Xenopus laevis eggs [15]. Two protein
kinases of 85–90 kDa (S6KI and S6KII) were identified by
biochemical purification, which led to the cloning of cDNAs
encoding highly homologous proteins that were later renamed p90
RSKs or RSKs [16]. It was subsequently shown that S6KII was
phosphorylated and activated by the insulin-stimulated MAP2
Abbreviations used: aa, amino acids; AGC, protein kinase A, G and C; AMPK, AMP-activated protein kinase; APC, anaphase-promoting complex;
ATF, activating transcription factor; Bad, Bcl-2/Bcl-xL-antagonist, causing cell death; CAMK, Ca2 + /calmodulin-dependent protein kinase; CBP, CREBbinding protein; Cdc, cell division cycle kinase; CDK, cyclin-dependent kinase; C/EBP, CCAAT/enhancer-binding protein; CLS, Coffin–Lowry syndrome;
CREB, cAMP-response-element-binding protein; CTKD, C-terminal kinase domain; DAPK, death-associated protein kinase; eEF2K, eukaryotic elongation
factor-2 kinase; EGF, epidermal growth factor; eIF, eukaryotic initiation factor; 4E-BP1, eIF4E-binding protein 1; Emi, early mitotic inhibitor; ERα, oestrogen
receptor α; ERK, extracellular-signal-regulated kinase; Erp1, Emi1-related protein; EST, expressed sequence tag; ETS, E twenty-six; Elk1, ETS-like kinase 1;
FGFR3, fibroblast growth factor receptor 3; FMK, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; GLUT4, glucose transporter 4; GSK3, glycogen
synthase kinase-3; IκB, inhibitor of nuclear factor κB; IEG, immediate-early gene; IFN, interferon; KIM, kinase interaction motif; LKB1, liver kinase B1; Mad,
mitotic arrest-deficient protein; MAP, microtuble-associated protein; MAPK, mitogen-activated protein kinase; MDCK cell, Madin–Darby canine kidney
cell; MEK, MAPK/ERK kinase; MK, MAPK-activated protein kinase; MSK, mitogen- and stress-activated kinase; mTOR, mammalian target of rapamycin;
Myt1, myelin transcription factor 1; NF-κB, nuclear factor κB; NOS, nitric oxide synthase; nNOS, neuronal NOS; NTKD, N-terminal kinase domain; p27kip1 ,
cyclin-dependent kinase inhibitor 1B; PAK1, p21-activated kinase-1; PDK1, 3 -phosphoinositide-dependent protein kinase 1; PEA-15, phosphoprotein
enriched in astrocytes, 15 kDa; PIF, PDK1-interacting fragment; PKA, protein kinase A; RanBP3, Ran-binding protein 3; RNAi, RNA interference; rpS6,
ribosomal protein S6; RSK, 90 kDa ribosomal S6 kinase; S6K, S6 kinase; SGK, serum-and glucocorticoid-induced protein kinase; SOS, son of sevenless;
SRF, serum-response factor; TSC2, tuberous sclerosis complex 2; YB-1, Y-box binding protein-1.
1
To whom correspondence should be addressed (email [email protected]).
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Figure 1
Y. Romeo, X. Zhang and P. P. Roux
The Ras/MAPK pathway leads to activation of the RSK family of protein kinases
Growth factor binding to their cognate receptor tyrosine kinase promotes GTP-loading of the Ras isoforms, which subsequently activate isoforms of the Raf family of serine/threonine kinases.
Activated Raf promotes activation of the MAPK pathway by direct phosphorylation of MEK1/2, which then phosphorylate and activate ERK1/2. The RSK protein kinases are directly phosphorylated by
ERK1/2 and PDK1, resulting in their activation. This leads to phosphorylation of functionally diverse RSK substrates in the cytosol as well as nuclear translocation of RSK, where it phosphorylates
transcription factors, including those of the IEG response. Whereas PD184352, U0126 and PD98059 are specific MEK inhibitors, BI-D1870, SL0101 and FMK specifically inhibit RSK activity. Grb2,
growth-factor-receptor-bound protein 2; NF1, neurofibromin.
(microtubule-associated protein-2) kinase, which was renamed
ERK2 [17] and provided the first evidence that S6KII (or RSK)
is activated downstream of the ERK/MAPK cascade.
TO BE, OR NOT TO BE, AN S6 KINASE
As described above, RSK was originally identified as an in vitro
rpS6 kinase [15,18]. However, two related protein kinases,
termed 70 kDa ribosomal S6 kinases (S6K1 and S6K2), were
later shown to be the predominant rpS6 kinases operating in
somatic cells [19,20]. Inhibition of mTOR (mammalian target of
rapamycin) activity and thus S6K1/2 activation using rapamycin
was shown to completely prevent rpS6 phosphorylation induced
by insulin [20,21], which suggested at the time that the
RSKs played negligible roles in rpS6 phosphorylation. This
conclusion was also suggested by experiments performed in cells
derived from S6k1 − / − S6k2 − / − double-knockout mice, where
absence of S6K1/2 is concomitant with an almost complete
loss of rpS6 phosphorylation [22]. Interestingly, low levels of
rpS6 phosphorylation were found to persist in S6K1/2-deficient
cells and to depend on the MAPK pathway, suggesting some
involvement on the part of the RSKs. The specific role of the
RSK isoforms in rpS6 phosphorylation was later confirmed in vivo
using RNAi (RNA interference) and pharmacological inhibitors
[23]. Indeed, both RSK1 and RSK2 were found to contribute to
rpS6 phosphorylation in response to agonists or oncogenes that
activate the MAPK pathway. Interestingly, whereas S6K1/2 were
found to phosphorylate all sites on rpS6 (Ser235 , Ser236 , Ser240 and
c The Authors Journal compilation c 2012 Biochemical Society
Ser244 ), the RSKs were shown to specifically phosphorylate Ser235
and Ser236 [23]. The role of this specific regulation remains elusive,
but growing evidence suggests that rpS6 phosphorylation may be
involved in fine-tuning the cellular response elicited by certain
growth signals [12]. The specific evaluation of the RSK- and
S6K-specific phosphorylation events on rpS6 will be necessary to
determine the complexities of its regulation and function.
STRUCTURE AND CONSERVATION
The vertebrate RSK family contains four isoforms, termed RSK1
[24], RSK2, RSK3 [25] and RSK4 [26]. The RSKs are 73–80 %
identical with each other and are mostly divergent in their N- and
C-terminal sequences (Figure 2). The most striking feature of the
RSK family is the presence of two functional and non-identical
phosphotransferase domains within the same polypeptide [16,27].
