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CHAPTER TWO
Ribosomal Protein S6
Phosphorylation: Four
Decades of Research
Oded Meyuhas
Department of Biochemistry and Molecular Biology, Institute for Medical Research eIsrael-Canada,
Hebrew University-Hadassah Medical School, Jerusalem, Israel
E-mail: meyuhas@cc.huji.ac.il
Contents
1. Introduction 42
2. rpS6 as an Indispensable Ribosomal Protein 43
3. Phosphorylation of rpS6 44
3.1 S6 Kinase (S6K1 and S6K2) 45
3.2 90-kDa rpS6 Kinase (RSK1eRSK4) 46
3.3 Protein Kinase A 47
3.4 Casein Kinase 1 48
3.5 rpS6 Dephosphorylation 48
4. Signals to rpS6 Phosphorylation 49
4.1 Growth Factors 49
4.1.1 PI3K/Akt/TSC/Rheb/mTORC1/S6K pathway 49
4.1.2 Ras/Raf/MEK/ERK/RSK pathway 50
4.2 Amino Acid Sufficiency 51
4.3 Energy Balance 52
4.4 Oxygen Supply 54
4.5 Osmolarity 55
5. Physiological Roles of rpS6 Phosphorylation 56
5.1 Global Protein Synthesis 56
5.2 Cell Size Regulation 57
5.3 Normal Muscle Function 59
5.4 Hypertrophic Responses 59
5.5 Cell Proliferation 59
5.6 Clearance of Apoptotic Cells 60
5.7 Tumorigenicity 60
5.8 Glucose Homeostasis 62
5.9 rpS6 Phosphorylation as Diagnostic Marker 62
6. Concluding Remarks and Future Perspectives 63
References 64
International Review of Cell and Molecular Biology, Volume 320
ISSN 1937-6448
http://dx.doi.org/10.1016/bs.ircmb.2015.07.006
©2015 Elsevier Inc.
All rights reserved. 41
j
Abstract
The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time
about four decades ago. Since then, numerous studies have shown that this modifica-
tion occurs in response to a wide variety of stimuli on five evolutionarily conserved
serine residues. However, despite a large body of information on the respective kinases
and the signal transduction pathways, the physiological role of rpS6 phosphorylation
remained obscure until genetic manipulations were applied in both yeast and mam-
mals in an attempt to block this modification. Thus, studies based on both mice and
cultured cells subjected to disruption of the genes encoding rpS6 and the respective
kinases, as well as the substitution of the phosphorylatable serine residues in rpS6,
have laid the ground for the elucidation of the multiple roles of this protein and its
posttranslational modification. This review focuses primarily on newly identified kinases
that phosphorylate rpS6, pathways that transduce various signals into rpS6 phosphor-
ylation, and the recently established physiological functions of this modification. It
should be noted, however, that despite the significant progress made in the last
decade, the molecular mechanism(s) underlying the diverse effects of rpS6 phosphor-
ylation on cellular and organismal physiology are still poorly understood.
1. INTRODUCTION
The higher eukaryotic ribosomes are composed of two subunits desig-
nated as 40S (small) and 60S (large) subunits. The mammalian 40S subunit is
composed of a single molecule of RNA, 18S ribosomal (r) RNA, and 33
proteins, whereas the 60S subunit contains three RNA molecules, 5S,
5.8S, and 28S rRNAs, and 46 proteins (Wool et al., 1996). Of all ribosomal
proteins it is ribosomal protein S6 (rpS6) that has attracted much attention,
since it is the first, and was for many years the only one, that has been shown
to undergo inducible phosphorylation.
The ribosome biogenesis takes place in the nucleolus starts with the syn-
thesis of 5S and 45S pre-rRNA by distinct RNA polymerases and requires
the import of ribosomal proteins from the cytoplasm. A complex pathway
that involves both endo- and exonucleolytic digestions enables the release
of mature rRNAs from the pre-rRNA. Concomitantly, the rRNAs are
extensively modified and bound by the ribosomal proteins before the assem-
bled pre-40S and pre-60S subunits are exported separately to the cytoplasm
(Fromont-Racine et al., 2003; Zemp and Kutay, 2007). High-resolution
cytological analysis has disclosed the fate of rpS6 from its biosynthesis site
in the cytoplasm to the pre-40S subunit. Thus, rpS6 enters the nucleus of
HeLa cells, reaches, via Cajal bodies, the nucleolus, where it is assembled
with other proteins and rRNA into pre-40S subunit. The latter is then
42 Oded Meyuhas
released to the nucleoplasm prior to its export through the nuclear pores to
the cytoplasm (Cisterna et al., 2006). Interestingly, the nuclear import, as
well as the nucleolar localization of human rpS6 and yeast rpS6A, relies
on motifs, whose number, nature, and position are evolutionary conserved
(Lipsius et al., 2005; Schmidt et al., 1995).