It is thought that, during evolution, the genes for two distinct
protein kinases have fused, generating a single protein kinase
capable of receiving an upstream activating signal from ERK1/2
to its CTKD (C-terminal kinase domain) and transmitting, with
high efficiency and fidelity, an activating input to its NTKD (Nterminal kinase domain). The kinase domains are connected by a
linker region of approximately 100 aa (amino acids) containing
essential regulatory domains, including hydrophobic and turn
motifs, involved in activation of the NTKD (Figure 3). Because
they possess two separate catalytic domains, the RSKs are part
of two different protein kinase families. The NTKD belongs
to the AGC (protein kinase A, G and C) family, which also
The RSK family of protein kinases
Figure 2
555
Alignment of the amino acid sequence of the RSK isoforms (RSK1–4)
Human RSK (hRSK) sequences comprising the NTKDs and CTKDs of RSK1–4 are indicated by blue and dark brown lines respectively. The activation loop (T-loop) of each kinase domain is indicated
with a yellow line. The conserved ERK1/2-binding sequence and PDZ-binding motifs are indicated by orange and mid-brown lines respectively. The conserved phosphorylation sites involved in RSK
activation are indicated by black circles.
includes Akt (also known as protein kinase B), SGK (serumand glucocorticoid-induced protein kinase) and S6K1/2. In that
respect, the NTKD of RSK is most similar to the kinase domain
of S6K1, with 57 % aa identity. The CTKD of RSK belongs to
the CAMK (Ca2 + /calmodulin-dependent protein kinase) family
which also includes AMPK (AMP-activated protein kinase),
MARK (MAP-regulating kinase/microtubule affinity-regulating
kinase) and DAPK (death-associated protein kinase). In addition,
the RSKs also belong to a functional subgroup of CAMKs
termed the MAPKAPK (MAPK-activated protein kinase) family
[8], which comprises MSK1/2 [9], MNKs (MAPK-interacting
kinases) [28], MK2/3 (MAPK-activated protein kinase 2/3) [29]
and MK5 [30]. This group is characterized by the fact that they
are activated by different MAPKs, and also display substantial
homology in their kinase domains [7]. Whereas the NTKD of the
RSKs is responsible for downstream substrate phosphorylation,
the only known function of the CTKD is to activate the NTKD via
autophosphorylation of the hydrophobic motif [27,31,32], and no
exogenous substrates have yet been ascribed to this domain [33].
Although this is true, the idea that the CTKD could phosphorylate
heterologous substrates in trans remains an interesting
possibility.
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556
Figure 3
Y. Romeo, X. Zhang and P. P. Roux
Schematic representation of RSK1 with its major functional domains
RSK is characterized by the presence of two functional domains, the NTKD and the CTKD, which are connected by a linker region of approximately 100 aa. Activation of RSK is associated with
increased phosphorylation at six sites (each shown as black or white circles). These phosphorylation sites are highly conserved in RSK1–4 and were shown to be either essential (black circles) or
accessory (white circles) for RSK activation. The C-terminal end of the protein contains an ERK1/2-docking domain resembling a KIM motif, and the extreme C-terminus comprises a PDZ-binding
motif. Amino-acid numbering refers to human RSK1.
All RSK isoforms interact with ERK1/2 through their
C-terminally located docking domain (Figure 3). This domain
differs from classical D-type ERK1/2 docking domains as it does
not match the best-characterized consensus sequence for such
motifs [34]. Instead, docking domains found in RSKs appear to fit
the KIM (kinase interaction motif) consensus sequence [35]. This
D domain-related motif corresponds to a hydrophobic residue
closely followed by two positively charged lysine or arginine
residues (Leu-Xaa2 -Lys/Arg-Lys/Arg-Xaa5 -Leu). A similar motif
has also been reported in several other MAPK substrates,
including the protein tyrosine phosphatase PTP-SL [36] and the
cAMP-specific phosphodiesterase PDE4D [35]. This domain is
required for ERK1/2 binding to RSKs [37,38], and was shown
to be essential for ERK1/2-mediated activation [39]. D domains
usually increase the affinity of MAPKs for their substrates and
thus the efficiency of their phosphorylation. In some cases, D
domains are responsible for pre-existing complexes between
inactive MAPKs and their substrates. In the case of RSK1
and RSK2, these isoforms were shown to have higher affinity
towards ERK1/2 when in their inactive state than following
mitogen stimulation [39]. Indeed, a complex between RSK
and ERK was found to transiently dissociate for the duration
of ERK1/2 activation and to reassemble upon signal termination
[39]. Autophosphorylation of a serine residue near the ERK1/2
docking domain of RSK1 was found to promote its dissociation
from ERK1/2 [39], providing a mechanism by which active
ERK and RSK dissociate to find their respective substrates.
These finding also suggest that an unknown serine/threonine
phosphatase dephosphorylates the C-terminal domain of RSK,
thereby promoting ERK1/2 binding. In addition to a KIM motif,
the C-terminal tails of all four RSK isoforms contain a type 1
PDZ domain binding motif (Ser-Thr-Xaa-Leu). Although this
motif is not conserved in RSK orthologues from Drosophila
and Caenorhabditis elegans, it was shown to be functional and
c The Authors Journal compilation c 2012 Biochemical Society
probably helps in bringing RSK closer to certain of its targets
[40].
ADULT AND EMBRYONIC TISSUE EXPRESSION
With the exception of RSK4, expression of the RSK mRNAs has
been shown to be ubiquitous in every human tissue tested [41].
This is consistent with approximate expression patterns inferred
from EST (expressed sequence tag) sources from both mouse and
human origins, which suggest that RSK4 is in low abundance
in both embryonic and adult tissues (Figure 4A). Although
these results support functional redundancy, available Affymetrix
microarray data demonstrate important tissue variations between
the RSK isoforms (BioGPS) [42], suggesting at least some
isoform-specific functions (Figure 5). Consistent with this,
more targeted studies have shown that the RSK1 mRNA is
predominantly found in the lung, kidney and pancreas, whereas
both RSK2 and RSK3 mRNAs are more abundant in skeletal
muscle, heart and pancreas [41,43,44]. In the brain, the RSK1
mRNA was found to be expressed in the granular cell layer of the
cerebellum, whereas RSK2 was more present in the neocortex,
hippocampus and cerebellum. RSK3 is also very abundant in
the central nervous system, with high mRNA expression in the
cerebral cortex, the dentate gyrus and the amygdala [41,45]. With
regards to RSK4, its expression is much lower than RSK1–3,
but Northern blot analysis has revealed the presence of the RSK4
mRNA in the brain, heart, cerebellum, kidney and skeletal muscle,
whereas other tissues such as lung, liver, pancreas and adipose
tissue showed no detectable RSK4 expression [46].
Developmental expression of the RSK isoforms has also been
determined. The mouse RSK2 mRNA was found to be expressed
at a very low level compared with RSK3, whose mRNA is
very abundant in fetal tissues [47]. RSK3 expression can be
The RSK family of protein kinases
557
isoforms, RSK4 does not significantly accumulate in the nucleus
following mitogenic stimulation. The mechanisms involved in
RSK translocation to the nucleus remain elusive, but the small
death effector domain protein PEA-15 (phosphoprotein enriched
in astrocytes, 15 kDa) has been shown to interact with RSK2
and inhibit its nuclear translocation [53]. Interestingly, RSK2 was
recently found to associate with TIA-1 (T-cell-restricted
intracellular antigen-1) and localize to stress granules upon
oxidative stress [54], suggesting the possibility that PEA-15 may
also localize to these structures. RSK3 is the only isoform to
possess a classical NLS (nuclear localization signal), consisting of
Lys-Lys-Xaa10 -Leu-Arg-Arg-Lys-Ser-Arg, but the functionality
of this domain has never been tested. Compared with other
isoforms, RSK3 is often found associated to detergent-resistant
cellular fractions, indicating that this isoform may localize to
distinct cellular compartments (P. P. Roux, unpublished work).
At the moment, very little is known about isoform-specific
functions of the RSKs, but apparent differences in their subcellular
localization probably determine their respective roles.
CANONICAL PATHWAY OF ACTIVATION
Figure 4
Approximate expression patterns of the RSK isoforms
Relative expression of RSK1–4 is indicated using approximate expression patterns inferred
from EST sources from mouse or human origins. Data were retrieved from the UniGene
database (http://www.ncbi.nlm.nih.gov/unigene) and are shown as transcripts per million.
(A) The abundance of each RSK isoform was averaged between different human organs and
compared with levels found in embryonic tissues. (B) Expression of the RSK isoforms in normal
tissues was compared with levels found in tumour specimens. The latter was averaged from 15
different tumour types and compared with normal tissues.
detected in the ventricular zone, a site of high proliferative activity.
Conversely, RSK1 expression is strongest in the neuroepithelium
of the forming neural tube, whereas at later stages it decreases
dramatically and becomes undetectable in the nervous system.
These results are consistent with a temporal regulation of RSK1
and RSK3 expression and support the requirement of
RSK1 in early development and RSK3 in later development
of the nervous system. These results are also corroborated
with approximate expression patterns inferred from EST sources
(Figure 6). At late stages of development, RSK1 is highly
expressed in regions harbouring highly proliferating cells. These
include liver, lung, thymus, olfactory and gut epithelia. RSK4
was found to be ubiquitously expressed throughout development
[47], and approximate expression patterns from EST sources
suggest that RSK4 is more highly expressed in specific phases
of embryogenesis (Figure 6). Whereas both Rsk1 and Rsk3
genes give rise to only one transcript, Northern blot analysis
of Rsk2 expression revealed the alternative use of two different
polyadenylation sites giving rise to two transcripts of 3.5 and
8.5 kb [41]. Similarly, two secondary Rsk4 transcripts (5 and 9 kb)
also exist, but whether they result from alternative splicing or
alternative polyadenylation remains unknown [48].
SUBCELLULAR LOCALIZATION
At the subcellular level, RSK1–3 are usually present in
the cytoplasm of quiescent cells, but upon stimulation, a
significant proportion of these proteins translocates to the nucleus
(Figure 1) [49–53]. Within minutes of stimulation, RSK1 was
shown to accumulate transiently at the plasma membrane,
where it presumably receives additional inputs necessary for
activation before nuclear translocation [52]. RSK4 appears to
be predominantly cytoplasmic [46], and contrary to other RSK
Understanding the mechanisms underlying RSK activation was
several years in the making, mostly due to the complex nature
by which several kinases induce sequential phosphorylation
events (Figure 7). All RSK isoforms, including C. elegans and
Drosophila melanogaster RSK orthologues, contain the four
essential phosphorylation sites (Ser221 , Ser363 , Ser380 and Thr573
in human RSK1) responsive to mitogenic stimulation [55].
Phosphorylation of Ser221 is mediated by PDK1 (3 phosphoinositide-dependent protein kinase 1) [56,57], a
constitutively active serine/threonine kinase that regulates several
other AGC family members, including Akt, SGK and S6K [58].
Consistent with this, mitogens do not stimulate RSK1–3 activity
in PDK1-deficient cells [59], and PI3K (phosphoinositide 3kinase) inhibitors do not affect RSK1–3 activation. Although
PDK1-dependent activation of Akt was shown to require a
functional PH (pleckstrin homology) domain on PDK1, activation
of RSK1–3 can still occur in the absence of this domain
[56,60], indicating that RSK activation is not restricted to PIP3
(phosphatidylinositol 3,4,5-trisphosphate)-containing membrane
domains. Interestingly, whereas PDK1 is required for mitogenic
stimulation of RSK1–3, RSK4 does not appear to require PDK1
to maintain its high basal activity [46].
Ser363 and Ser380 are located in the linker region within
sequences that are conserved among most AGC family kinases.
These sequences are termed the ‘turn motif’ and the ‘hydrophobic
motif’ respectively [61]. Ser363 is followed by a proline residue
and has been shown to be phosphorylated by ERK1/2, but whether
it also undergoes autophosphorylation and/or phosphorylation by
a heterologous kinase remains unclear [57]. The hydrophobic
motif containing Ser380 is phosphorylated by the CTKD [32],
but the involvement of the NTKD or a heterologous kinase in
Ser380 phosphorylation cannot be excluded. The phosphorylated
hydrophobic motif of RSK serves as a docking site for PDK1
by binding to the PIF (PDK1-interacting fragment) pocket of
PDK1 [62]. For RSK2, this interaction has been shown to increase
the catalytic activity of PDK1 severalfold, indicating that this
motif functions to both recruit and activate PDK1 [62]. PDK1
binding promotes its ability to phosphorylate the RSK NTKD
activation loop (Ser221 ), a phosphorylation event that leads to full
activation of this kinase domain in collaboration with binding of
the phosphorylated hydrophobic motif to the PIF pocket in the
RSK NTKD [63].
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Figure 5
Y. Romeo, X. Zhang and P. P. Roux
Expression pattern of the RSK1–4 mRNAs across human and mouse tissues
Relative expression of the RSK1–4 mRNAs derived from fluorescence intensities of multiple probes for each transcript arrayed on Affymetrix microarray chips. Intensity values were summarized
using the data-processing algorithm gcrma, and data were retrieved from the BioGPS database.
c The Authors Journal compilation c 2012 Biochemical Society
The RSK family of protein kinases
Figure 7
Figure 6 Approximate expression patterns of the RSK isoforms during
mouse development
Relative expression of RSK1–4 is indicated using approximate expression patterns inferred from
EST sources from mouse embryonic tissues. Data were retrieved from the UniGene database
(http://www.ncbi.nlm.nih.gov/unigene) and are shown as transcripts per million.
Thr573 is also followed by a proline residue and is located in
the activation loop of the CTKD. This residue is phosphorylated
by ERK1/2 [37,64] and might contribute to RSK translocation to
the plasma membrane [57], where it can be further activated by a
membrane-associated kinase. This hypothesis is supported by the
discovery that a myristoylated RSK1 mutant that cannot associate
with ERK1/2 displays increased phosphorylation on Ser363 and
Ser380 [57], which suggests that membrane translocation of RSK1
might have an important role in its activation.
The use of phosphomimetic mutations introducing a negative
charge that functionally mimicked phosphorylation of Ser363 ,
Ser380 and Thr573 residues led to the prediction of a hierarchical
phosphorylation cascade for the activation of RSK [55]. The
current model of RSK activation is that ERK1/2 (and potentially
ERK5) phosphorylate the activation loop of the CTKD on
Thr573 [37,65,66]. This phosphorylation event requires ERK
docking to the KIM domain of RSK and a proline-directed
ERK consensus sequence in the RSK activation loop. ERK1/2
also phosphorylate Thr359 /Ser363 in the linker region [55].
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Model of RSK activation by sequential phosphorylation
The KIM motif on RSK promotes its interaction with ERK and facilitates RSK activation by
the MAPK pathway (step 1). ERK activation results in the phosphorylation of Thr573 in the
CTKD of RSK (step 2). The activated CTKD autophosphorylates RSK at its hydrophobic motif,
Ser380 (step 3). ERK might also phosphorylate Thr359 and Ser363 in the linker region. PDK1,
a constitutively active serine/threonine kinase, docks at the phosphorylated hydrophobic motif
and in turn phosphorylates Ser221 in the NTKD activation loop sequence, resulting in complete
RSK activation (step 4). This is followed by autophosphorylation of Ser737 by the NTKD, which
in the case of RSK1 and RSK2 results in dissociation of ERK from the KIM motif of these RSK
isoforms (step 5). Finally, activated RSK phosphorylates substrates located throughout the cell
using its NTKD (step 6), as no exogenous substrates have yet been identified for the CTKD.
Phosphorylation of Ser363 stimulates the NTKD catalytic activity
by an unknown mechanism. Phosphorylation of Thr573 activates
CTKD autophosphorylation on Ser380 , which is located within
the hydrophobic motif of aromatic residues in the linker [32].