The phosphorylation of rpS6 has attracted much attention in numerous
labs since its discovery in 1974 (Gressner and Wool, 1974). However, it is
only during the last decade that the role of rpS6 and its posttranslational
modification has started being unveiled by genetic targeting of the rpS6
gene and of the respective kinases. Hence, this review includes an account
the critical role of rpS6 for mouse development, on the enzymes that
conduct its phosphorylation, the cues that affect it, the pathways that trans-
duce various signals into rpS6 phosphorylation, and the physiological role of
this modification.
2. rpS6 AS AN INDISPENSABLE RIBOSOMAL PROTEIN
rpS6 is an evolutionary conserved protein that spans 236e253 residues
in species as remote as yeast, plants, invertebrates, and vertebrates (Meyuhas,
2008), yet no homology with any ribosomal protein in Escherichia coli or
archaebacteria has been detected (Wool et al., 1996).
The role of rpS6 was first addressed by conditional knockout of the
respective gene in adult mouse liver (Volarevic et al., 2000). Hepatocytes
that lacked rpS6 gene failed to synthesize the 40S ribosomal subunit and
consequently to proliferate following partial hepatectomy. This failure to
progress through the cell cycle correlated with a block in expression of cyclin
E gene. Nonetheless, the expression of rpS6 gene was not required for liver
growth when starved mice were refed. Moreover, the relative engagement
of liver ribosomes in translation, as exemplified by their polysomal associa-
tion, was indistinguishable between rpS6-containing and lacking hypertro-
phying livers (Volarevic et al., 2000).
The critical role of rpS6 is not confined to the regenerating liver, as
thymus-specific knockout of both rpS6 alleles, but not conditional deletion
of one allele, had devastating effect on the gland development (Sulic et al.,
2005). rpS6 heterozygosity (rpS6
wt/del
), however, had a remarkable effect on
the number of mature T cells in peripheral lymphoid organs (spleen and
lymph nodes). The deficiency of one rpS6 allele led to a proportional dimi-
nution in the abundance of rpS6 and ribosome content in purified rpS6
wt/del
Ribosomal Protein S6 Phosphorylation 43
T cells, yet with no effect on their total protein content or their ability to
undergo normal stimulated cell growth (Sulic et al., 2005). Likewise, 30e
50% reduction in rpS6 content of HeLa cells by siRNA only mildly affected
global protein synthesis (Montgomery et al., 2006). Nevertheless, while
wild-type T cells progressed in vitro through several divisions upon mito-
genic stimulation, their rpS6
wt/del
counterparts failed to proliferate as a result
of a block at the G1/S checkpoint of the cell cycle, and partially due to
increased apoptosis. Interestingly, deletion of both p53 alleles almost
completely resumed the proliferative capacity of stimulated rpS6
wt/del
T cells.
These observations strongly support the notion that impaired ribosome
biogenesis, associated with rpS6 deficiency, activates a p53-dependent
checkpoint to eliminate defective T cells (Sulic et al., 2005).
rpS6
wt/del
embryos died during gastrulation, at day 8.5. However,
already at day 6.5 their cells failed to show dephosphorylation and activation
of Cdk1 and to enter mitosis. Moreover, the embryonal death was preceded
by induced apoptosis. The fact that p53 gene knockout enabled rpS6
wt/del
embryo to develop past gastrulation stage, suggests that rpS6 heterozygosity
triggers a p53-mediated checkpoint during gastrulation. Interestingly, ribo-
some biogenesis is defective in rpS6
wt/del
/P53
/
embryo, as well as in the
corresponding MEFs. However, while neither cell cycle progression nor
cell growth is impaired in rpS6
wt/del
/P53
/
MEFs, compromised cell pro-
liferation was observed in the liver from rpS6
wt/del
/P53
/
embryo. This
decreased in hepatic proliferation might be explained by the relative defi-
ciency of cyclins D1 and D3, observed in this organ (Panic et al., 2006).
Interestingly, unlike mammalian rpS6, lesions in Drosophila rpS6 gene
expression, due to insertion of a P element upstream of the transcription
initiation site, had a mixed response: Hyperplasia of lymphglands on the
one hand, and growth inhibition of most larval organs on the other hand
(Stewart and Denell, 1993; Watson et al., 1992). The latter response is
consistent with rpS6 having a tumor suppressor like function in Drosophila.
3. PHOSPHORYLATION OF rpS6
A pioneer study conducted by David Kabat showed that a 33-kDa
protein, termed F protein, which resided in the small ribosomal subunit un-
dergoes phosphorylation in rabbit reticulocytes (Kabat, 1970). Later, it was
identified as rpS6, and that it is the only ribosomal protein that undergoes
phosphorylation during rat liver regeneration (Gressner and Wool, 1974).