Phosphorylation of Ser380 then creates a docking site for PDK1
[62], which, in turn, phosphorylates Ser221 in the activation
loop of the NTKD [56,57]. Once PDK1 dissociates from RSK,
phosphorylated Ser380 binds a phosphate-binding site in the
NTKD, resulting in a stable association between the hydrophobic
motif and a proximal hydrophobic pocket within the NTKD. The
αC-helix then collaborates with phosphorylated Ser221 to stabilize
NTKD in an active conformation, resulting in synergistic full
activation of RSK. Phosphorylation of RSK at its C-terminus
releases ERK1/2 and thereby allows active RSK to translocate to
its many cytoplasmic and nuclear substrates (Figure 7).
ALTERNATIVE MECHANISMS OF ACTIVATION
Recent evidence indicates that RSK2 is also phosphorylated on
tyrosine residues in response to FGF (fibroblast growth factor)
receptor [67] and Src activation [68]. These phosphorylation
c The Authors Journal compilation c 2012 Biochemical Society
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Figure 8
of RSK
Y. Romeo, X. Zhang and P. P. Roux
Chemical structures of recently described selective inhibitors
Whereas the kaempferol glycoside SL0101 and the dihydropteridinone BI-D1870 are
ATP-competitive inhibitors of the RSK NTKD, the pyrrolopyrimidine FMK is a covalent inhibitor
of the CTKD.
events were found to activate RSK2 by stabilizing the association
between ERK1/2 and RSK2, suggesting an alternative mechanism
for RSK activation in human tumours with activated FGFR3
(fibroblast growth factor receptor 3) signalling and in response
to normal EGF (epidermal growth factor) receptor activation.
Ser380 phosphorylation may also occur in a CTKD-independent
manner [52,69], since mutational inactivation of the CTKD
only partially inhibits activation of the NTKD of RSK1
[33,39]. Interestingly, the related MK2/3 enzymes were found
to phosphorylate Ser380 in several cell types, which may explain
how various p38-mediated stresses lead to RSK activation [70].
In addition to phosphorylating RSK, ERK1/2 may also promote
RSK1 activation by facilitating its recruitment to the plasma
membrane, as suggested by the apparent constitutive activation
of the myristoylated RSK mutant [52].
A new point of cross-talk between PKA (protein kinase A) and
ERK1/2 signalling pathways was recently identified and shown
to be involved in both PKA activity and the cellular distribution
of active RSK1 [71,72]. Inactive RSK1 was found to interact
with the PKA regulatory I subunit and thereby stimulate cAMPdependent PKA function, whereas activated RSK1 was shown to
interact with the PKA catalytic subunit, thereby decreasing the
ability of cAMP to stimulate PKA. RSK inactivation may require
PP2Cδ (protein phosphatase 2Cδ), which was shown to associate
with RSK1–4 [73]. Inactivation of RSK activity may also involve
its autophosphorylation at Ser732 , which was found to promote
ERK/RSK dissociation and correlate with reduced RSK kinase
activity [39].
(Figure 8) have been identified [74]. The dihydropteridinone
BI-D1870 is a reversible inhibitor that competes with ATP by
binding to the NTKD ATP-interacting sequence. BI-D1870 is
remarkably selective for RSK relative to other AGC kinases [75],
and its in vitro IC50 was shown to be approximately 15–30 nM
at an ATP concentration of 100 μM [76]. In vivo results indicate
that to completely inhibit the phosphorylation of RSK substrates
in cells, a concentration of 10 μM BI-D1870 is required [76].
The kaempferol glycoside SL0101 is another ATP-competitive
inhibitor of the NTKD, and was in fact the first identified specific
inhibitor of RSK [77]. SL0101 is a natural product obtained from
the tropical plant Fosteronia refracta and was reported to inhibit
RSK2 with an IC50 of 90 nM at an ATP concentration of 10 μM.
The EC50 of SL0101 was found to be approximately 50 μM in
intact cells [77], indicating that the activity of SL0101 in intact
cells is significantly weaker than in vitro. [39]. Like BI-D1870,
SL0101 was tested against a panel of purified kinases and was
found to be relatively specific to the RSK [75]. In contrast, the
pyrrolopyrimidine FMK (Z-VAD-FMK, benzyloxycarbonyl-ValAla-DL-Asp-fluoromethylketone) is an irreversible inhibitor that
covalently modifies the CTKD of RSK1, RSK2 and RSK4 [78].
FMK is a potent and specific inhibitor of RSK, and was shown
to inhibit RSK2 with an in vitro IC50 of 15 nM and an EC50 of
200 nM. FMK contains a reactive electrophile in its fluoromethylketone motif and, in this instance, derives its potent kinase activity
from the covalent addition of its chloromethylketone functionality
to the thiol group of Cys436 located in the ATP-binding pocket of
the RSK2 CTKD. Despite having a mechanism of action involving
covalent addition to the molecular target, FMK was shown to
be remarkably RSK-specific. In human epithelial cell lysate,
containing thousands of proteins, biotin-labelled FMK was found
to only react with RSK1 and RSK2, which were identified using
quantitative immunodepletion with specific antibodies. Therefore,
there are now three potent and highly specific inhibitors of RSK,
which can be used as chemical probes for dissecting the complex
signalling events associated with RSK, and additionally, their
potential as therapeutic candidates.
SUBSTRATE RECOGNITION
The substrate specificity of RSK1 for target phosphorylation
was originally determined using synthetic peptides and found
to require the minimum motifs Arg/Lys-Xaa-Arg-Xaa-XaapSer/Thr or Arg-Arg-Xaa-pSer/Thr [79]. Recently, we have used
an arrayed positional scanning peptide library to determine
the consensus phosphorylation motif of RSK1 and also found
a stronger requirement for the − 3 position arginine residue
compared with the − 5 position residue (P. P. Roux, unpublished
work). In addition, RSK1 appeared to greatly prefer arginine
residues to lysine, which is in agreement with the majority of
RSK substrates identified to date (Table 1 and Figure 9). These
analyses also indicated a propensity for RSK to phosphorylate
serine rather than threonine residues by a factor of at least fivefold, and, consistently, the majority of identified RSK substrates
have a serine residue as the phosphoacceptor site (Figure 9).
Overall, only three putative RSK substrates were shown to be
phosphorylated on threonine residues, casting some doubt as to
whether these proteins are bona fide RSK substrates in cells.
PHARMACOLOGICAL INHIBITORS
RSK activation is closely linked to ERK1/2 activity, and
therefore MEK1/2 inhibitors (U0126, PD98059 and PD184352)
have been and are still widely used to study RSK function
(Figure 1). To date, three different classes of RSK inhibitors
c The Authors Journal compilation c 2012 Biochemical Society
BIOLOGICAL FUNCTIONS
Although a certain number of RSK functions can be deduced
from the nature of its substrates, accumulating evidence suggests
that the RSK isoforms regulate nuclear signalling, cell-cycle
The RSK family of protein kinases
Table 1
561
Phosphorylation substrates of the RSK isoforms
Phosphorylated residues are in bold. * indicates the end of the sequence and underlined residues indicate basic residues forming the RSK consensus sequences. Abbreviations: CCTβ, chaperonin
containing TCP-1 (T-complex polypeptide 1), β subunit; CRHSP24, calcium-regulated heat-stable protein of apparent molecular mass 24 kDa; GAP, guanine nucleotide-activating protein; h, human;
m, mouse; MEF2C, myocyte enhancer factor 2C; METTL1, methyltransferase-like protein 1; mTORC1, mTOR complex 1; NFAT3, nuclear factor of activated T-cells 3; r, rat; SHANK, Src homology 3
domain and ankyrin repeat-containing protein-1; SRE, sterol-regulatory element; x, Xenopus ; YB-1, Y-box-binding protein-1.
Substrate
Phosphorylation site(s)
Proposed biological functions
Isoform(s)
References
GSK3β
c-Fos
NHE-1
MITF
ERα
LKB1
p27kip1
SOS1
RanBP3
YB-1
Mad1
Myt1
Bub1
Erp1
eEF2K
eIF4B
rpS6
TSC2
METTL1
Bad
DAPK
C/EBPβ
nNOS
CREB
SRF
ER81
Nur77
IκBα
NFATc4
NFAT3
TIF-1A
ATF4
SH3P2
ATF1
MEF2c
Filamin A
AS160
Raptor
CCTβ
CRHSP24
SHANK
RPRTTS9 FAEPG (r)
AHRKGS362 SSNEP (r)
RARIGS703 DPLAY (h)
SSRRSS409 MSAEE (h)
RERLAS167 TNDKG (h, r)
KIRRLS431 ACKQQ (m)
LRRRQT198 * (h)
RRRPES1161 APAES (h)
RERTSS58 LTHSE (m)
RKYLRS102 VGDGE (h)
RIRMDS145 IGSTV (h)
Serine residues (x)
Not determined
RCRRLS335 T336 LRER (x)
RVRTLS366 GSRPP (h)
RSRTGS422 ESSQT (h)
KRRRLS235 S236 LRAST (h)
RKRLIS1798 SVEDF (h)
RQRAHS27 NPMAD (h)
RSRHSS112 YPAGT (h, r)
LSRKAS289 AVNME (h)
PSKKPS105 DYGYV (r) AKAKKT217 VDKLS (m)
KVRFNS847 VSSYS (r)
LSRRPS133 YRKIL (h, m)
LKRSLS103 EMEIG (h)
FRRQLS191 EPCNS (h, m) YQRQMS216 EPNIP (h, m)
RGRLPS354 KPKQP (m)
DDRHDS32 GLDSM (r)
GRRKRS676 PTQSF (h, m)
Serine residues (h, m)
LYMQPS649 PL* (h)
PSPGGS251 RGSPR (m) GSPNRS245 LPSPG (h)
AVRTLS202 NAEDY
LARRPS63 YRKIL (h)
LHRNVS192 PGAPQ (r)
RRRAPS2152 VANVG (h, r)
RRRHAS341 APSHV (h) RSRCSS318 VTGVQ (h) RGRLGS588 VDSFE (h) RKRTSS751 TCSNE (h)
TPRLRS719 VS721 S722 YGNI (h)
RVRVDS260 TAKVA (h)
RTRTFS52 ATVRA (h)
Not determined
Inhibits GSK3β activity
Stabilizes c-Fos and increases its activity
Promotes cell proliferation
Increases transactivation potential
Regulates AF1 transcriptional activation
Required for LKB1 growth suppression
Promotes cytoplasmic retention
Potential negative feedback
Regulation of nuclear transport
Promotes transcriptional activation
Promotes its ubiquitination and degradation
Inactivating
Promotes its kinase activity
Stabilizes and increases activity
Inhibits its kinase activity
Enhances interaction to eIF3
Promotes rpS6 binding to the mRNA cap
Inhibits TSC2 activity
Reduces its tRNA methylase activity
Suppresses Bad-mediated apoptosis
Suppresses its pro-apoptotic function
Stimulates hepatocyte proliferation
Blocks toxic accumulation of NO
Stimulates its transcriptional activity
Enhances its affinity for the SRE motif
Enhances its transactivation potential
No clear function
Increases NF-κB activity
promotes DNA binding
Induces NFAT3 nuclear accumulation
Enhances cellular pre-rRNA synthesis
Promotes collagen synthesis
Promotes cell motility
Induces ATF1 transcriptional activity
Stimulates MEF2C transcriptional activity
Controls actin cytoskeletal dynamics
14-3-3 binding and inactivation of its GAP activity
Increases mTORC1 activity
Plays a role in cytoskeletal reorganization
No clear function
Enhances synaptic transmission
RSK2
RSK1/2
RSK1/2
RSK1
RSK1/2
RSK1
RSK1/2
RSK2
RSK1/2
RSK1/2
RSK1/2
RSK1
RSK1
RSK2
RSK1
RSK1/2
RSK1/2
RSK1
RSK
RSK1/2
RSK1/2
RSK1
RSK1
RSK1
RSK1
RSK1
RSK2
RSK1
RSK1
RSK2
RSK2
RSK2
RSK1
RSK2
RSK2
RSK1/2
RSK1
RSK1/2
RSK1/2
RSK1/2
RSK2
[113,180]
[88,91,109]
[162]
[97]
[94]
[117]
[111]
[166]
[163]
[164]
[115]
[121]
[127]
[129,131]
[146]
[147]
[23]
[140,181]
[182]
[150,153]
[155]
[152]
[161]
[82–84,150]
[81,87]
[96]
[27,89,90]
[99,100]
[183]
[184]
[93]
[107,185]
[159]
[186]
[187]
[156]
[165]
[142]
[188]
[189]
[40]
Figure 9
WebLogo frequency plot of the target sequences in known RSK substrates
RSK phosphorylation sites in substrates show a strong preference for arginine residues in the − 5 and − 3 positions relative to the phosphoacceptor site indicated by an asterisk. The latter is more
often a serine residue, as very few validated RSK substrates were shown to be phosphorylated on threonine residues.
c The Authors Journal compilation c 2012 Biochemical Society
562
Y. Romeo, X. Zhang and P. P. Roux
progression and cell proliferation, cell growth and protein
synthesis, cell migration and cell survival (Table 1). RSK2 has
been extensively studied and, to date, most substrates have been
identified for this isoform. Nevertheless, most studies have not
determined isoform selectivity. Therefore many known RSK2
substrates may be shared by other RSK family members and
vice versa. Thus more effort will be necessary to assess potential
overlapping functions between the RSK isoforms. In addition,
the RSK consensus phosphorylation motif is shared by other
AGC family members, such as Akt and S6K1, suggesting that
an important functional overlap may exist between these related
basophilic protein kinases. Indeed, several RSK substrates
have been shown to be also targeted by Akt [TSC2 (tuberous
sclerosis complex 2), Bad (Bcl-2/Bcl-xL-antagonist, causing
cell death), GSK3 (glycogen synthase kinase-3) and p27kip1
(cyclin-dependent kinase inhibitor 1B)] and S6K1 [eEF2K
(eukaryotic elongation factor-2 kinase), rpS6, GSK3 and eIF4B
(eukaryotic initiation factor 4B)]. This needs to be kept in mind
when analysing the phenotype of knockout mice as compensation
may occur between these highly related kinases.
Transcriptional regulation
Global profiling analysis of RSK has been reported and identified
RSK as an important effector of ERK in global transcriptional
regulation [80]. RSK was found to regulate ∼ 20 % of mRNAs
controlled by ERK in MDCK cells (Madin–Darby canine kidney
cells) through direct and indirect mechanisms, such as induction
of transcription factors. Indeed, activated RSK was shown
to phosphorylate several transcription factors, some of which
contribute to the IEG (immediate-early gene) response or are
IEG products themselves. Two studies using human cells from
CLS (Coffin–Lowry Syndrome) patients and primary fibroblasts
isolated from Rsk2 − / − mice showed that RSK2 mediates mitogeninduced c-Fos transcription by activating both the Elk1 [ETS (E
twenty-six)-like kinase 1]/SRF (serum-response factor) complex
and CREB (cAMP-response-element-binding protein) [81,82].