44 Oded Meyuhas
The phosphorylation sites in rpS6 in mammals and Xenopus laevis have been
mapped to five clustered residues, S
235
,S
236
,S
240
,S
244
, and S
247
(Bandi
et al., 1993; Krieg et al., 1988; Wettenhall et al., 1992), whose location at
the carboxy terminus of higher eukaryotes is evolutionarily conserved
(Meyuhas, 2008). It has been proposed that phosphorylation progresses in
an ordered fashion, with Ser236 as the primary phosphorylation site (Flotow
and Thomas, 1992; Wettenhall et al., 1992). A similar organization of phos-
phorylation sites, relative to the carboxy terminus was described for
Drosophila melanogaster rpS6 (Radimerski et al., 2000).
The first report on the phosphorylation of rpS6 (S10 according to an
older nomenclature) in Saccharomyces cerevisiae lagged behind that of its
mammalian counterpart (Hebert et al., 1977). Yeast rpS6 is phosphorylated
after transfer of a stationary culture to fresh nutrient medium, as well as at an
early stage of germination, and as in other eukaryotes, the protein is dephos-
phorylated during heat shock (Jakubowicz, 1985; Szyszka and Gasior, 1984).
However, yeast rpS6, unlike higher eukaryotes, bears only two phosphory-
latable serine residues (Ser232 and Ser233) that correspond to Ser235 and
Ser236 in the mammalian protein.
Numerous reports have demonstrated that rpS6 is subject to phosphor-
ylation in response to multiple physiological, pathological, and pharmaco-
logical stimuli ((Meyuhas, 2008) and references therein). Notably, this
modification can be detected in both the cytosol and the nucleus (Pende
et al., 2004). However, distinct nuclear/cytoplasmic distribution of rpS6
phosphorylated at different sits has been noticed for primary human cells
(Rosner et al., 2011), yet the physiological significance of this compart-
mental preference is not clear.
3.1 S6 Kinase (S6K1 and S6K2)
Characterization of an S6 kinase at a molecular level was first achieved in
Xenopus oocytes, wherein the dominant form of S6 kinase detected after
mitogenic stimulation had been purified as a 90 kDa polypeptide (Erikson
and Maller, 1985), later termed as p90 rpS6 kinase (RSK, also known as
p90
RSK
). Purification of the avian and mammalian major rpS6 kinase recov-
ered 65- to 70-kDa polypeptides (Blenis et al., 1987; Jen€o et al., 1988) that
are currently referred to as S6K.
Mammalian cells contain two forms of S6K, S6K1 and S6K2 (also
known as S6Kaand S6Kbrespectively), which are encoded by two different
genes and share a very high level of overall sequence homology (Meyuhas
and Dreazen, 2009). S6K1 has cytosolic and nuclear isoforms (p70 S6K1
Ribosomal Protein S6 Phosphorylation 45
and p85 S6K1, respectively), whereas both S6K2 isoforms (p54 S6K2 and
p56 S6K2) are primarily nuclear (Martin et al., 2001) and references therein)
and partly associated with the centrosome (Rossi et al., 2007). Analysis of
rpS6 phosphorylation in mouse cells deficient in either S6K1 or S6K2 sug-
gests that both are required for full S6 phosphorylation, with the predomi-
nance of S6K2 (Pende et al., 2004). Notably, the phosphorylation of the
evolutionary conserved sites of Drosophila rpS6 is carried out by dS6K that
is encoded by a single gene (Watson et al., 1996).
Two putative S6K homologs, originally named atpk1/ATPK6 and
atpk2/ATPK19, sharing 87% sequence homology were identified in Arabi-
dopsis (Mizoguchi et al., 1995; Zhang et al., 1994). They were later referred
to as atS6K1 and atS6K2, respectively (Turck et al., 1998), of which, atS6K2
was suggested to be an ortholog of the mammalian S6Ks, since this kinase,
and not atS6K2, was able to phosphorylate rpS6 (Zhang et al., 1994). Phos-
phorylation of yeast rpS6 has been known for nearly four decades (Hebert
et al., 1977), yet the identity of the respective kinase remained elusive until
recently. Initially, the Sch9 kinase was proposed to comprise the yeast S6K
(Urban et al., 2007). However, recently this role has been assigned instead,
to Ypk3, a kinase that exhibits high homology to human S6K (Gonzalez
et al., 2015). This latter notion is supported by the observation that rpS6
phosphorylation is completely abolished in cells lacking Ypk3, ypk3D,
whereas Sch9 is dispensable for rpS6 phosphorylation. Furthermore,
complementation of ypk3Dcells with human S6K restored rpS6 phosphor-
ylation in a rapamycin-sensitive manner (Gonzalez et al., 2015)(Figure 1).