Although RSK2 was shown to phosphorylate CREB at Ser133
[83,84], more recent evidence indicated that the related MSK1/2
protein kinases are the predominant CREB kinases following
mitogenic stimulation or stresses in somatic cells [85]. Residual
CREB phosphorylation in cells derived from Msk1 − / − Msk2 − / −
mice suggested that RSK might co-operate with MSK1/2 for
the phosphorylation of CREB induced by mitogens, but not
cellular stresses, although more work will be needed to strengthen
this hypothesis. Consistent with this idea, histone H3 was also
initially found to be regulated by RSK, but convincing evidence
indicated that MSK1/2 are the predominant histone H3 kinases
operating in response to stress or mitogenic stimulations [86]. In
addition, RSK1 was shown to participate in the post-translational
modification of the IEG products SRF [87], c-Fos [88] and Nur77
[27,89,90]. Regulation of c-Fos by RSK2 was shown to play
important roles in bone homoeostasis and tumorigenesis [91,92].
Data from several groups point towards a role for RSK in
regulating growth-related transcription initiation. Upon serum
stimulation, RSK2 phosphorylates one serine residue important
for the function of TIF-1A, a transcription initiation factor
required for RNA polymerase I transcription and rRNA synthesis
[93]. Growth factor-activated RSK1 also associates with and
phosphorylates ERα (oestrogen receptor α) on Ser167 , which
increases ERα-mediated transcription [94,95]. RSK may also
be responsible for transcriptional control of genes involved in
homoeostasis, signalling response and development, as RSK
interacts with the ETS transcription factor ER81 and enhances
ER81-dependent transcription by phosphorylating Ser191 and
c The Authors Journal compilation c 2012 Biochemical Society
Ser216 [96]. The RSKs were also found to phosphorylate the
microphthalmia (mi) transcription factor [97], which has been
linked to malignant melanoma [98].
RSK regulates the activity of nuclear proteins involved in
the inflammatory response, such as the transcription factor
NF-κB (nuclear factor κB). RSK1 phosphorylates the inhibitory
proteins IκBα (inhibitor of NF-κB α) and IκBβ on sites that
promote their degradation, thereby stimulating NF-κB activity
[99–101]. Related to this, RSK was also suggested to promote
p65 phosphorylation at Ser536 [102,103], but the involvement
of RSK in Ser65 phosphorylation is still unclear [104]. Activated
RSK1 binds to the transcriptional co-activators CBP (CREBbinding protein) and p300 [105,106], but the exact outcome of
this association remains to be determined. Interestingly, CBP
also interacts with transcription factors that are phosphorylated
by RSK1 and RSK2, such as CREB, c-Fos, ER81, ERα and
NF-κB, suggesting that RSK may help in the recruitment of CBP
and p300 cofactors to promoters regulated by these transcription
factors. The CREB family member ATF4 (activating transcription
factor-4) is a transcription factor required for the timely onset
of osteoblast differentiation during development. RSK2 was
found to phosphorylate and thereby control ATF4 activity [107],
suggesting a mechanism by which loss of RSK2 may lead to
CLS-associated skeletal abnormalities.
Cell-cycle progression and cell proliferation
RSK has been directly involved in the regulation of cell-cycle
progression and cell proliferation. Treatment with the SL0101
inhibitor or RNAi against RSK1 and RSK2 was shown to inhibit
proliferation of cells derived from human prostate and breast
tumours, where both isoforms are also overexpressed (P. P. Roux,
unpublished work) [77,108]. These data indicate that RSK1 and
RSK2 positively regulate cancer-cell proliferation, and consistent
with this affirmation, approximate expression patterns inferred
from EST sources indicate that RSK1 is often found in higher
abundance in tumours compared with normal tissues (Figure 4B).
Although this is not the case for RSK2, it is also interesting to
note that RSK3 and RSK4 are usually found in lower abundance
in tumour specimens (Figure 4B).
RSK controls cell proliferation through the regulation of
mediators of the cell cycle. RSK2 was shown to promote cellcycle progression by phosphorylating c-Fos, a transcription factor
that positively regulates expression of cyclin D1 during G1 /S
transition. RSK2-mediated phosphorylation of c-Fos at Ser362
promotes its stability and oncogenic properties, and was found
to be essential for osteosarcoma formation in mice [91,109,110].
RSK1 and RSK2 may also regulate G1 phase progression by
controlling the activity of the CDK2 (cyclin-dependent kinase
2) inhibitor p27kip1 [111]. RSK-mediated phosphorylation of
p27kip1 on Thr198 prevents its translocation to the nucleus by
promoting its association with 14-3-3 [111,112]. RSK also
mediates cell-cycle progression by negatively regulating GSK3
[113], which targets c-Myc and cyclin D1 for degradation
[114]. More recently, Mad1 (mitotic arrest-deficient protein 1), a
suppressor of Myc-mediated cell proliferation and transformation,
was shown to be phosphorylated by RSK on Ser145 [115]. This
phosphorylation event results in increased degradation of Mad1
by the ubiquitin–proteasome system, leading to enhanced Mycdependent transcription and cell proliferation. Inhibition of RSK
activity was shown to prevent the kinetochore localization of
Mad1 and Mad2, suggesting that RSK may participate in the
spindle assembly checkpoint [116]. In addition, RSK2-mediated
phosphorylation on Ser431 of the tumour suppressor LKB1
(liver kinase B1), a kinase found mutated in the cancer-prone
The RSK family of protein kinases
Peutz–Jeghers syndrome [117], inhibits LKB1-mediated growth
suppression in melanomas through an unknown mechanism [118].
The role of RSK in G2 /M progression has been established
using the preferred model of Xenopus oocyte maturation.
Immature oocytes are arrested in late G2 phase of the first
meiotic cell division. Upon progesterone exposure, the synthesis
of the MAPKKK (MAPK kinase kinase) c-Mos activates
the MEK1/ERK/RSK cascade leading to M phase entry and
subsequent maturation to an unfertilized egg. M phase entry is
controlled in part by Cdc2 (cell division cycle 2 kinase), a CDK
that is regulated through phosphorylation on both Thr14 and Tyr15
by the inhibitory kinase Myt1 (myelin transcription factor 1).
Among the RSK isoforms, RSK2 is the most highly expressed in
Xenopus oocytes [119] and was shown to participate in the control
of critical stages of the meiotic cell cycle [120], including G2 /M
progression during meiosis I by phosphorylating and inhibiting
the Myt1 kinase [121–123]. In fact, Myt1 was the first identified
substrate of RSK [121], providing a direct link between RSK and
the cell cycle. RSK2 was also found to promote Cdc2 activation
by directly phosphorylating and activating Cdc25C, an enzyme
involved in the activation of Cdc2 complexed to cyclin B [124].
RSK also modulates the meiotic cell cycle in Xenopus through
ERK-mediated metaphase II arrest, an activity known as CSF
(cytostatic factor) [125,126]. RSK1 is thought to participate until
metaphase II arrest in part by phosphorylating and activating the
kinase Bub1, a mediator of APC (anaphase-promoting complex)
inhibition [127,128]. More recently, Emi2 [early mitotic inhibitor
2, also called Erp1 (Emi1-related protein)], another APC inhibitor,
has been identified as a RSK substrate. Phosphorylation of
Emi2 by RSK promotes Emi2–PP2A (protein phosphatase 2A)
association, facilitating Emi2 dephosphorylation at specific Cdc2
phosphorylation sites, which in turn enhances Emi2 stability and
function [129–131].
RSK1 and RSK2 recently emerged as essential regulators of
tumorigenesis. Ectopic expression of RSK2 was demonstrated
to increase proliferation as well as anchorage-independent
transformation [132]. In addition, RSK2 is thought to
mediate FGFR3-dependent transformation of haemopoietic cells,
as FGFR3 was shown to activate RSK2 by facilitating both
ERK–RSK2 interaction and subsequent phosphorylation of RSK2
by ERK in these cells [67]. In contrast to what was initially
believed [51], RSK3 was recently shown to act as a potential
tumour suppressor in ovarian cancer [133]. RSK3 expression
was found to be downregulated in ovarian cancer, and cells
overexpressing RSK3 displayed reduced proliferation due to G1
arrest and increased apoptosis. At the moment, the mechanisms
by which RSK3 negatively regulates cell proliferation are still
unknown. The role of RSK4 on proliferation is even more
enigmatic. As for RSK3, several lines of evidence suggest that
RSK4 behaves as a negative regulator of cell proliferation.
Exogenous expression of RSK4 resulted in decreased breast
cancer cell proliferation and increased accumulation of cells in
G0 /G1 phase of the cell cycle [134]. In addition, RSK4 was found
to play an inhibitory role during embryogenesis by suppressing
RTK (receptor tyrosine kinase) signalling [135], as well as to
participate in p53-dependent cell growth arrest [136] and in
oncogene-induced cellular senescence [137]. Clearly, RSK3 and
RSK4 appear to play separate functions to their brethren, and
more in-depth studies will be required to fully understand the
underlying mechanisms.
Cell growth and protein synthesis
The first evidence linking RSK to translational control came
from the identification of RSK1 as an in vitro ribosomal protein
563
(rp) S6 kinase [15,18]. However, S6K1 and S6K2 were later
shown to be the predominant rpS6 kinases operating in somatic
cells [20]. A few years later, RSK was found to associate with
polysomes [138], which are ribosomes that are actively translating
mRNAs. More recently, studies using S6k1 − / − S6k2 − / − [22] or
rapamycin-treated [23] cells demonstrated that the RSK isoforms
participate in rpS6 phosphorylation in vivo. Upon Ras/MAPK
pathway activation, RSK1/2-mediated phosphorylation of rpS6
at both Ser235 and Ser236 was found to promote assembly of
the cap-binding complex and to correlate with increased capdependent translation [23]. These findings strongly support RSK
as an important regulator of mRNA translation that provides
an additional oncogene- and mitogen-regulated input into the
regulation of translation initiation. These data also indicate
that rpS6 phosphorylation can occur in an mTOR-independent
manner.
mTOR is an essential regulator of ribosome biogenesis, mRNA
translation and cell growth, and its activity is controlled by several
growth-regulating pathways. Activated RSK promotes mTOR
signalling through the phosphorylation of TSC2 on Ser1798 , which
prevents its GAP (guanine nucleotide-activating protein) activity
towards the small GTPase Rheb [139–141]. More recently, RSK
was shown to phosphorylate Raptor, an important mTORC1
(mTOR complex 1) scaffolding protein, providing another link
between the Ras/MAPK and mTOR signalling pathways [142]. It
is becoming apparent that certain cell types are more dependent
on RSK activity for the regulation of mTOR signalling than
others, such as cancer cells harbouring activating mutations in
components of the Ras/MAPK pathway (P. P. Roux, unpublished
work).
RSK may also regulate mRNA translation through the
phosphorylation of GSK3β [113,138]. Phosphorylation by RSK
on Ser9 inhibits GSK3β kinase activity and thereby releases
inhibition of the translation-initiation factor eIF2B [143].
Interestingly, activated GSK3β and the LKB1-activated kinase
AMPK also stimulates TSC2 activity [144,145], suggesting that
RSK regulates TSC2 directly or indirectly. In addition, RSK
phosphorylates eEF2K [146] and the translation-initiation factor
eIF4B [147]. RSK-mediated phosphorylation of eIF4B stimulates
its recruitment to the translation-initiation complex and thereby
probably promotes translation of mRNAs encoding proteins
involved in cell growth and survival [148]. These findings also
suggest that RSK may control several biological processes by
altering the pattern of mRNA translation. Recently, RSK was
found to play a role in mRNA translation mediated by type III IFN
(interferon) (IFNλ) receptors, a novel family of cytokines involved
in certain viral infections and malignancies [149]. An inactive
form of RSK1 was found to associate with 4E-BP1 (eIF4Ebinding protein 1), a protein that binds eIF4E and thereby prevents
initiation of mRNA translation. IFNλ-mediated activation
of RSK1 was found to promote 4E-BP1 phosphorylation
on Thr37 /Thr46 , thereby promoting selective cap-dependent
translation of growth-inhibitory genes whose transcription is
induced during engagement of the IFNλ receptor [149].
Cell survival
RSK1 and RSK2 have been shown to positively regulate cell
survival through transcription-dependent mechanisms. RSK2 was
shown to promote survival of primary neurons by increasing
CREB-dependent transcription of survival-promoting genes,
including Bcl-2, Bcl-xL and Mcl1 (myeloid cell leukaemia
sequence 1) [84,150]. More recently, RSK1 was found to
promote survival through the activation of the transcription
factor NF-κB [99,100,151]. RSK also promotes cell survival
c The Authors Journal compilation c 2012 Biochemical Society
564
Y. Romeo, X. Zhang and P. P. Roux
through post-translation mechanisms. Indeed, RSK1-mediated
phosphorylation of C/EBPβ (CCAAT/enhancer-binding protein
β) promotes survival of hepatic stellate cells in response to the
hepatotoxin CCI4 [152]. Phosphorylation on Thr217 was suggested
to create a functional XEVD caspase-inhibitory box that binds
and inhibits caspases 1 and 8. In addition, RSK1 and RSK2
phosphorylate the pro-apoptotic protein Bad on Ser112 , thereby
enhancing its ability to bind, and be inactivated by, cytosolic
14-3-3 proteins [150,153]. More recently, RSK1 and RSK2
were shown to inactivate DAPK. DAPK behaves as a tumour
suppressor and its expression is commonly silenced in cancer
through DNA methylation [154]. RSK-mediated phosphorylation
on Ser289 was found to prevent DAPK pro-apoptotic activity,
resulting in increased cell survival in response to mitogenic
stimulation [155].
Cell migration
A potential role for RSK in cell migration was first suggested by
the identification of the cytoskeleton-associated protein filamin A
as a phosphorylation substrate of both RSK1 and RSK2 [156].
Indeed, these RSK isoforms were suggested to play a role in
membrane ruffling by phosphorylating filamin A on Ser2152 [156],
a site previously shown to be phosphorylated by PAK1 (p21activated kinase-1) and to be necessary for membrane ruffling in
response to PAK1 activation [157]. More recently, RSK1/2 were
identified as key effectors of Ras/ERK-mediated EMT (epithelial–
mesenchymal transition) [80]. RSK1/2 were found to stimulate
motility and invasion by activating a transcriptional programme
that co-ordinately modulates the extracellular environment,
the intracellular motility apparatus and receptors mediating
communication between these compartments. Therefore RSK1
and RSK2 were shown to be important ERK1/2 effectors of
Raf1-dependent migration in MDCK cells [80]. Accordingly,
a genome-wide RNAi screen highlighted a prominent role for
RSK as a common effector for multiple migratory stimuli [158].
Pharmacological inhibitors of all RSK isoforms were used in this
study, suggesting that several RSK isoforms could be involved in
the migratory phenotype [158]. More recently, RSK was shown
to phosphorylate the SH3 (Src homology 3) domain-containing
protein SH3P2 on a residue that inhibits its function [159].
This protein appears to be a negative regulator of cell motility,
indicating a new mechanism by which RSK activation promotes
cell migration.
Other substrates and functions
In addition to their role in cell growth, proliferation, survival and
migration, the RSK isoforms have been shown to phosphorylate
many more substrates involved in diverse cellular processes.