3.2 90-kDa rpS6 Kinase (RSK1eRSK4)
RSKs are central mediators of extracellular signal-regulated kinase (ERK
(for further details on this pathway see Section 4.1.2.)) in regulation of
cellular division, survival, and differentiation via phosphorylation of
numerous intracellular proteins ((Romeo et al., 2012) and references
therein). Four RSK genes (RSK1eRSK4) have been identified in mammals,
and RSK orthologs have been described in D. melanogaster and Caenorhabditis
elegans, but not in yeast and plants (Hauge and Fr€odin, 2006; Lara et al.,
2013).
The discovery that S6K is the predominant rpS6 kinase in somatic cells
(Ballou et al., 1991; Chung et al., 1992) has led to a widely accepted belief
that RSK, despite its name, is physiologically irrelevant for rpS6 phosphor-
ylation. However, later observations have challenged this dogma. Thus,
phosphorylation of rpS6 at Ser235 and Ser236 (Ser235/236) can still be
46 Oded Meyuhas
detected, albeit at a much lower level, in cells lacking both S6K1 and S6K2.
This phosphorylation is abolished by treatment by either U0126 (a MAP
and ERK kinases (MEK) inhibitor) or PD184352 (an ERK inhibitor), indi-
cating the involvement of a MEK/ERK-dependent kinase (Pende et al.,
2004). Likewise, Ser235/236 remained partly phosphorylated in cells treated
with rapamycin, which completely inhibits S6K through inhibiting
mammalian target of rapamycin (mTOR), indicating the presence of an
mTOR-independent pathway leading to rpS6 phosphorylation at these
sites. Moreover, it has been shown that this phosphorylation is carried
out, both in vitro and in vivo, by RSK, which phosphorylates rpS6
exclusively at Ser235/236 in a response to serum, growth factors, tumor-
promoting phorbol esters, and oncogenic Ras (Roux et al., 2007).
The consensus recognition sequences of S6K and RSK are similar,
RxRxxS and R/KxRxxS respectively, where x represents any amino acid
and the carboxy-terminal S is the phosphorylated serine residue (Flotow
and Thomas, 1992; Hauge and Fr€odin, 2006). Notably, however, the
sequence context of serine 236 in rpS6 is the only one, among the phos-
phorylatable serine residues, that conforms to the consensus recognition
sequence of these enzymes (Figure 1).
3.3 Protein Kinase A
Protein kinase A (PKA) is a family of enzymes, whose activity is dependent
on cellular levels of cyclic AMP (cAMP) and phosphorylates a large number
of cytosolic and nuclear proteins (Kirschner et al., 2009). Agents that induce
increases in cAMP level in the pancreatic bcell line MIN6 (mouse insuli-
noma cell line 6) and in mouse islets of Langerhans lead to the phosphory-
lation of rpS6 that is confined to Ser235/236 via a pathway that is sensitive to
inhibitors of PKA. PKA was also shown to exclusively phosphorylate
recombinant rpS6 on Ser235/236 in vitro, and is likely to phosphorylate
Figure 1 Sites of rpS6 phosphorylation by different kinases and of dephosphorylation
by PP1. Arrows represent phosphorylation and the dashed lineddephosphorylation.
See text for details.
Ribosomal Protein S6 Phosphorylation 47
rpS6 on Ser235/236 in a number of other mammalian cell lines, such as
fibroblasts, pheochromocytoma, neuroblastoma, and kidney cells (Moore
et al., 2009).
Furthermore, treatment of mice with haloperidol, a typical antipsychotic
drug, increased rpS6 phosphorylation at Ser235/236 in medium spiny
neurons of the striatum. This phosphorylation is carried out through activa-
tion of the cAMP signaling, as was indicated by elevated Ser235/236 phos-
phorylation upon stimulation of PKA in cultured striatal neurons (Valjent
et al., 2011). Likewise, stimulation of cAMP production by forskolin, an
adenylate cyclase activator, in mouse striatal slices led to a marked increase
in the phosphorylation of S235/236, which was attenuated by pretreatment
with Rp-cAMP, a PKA inhibitor (Biever et al., 2015)(Figure 1).
3.4 Casein Kinase 1
Members of the casein kinase 1 (CK1), a family of serine/threonine kinases,
are ubiquitously expressed and regulate diverse cellular processes, through
phosphorylation of a variety of proteins, which are preferentially “primed”
prephosphorylated substrates (Cheong and Virshup, 2011). CK1 has recently
been shown to selectively phosphorylate rpS6 at Ser247 (Hutchinson et al.,
2011). The identification of CK1 and its specificity toward Ser247 was based
on both pharmacological inhibition of all members of the casein kinase family
and on knockdown experiments with CK1 siRNAs. However, the ability of
recombinant CK1 to phosphorylate Ser247 in in vitro kinase assay was indis-
tinguishable from that of its capacity to phosphorylate Ser235/236 or Ser240/
244 in rpS6. This apparent lack of specificity might be an artifact of the in
vitro assay (Hutchinson et al., 2011)(Figure 1).
3.5 rpS6 Dephosphorylation
The steady state level of rpS6 phosphorylation is the product of a dynamic
equilibrium between the activities of the respective kinases and the opposing
phosphatases. Nonetheless, the fluctuations in rpS6 phosphorylation have
been attributed, in nearly all the relevant reports, to parallel changes in
the kinase(s) activity. In a few cases, however, the phosphorylation status
of rpS6 has been primarily ascribed to the activity of a phosphatase rather
than a kinase. Thus, Rous sarcoma virus-transformed chick embryo
fibroblasts show attenuated dephosphorylation of rpS6 during mitosis and
a parallel decrease in the activity of the protein phosphatase type 1 (PP1).
This observation suggests that it is the PP1 activity that might control
rpS6 phosphorylation under these circumstances (Belandia et al., 1994).
48 Oded Meyuhas
Conversely, rpS6 phosphorylation is not detectable in murine erythroleuke-
mia or other hematopoietic cells, and this constitutive dephosphorylation
state appears to be due to the action of a phosphatase that is likely to act
directly on rpS6 (Barth-Baus et al., 2002).
The demonstration that rpS6 phosphorylation is enhanced upon treat-
ment of cells with tautomycetin, a PP1 inhibitor, and that rpS6 is coimmu-
noprecipitated with anti-PP1Caantibody (Li et al., 2012), provided the first
indication that rpS6 might indeed be a PP1 substrate. This contention has
been further supported by genetic manipulations. Thus, expression of
PP1D95N, a dominant-negative PP1 catalytic subunit, caused a marked
elevation in rpS6 phosphorylation at all five phosphorylatable sites of
rpS6. Likewise, knockdown of PP1 catalytic subunit aresulted in similar
consequences (Hutchinson et al., 2011). Collectively, these observations
imply that PP1 is the primary phosphatase of rpS6 (Figure 1).
4. SIGNALS TO rpS6 PHOSPHORYLATION
Following the initial wave of descriptive reports on rpS6 phosphory-
lation, much effort has been invested in an attempt to establish the pathways
that transduce various signals into activation or inhibition of the respective
kinases. The pathways discussed below are only those that have been
documented to exert a parallel effect on both rpS6 phosphorylation and
the activity of the respective kinase.
4.1 Growth Factors
4.1.1 PI3K/Akt/TSC/Rheb/mTORC1/S6K pathway
Signaling to S6 phosphorylation by growth factors starts by activation of the
respective receptor tyrosine kinase. This in turn, leads to activation of class I
phosphatidylinositol 3-kinase (PI3K), either through direct binding to the
phosphorylated receptor or through tyrosine phosphorylation of scaffolding
adaptors, such as insulin receptor substrate, which then binds and activates
PI3K (Cantrell, 2001). PI3K converts the lipid phosphatidylinositol-4,5-
P2 (PIP2) into phosphatidylinositol-3,4,5-P3 (PIP3), in a reaction that can
be reversed by the PIP3 phosphatase PTEN (phosphatase and tensin homo-
log deleted from chromosome 10) (Leslie and Downes, 2002). PIP3 recruits
both 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt (also known
as protein kinase B (PKB)) to the plasma membrane (Brazil and Hemmings,
2001; Lawlor and Alessi, 2001), and PDK1 phosphorylates and activates Akt
at T308 (Belham et al., 1999). Activated Akt phosphorylates tuberous
Ribosomal Protein S6 Phosphorylation 49
sclerosis complex 2 (TSC2) at multiple sites (Inoki et al., 2002; Manning
et al., 2002; Potter et al., 2002). This phosphorylation blocks the ability of
TSC2, while residing within the TSC1eTSC2 tumor suppressor dimmer
to act as a GTPase-activating protein (GAP) for Rheb (Ras-homolog
enriched in brain), thereby allowing Rheb-GTP to accumulate and operate
as an activator of the rapamycin-sensitive mammalian TOR complex 1
(mTORC1) (Avruch et al., 2006). The latter is consisting of target of rapa-
mycin (TOR), RAPTOR (regulatory associated protein of TOR) LST8
(also known as GbL), and PRAS40 (proline-rich Akt substrate 40 kDa)
(Bhaskar and Hay, 2007; Yang and Guan, 2007). Since it is mTORC1
that conveys signals to S6Ks and rpS6, it will be mentioned in the remainder
of this review, rather than mTOR, when transduction of signals is discussed.