Indeed, RSK2 was linked to neurite outgrowth, since it
phosphorylates the cell adhesion molecule L1 on Ser1152 [160], a
protein that becomes hyperphosphorylated during periods of high
neuronal activity. RSK may also be involved in the regulation
of nitric oxide function in the brain, since EGF exposure of
hippocampal and cerebellar neurons leads to RSK1-mediated
phosphorylation on Ser847 of nNOS [neuronal NOS (nitric oxide
synthase)] and inhibition of NOS activity [161]. Activated
RSK2 was shown to phosphorylate NHE-1 (Na + /H + exchanger
isoform-1) on Ser703 [162], thereby regulating mitogen-dependent
Na + /H + exchange and intracellular pH [162].
RanBP3 (Ran-binding protein 3) phosphorylation by RSK
modulates nucleocytoplasmic protein transport. Both RSK and
Akt phosphorylate Ser58 of RanBP3, and this phosphorylation
c The Authors Journal compilation c 2012 Biochemical Society
event was shown to contribute to the formation of a Ran
gradient essential for nucleocytoplasmic transport, kinetochore
function, spindle assembly, microtubule dynamics and other
mitotic events [163]. RSK-mediated phosphorylation of the
transcription/translation factor YB-1 (Y-box binding protein-1)
on Ser102 promotes its nuclear accumulation [164], suggesting
that RSK signalling may contribute to the oncogenic functions of
YB-1. RSK has been shown to phosphorylate AS160 (Akt
substrate of 160 kDa), a protein implicated in the translocation
of GLUT4 (glucose transporter 4) to the plasma membrane in
response to insulin [165]. Deregulation of GLUT4 translocation
occurs early in the pathophysiology of insulin resistance and
Type 2 diabetes, providing a potential mechanism by which insulin
signalling is altered.
In addition, accumulating evidence suggests the existence of a
RSK-initiated negative-feedback loop that restricts activation of
the Ras/ERK signalling cascade. More than a decade ago, RSK2
was shown to phosphorylate SOS (son of sevenless) in vitro,
a guanine-nucleotide-exchange factor for Ras [166]. Although
these results suggested that RSK may inhibit SOS function and
thereby reduce Ras/ERK pathway activation, the impact of SOS
phosphorylation and the exact sites of phosphorylation have not
been addressed. Interestingly, a subsequent study demonstrated
increased ERK1/2 phosphorylation in skeletal muscle tissue
from RSK2-deficient mice [167], suggesting that RSK is indeed
implicated in a feedback loop that limits ERK1/2 activation. More
recently, inhibition of RSK using the BI-D1870 inhibitor was
shown to increase ERK1/2 phosphorylation in several cell types
[76], suggesting that a normal function of RSK may be to prevent
hyperactivation of the MAPK pathway.
PHYSIOLOGICAL FUNCTIONS
The involvement of the RSK isoforms in physiological functions
was highlighted by the finding that inactivating mutations in
the Rps6ka3 gene (which encodes RSK2) were the cause of
CLS [168]. CLS is an X-linked mental retardation syndrome
characterized in male patients by severe psychomotor retardation,
and facial, hand and skeletal malformations [169]. Female
patients are usually more mildly and variably affected, and
thus more difficult to diagnose. In addition, individuals with
CLS consistently displayed markedly reduced total brain
volume, with the cerebellum and hippocampus being particularly
affected [170]. Rps6ka3 mutations are heterogeneous and most
often result in truncated forms of RSK2 that are devoid of
phosphotransferase activity [171]. RSK2-knockout mice have
been generated and extensively studied. These mice exhibit
deficient learning and cognitive functions, as well as poor coordination compared with wild-type littermates, suggesting that
RSK2 may be required for proper neuronal development and
function [167,172]. Consistently, a recent study demonstrated
that shRNA (small hairpin RNA)-mediated RSK2 depletion
perturbs the differentiation of neural precursors into neurons
and maintains them as proliferating radial precursor cells [173].
Although these findings indicated that other RSK isoforms do
not compensate for the loss of RSK2, the developmental role
of RSK2 in the nervous system remains unknown and will
require further investigation. Deletion of the single Drosophila
RSK isoform leads to learning and conditioning defects [174],
but whether these defaults result from the specific loss of RSK
activity or deregulated ERK activation or function remains to
be determined. RSK2-deficient mice also develop osteopenia, a
progressive skeletal disease caused by cell-autonomous defects in
osteoblast activity [91]. Findings have suggested that the ATF4
The RSK family of protein kinases
and c-Fos transcription factors may be in vivo RSK2 substrates
responsible for osteosarcoma development [91,107]. Accordingly,
ATF4 deletion in the mouse was found to partly phenocopy
the loss of RSK2 [107]. In addition, RSK2-knockout mice are
approximately 15 % smaller than their wild-type littermates, with
a specific loss of white adipose tissue that is accompanied by
reduced serum levels of the adipocyte-derived peptide, leptin
[175]. Although RSK1/RSK2/RSK3 triple-knockout mice are
viable, no other information regarding their phenotype has
yet been reported [176]. The Rps6ka6 gene, which encodes
RSK4, is located on chromosome X and was suggested to be
involved in non-specific X-linked mental retardation [48]. Recent
evidence suggests that Rps6ka6 deletion perturbs early embryonic
development [177], but further experiments will be required to
validate and characterize these preliminary results concerning the
role of RSK4 during development.
CONCLUDING REMARKS
On the basis of recent advances in our understanding of its
biological functions, RSK is emerging as a multifunctional
effector of the Ras/MAPK signalling cascade. These discoveries
have been facilitated by the use of small-molecule inhibitors
and RNAi, which have expanded the repertoire of biological
functions linked to the RSK family. As critical regulators of
several substrates involved in cell growth, proliferation, survival
and motility, it is becoming increasingly important to address the
role of individual RSK isoforms in these biological processes.
Towards this end, the development of knockout animals for the
other RSK isoforms will probably help to resolve their exact
biological and physiological functions. Recently, a large-scale
proteomics approach identified a large number of potential RSK
substrates [178], and such high-throughput approaches will help
define the exact targets and/or partners specific to each RSK
isoform.
Considering the growing number of cancers that exhibit
deregulated RSK expression and/or activity, the roles played by
each RSK family member in human diseases will also require
further investigation. In addition, the discovery of RSK-specific
inhibitors [74] will allow the testing of RSK as a potential
therapeutic target in pre-clinical cancer studies. Research in this
area will hopefully expand our knowledge of RSK signalling
in cancer and reveal novel therapeutic approaches for treatment.
Indeed, targeting RSK in cancer may be a valuable and more
specific alternative to the inhibition of upstream components of the
Ras/MAPK pathway [179], such as MEK1/2, but this interesting
hypothesis will have to be directly evaluated.
FUNDING
Research in the Roux laboratory is supported by the Canadian Cancer Society Research
Institute (CCSRI) [grant number 2011-700878], the Cancer Research Society (CRS) [grant
number DF121153] and a Career Development Award from the Human Frontier Science
Program (HFSP). P.R. holds the Canada Research Chair in Signal Transduction and
Proteomics. X.Z. is the recipient of a studentship from the China Scholarship Council
(CSC). The Institute for Research in Immunology and Cancer (IRIC) is supported in part
by the Canadian Centre of Excellence in Commercialization and Research, the Canada
Foundation for Innovation, and the Fonds de Recherche en Santé du Québec (FRSQ).
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Received 14 February 2011/11 August 2011; accepted 11 August 2011
Published on the Internet 21 December 2011, doi:10.1042/BJ20110289
c The Authors Journal compilation c 2012 Biochemical Society