Akt can also activate mTORC1 independently of TSC1eTSC2 by phos-
phorylating PRAS40, thereby relieving the PRAS40-mediated inhibition
of mTORC1 (Sancak et al., 2007; Vander Haar et al., 2007).
Active mTORC1 phosphorylates two translational regulators, S6Ks and
eukaryotic initiation factor 4E (eIF-4E)-binding protein (4E-BP1, 2, and 3)
(Hay and Sonenberg, 2004). Activation of S6Ks requires also phosphoryla-
tion by PDK1 in a reaction that does not need binding of PDK1 to PIP3
(Alessi et al., 1998). Finally, activated S6K phosphorylates rpS6, as well as
many other substrates (Meyuhas and Dreazen, 2009)(Figure 2).
4.1.2 Ras/Raf/MEK/ERK/RSK pathway
Activation of the second family of rpS6 kinases, the RSKs involve a distinct
signaling pathway, even though it might share the same initial event, namely
the ligand binding, with the PI3K/Akt/TORC1 pathway. Since RSK
seemed to be a minor rpS6 kinase, the pathway leading to its activation is
only very briefly described here. Thus, the binding of insulin, as well as of
many other growth factors, to their receptors induces the activation of the
small GTPase Ras and consequently the recruitment of Raf to the mem-
brane for subsequent activation by phosphorylation. Raf activates
mitogen-activated protein (MAP) kinase, kinases 1 and 2 (MEK1/2), which
in turn phosphorylates and activates the extracellular-signal-regulated
kinases (ERK1 and ERK2). Activated ERKs phosphorylate and activate a
vast array of substrates localized in all cellular compartments such as the
RSK family (McCubrey et al., 2007; McKay and Morrison, 2007).
Interestingly, TSC2 is repressed by the Ras/MAPK pathway in addition
to its downregulation by the PI3K/Akt pathway, as evidenced by the obser-
vation that activated Erk1/2 directly phosphorylates TSC2 at sites that differ
50 Oded Meyuhas
from the Akt target sites, thereby causing functional inactivation of the
TSC1eTSC2 complex (Ma et al., 2005). Moreover, the MAPK-activated
RSK1 also phosphorylates TSC2 at a unique site. This RSK1-mediated
phosphorylation inhibits the TSC1eTSC2 complex and thereby increases
mTORC1 signaling toward S6K1 (Roux et al., 2004)(Figure 2).
4.2 Amino Acid Sufficiency
Amino acid starvation, unlike serum starvation, fails to downregulate PI3K
or Akt (Hara et al., 1998; Wang et al., 1998), yet it results in a rapid dephos-
phorylation of S6K1 and rpS6. Furthermore, reintroduction of amino acids
restores the phosphorylation of these targets in an mTORC1-dependent
(rapamycin-sensitive) fashion (Tang et al., 2001) and references therein). It
was widely argued that the TSC1eTSC2 complex plays no role in trans-
ducing the negative signal resulting from amino acid starvation to
mTORC1 activity (Bar-Peled and Sabatini, 2014; Jewell et al., 2013).
However, recent studies have shown that inhibition of mTORC1 by amino
acid deprivation is indeed mediated by the TSC1eTSC2 complex
(Demetriades et al., 2014; Patursky-Polischuk et al., 2014). The latter is
Figure 2 Pathways transducing signals emanating from growth factors to rpS6 phos-
phorylation. Arrows represent activation and bars inhibition. See text for details.
Ribosomal Protein S6 Phosphorylation 51
required for the release of mTORC1 from its site of action, the lysosomal
membrane, and therefore, cells lacking TSC fail to efficiently turn off
mTORC1 and consequently their response to amino acid starvation is
compromised (Demetriades et al., 2014).
Amino acid starvation leads to rapid dephosphorylation of S6K1, which
can be restored upon readdition of amino acids in an mTORC1-dependent
fashion ((Kim and Guan, 2011) and references therein). Members of the Rag
subfamily of Ras small GTPases (RagA, B, C, and D) and the trimeric
complex, Ragulator, are essential transducers of amino acids signals to
mTORC1 activity (Duran and Hall, 2012). Amino acid stimulation elicits
movement of mTORC1 to the lysosomal surface, where Rheb and Ragu-
lator reside. The latter recruits Rag GTPases to the lysosomes in a p62- and
vacuolar H
þ
-ATPase-dependent manner (Duran et al., 2011; Zoncu et al.,
2011), and thereby participates in mTORC1 activation (Sancak et al., 2008,
2010). In contrast, the pentameric complex, GATOR1, inhibits the
mTORC1 pathway by functioning as a GAP for RagA, whereas the
trimeric complex, GATOR2 negatively regulates GATOR1 (Bar-Peled
et al., 2013).
Another mechanism whereby amino acids could affect mTORC1 activ-
ity is via hVPS34 (vacuolar protein sorting 34). This class III PI3K (converts
phosphatidylinositol to phosphatidylinositol-3-phosphate) has been shown
to transduce the signal of amino acid sufficiency to mTORC1 indepen-
dently of the TSC1eTSC2/Rheb axis (Byfield et al., 2005; Nobukuni
et al., 2005). Nevertheless, the mechanism by which hVPS34 regulates
mTOR is unknown (Figure 3).
4.3 Energy Balance
Starving mammalian cells of glucose or treating them with glycolytic (e.g.,
2-deoxyglucose (2-DG)) or mitochondrial (e.g., valinomycin, antimycin A,
oligomycin) inhibitors, depletes cellular energy and causes a concomitant
decrease in mTORC1 activity (Dennis et al., 2001; Inoki et al., 2003;
Kim et al., 2002). Activation of AMP-activated protein kinase (AMPK) is
currently the prevailing model used to explain how energy levels couple
to the regulation of mTORC1. AMPK acts as a sensor of cellular energy
status and is activated by increases in the cellular AMP: ATP ratio, caused
by metabolic stresses that either interfere with ATP production (e.g., depri-
vation for glucose or oxygen) or that accelerate ATP consumption (e.g.,
muscle contraction). Activation in response to increases in AMP levels in-
volves phosphorylation by an upstream kinase, the tumor suppressor
52 Oded Meyuhas
LKB1 (Towler and Hardie, 2007), since AMPK activation in response to
low energy conditions is blocked in LKB1 null cells (Corradetti et al.,
2004). Furthermore, LKB1 mutant cells exhibit hyperactive mTORC1
signaling (Corradetti et al., 2004; Shaw et al., 2004). Activation of AMPK
by 5-aminoimidazole-4-carboxyamide (AICAR), an AMP analog, inhibits
mTORC1-dependent phosphorylation of S6K1 (Bolster et al., 2002). Like-
wise, expression of an activated form of AMPK decreases S6K1 phosphor-
ylation, whereas a dominant-negative form of AMPK increases S6K1
phosphorylation (Kimura et al., 2003).
AMPK phosphorylates several targets to enhance catabolism and suppress
anabolism in response to low energy, and exerts this effect by directly phos-
phorylating and activating TSC2 and thereby downregulates mTORC1
(Inoki et al., 2003). Thus, the phosphorylation of S6K1 is more resistant
to glucose deprivation in TSC2
/
cells or cells, whose mutant TSC2
Figure 3 Amino acid signaling to rpS6 phosphorylation. Arrows represent activation,
bars inhibition and dotted lines putative links. See text for details.
Ribosomal Protein S6 Phosphorylation 53
cannot be phosphorylated by AMPK (Inoki et al., 2003). It appears, there-
fore, that energy depletion is sensed by AMPK and relayed to mTORC1
through the TSC1eTSC2 complex. However, it seems that cells can
convey energy stress signals to TSC2 also through upregulation of the
mRNA encoding REDD1 (Regulated in Development and Damage
Responses 1, also known as RTP801). REDD1 binds 14-3-3 and thereby
alleviates the 14-3-3-mediated inhibition of TSC1eTSC2 complex
(DeYoung et al., 2008) and consequently leads to inhibition of mTORC1
signaling to S6K1 (Sofer et al., 2005). Yet, the fact that mTORC1 is refrac-
tory to energy starvation in REDD1
/
cells, despite normal activation of
AMPK and AMPK-dependent activation of TSC2, suggests that the effect
of REDD1 on TSC2 is predominant over that of AMPK. This notion is
further supported by the observation that overexpression of REDD1 can
suppress mTORC1 activity even in the presence of dominant-negative
AMPK (Sofer et al., 2005). Taken together, these results imply that
REDD1 may act in parallel with, or downstream of AMPK toward TSC2
(Figure 4). It should be noted, however, that conflicting results have shown
that acute treatment of TSC2
/
with 2-DG leads to inactivation of S6K1
(Smith et al., 2005), suggesting that signals from energy starvation might be
transduced into suppression of mTORC1 also in a TSC1eTSC2 complex-
independent fashion.
4.4 Oxygen Supply
Hypoxia has a prominent inhibitory effect on mTORC1 activity, which is
mediated in part by REDD1 (Figure 4), as REDD1
/
mouse cells are
defective in hypoxia-mediated inhibition of S6K activation (Brugarolas
et al., 2004). This effect, however, relies on an intact TSC1eTSC2 com-
plex, as S6K phosphorylation is refractory to hypoxic treatment of
TSC1
/
or TSC2
/
mouse cells (Brugarolas et al., 2004; Miloslavski
et al., 2014). Transcriptional activation of REDD1 gene under conditions
of hypoxia (Shoshani et al., 2002) is mediated by HIF-1, the master regula-
tors of oxygen homeostasis (Brahimi-Horn et al., 2007). Hypoxia can also
inhibit mTORC1 independently of REDD1 via the induction of energy
stress, possibly due to reduced oxidative phosphorylation. AMPK is upregu-
lated under these conditions, thereby activates TSC2 and inhibits mTORC1
(Liu et al., 2006)(Figure 4). It should be noted, however, that prolonged
exposure to low oxygen leads to a reduced mTORC1 activity indepen-
dently of TSC2 by an unknown mechanism (Liu et al., 2006).
54 Oded Meyuhas
4.5 Osmolarity
An increase in the concentration of solutes outside the cell relative to that
inside is termed a hyperosmotic stress. Such a stress causes water to diffuse
out of the cell, resulting in cell shrinkage, which can lead to DNA and pro-
tein damage, cell cycle arrest, and ultimately cell death (Burg et al., 2007).
Hyperosmotic stress that is induced by treating cells with either sorbitol
(Kruppa and Clemens, 1984) or high salt concentration (Naegele and
Morley, 2004) elicits reversible dephosphorylation of rpS6 in mammals. A
closer look at the sorbitol effect has suggested the involvement of a phospha-
tase, since calyculin A, a phosphatase inhibitor, was able to prevent sorbitol-
induced suppression of S6K (Parrott and Templeton, 1999).
Figure 4 Pathways transducing energy balance and hypoxic signals to rpS6 phosphor-
ylation. Arrows represent activation and bars inhibition. See text for details.
Ribosomal Protein S6 Phosphorylation 55
Hyperosmotic-dependent S6K inhibition has been shown also in
mannitol-treated tobacco leaves. Downregulation of S6K activity appears
to have a protective effect against sustained osmotic stress, as Arabidopsis
plants expressing high levels of S6K were hypersensitive to mannitol treat-
ment (Mahfouz et al., 2006; Williams et al., 2003).
5. PHYSIOLOGICAL ROLES OF rpS6
PHOSPHORYLATION
5.1 Global Protein Synthesis
An early study has shown that substitution of the two phosphorylat-
able serine residues to alanines in yeast rpS6 had no detectable effect on yeast
growth, under a wide variety of nutritional conditions (Johnson and
Warner, 1987). This observation implies that rpS6 phosphorylation has no
obvious role in protein synthesis or other cellular functions in yeast. A similar
approach has also been applied to studying the role of rpS6 phosphorylation
in regulation of protein synthesis in rpS6 phosphorylation-deficient
mammalian cells (rpS6
P/
knockin MEFs). Thus, the relative rates of
global protein synthesis (incorporation of radio-labeled amino acids) and
of the accumulation of steady state levels of protein were significantly higher
in these cells, relative to wild-type MEFs (Ruvinsky et al., 2005). It appears
therefore, that protein synthesis, at least in this cell type, is downregulated by
rpS6 phosphorylation. Though slightly faster elongation rate was deter-
mined in rpS6
P/
MEFs, the augmentation in overall protein synthesis
in these cells is mainly attributable to enhanced translation initiation by an
as yet unknown mechanism.
A lack of stimulatory effect of rpS6 phosphorylation on global protein
synthesis has also been shown in mouse liver. Thus, monitoring the relative
proportion of ribosomes engaged in translation (associated with polysomes)
has demonstrated a similar proportion in the liver of both rpS6
P/
and
wild-type mice. Furthermore, this similarity was apparent even in regener-
ating liver, in which rpS6 undergoes extensive phosphorylation only in the
wild-type (Ruvinsky et al., 2005). Consistently, mice treated for 4 weeks
with rapamycin showed a dramatic reduction in the phosphorylation of
rpS6 in both liver and muscle, yet their translational activity was indistin-
guishable from that monitored in mice treated with just a vehicle (Garelick
et al., 2013).
Notably, a lack of a role in the regulation of global protein synthesis has
been reported also for S6K-deficient (S6K1
/
;S6K2
/
) mice. Thus,
56 Oded Meyuhas
myoblasts, hepatocytes, fibroblasts, and a whole liver from both wild-type
and S6K-deficient mice displayed a similar proportion of ribosomes engaged
in polysomes (Chauvin et al., 2014; Mieulet et al., 2007). Similarly,
measuring protein synthesis by methionine incorporation, showed no differ-
ence between wild-type and S6K1
/
;S6K2
/
mice (Mieulet et al., 2007).
Clearly, these results indicate that both rpS6 phosphorylation and S6K activ-
ity are dispensable for efficient global protein synthesis.
5.2 Cell Size Regulation
Previous reports have demonstrated that the mTORC1 pathway is an inte-
gral cell growth regulator (Lee et al., 2007). Thus, treatment of mammalian
cells by rapamycin decreases their size. This mTOR-dependent regulation
of the cell size involves its downstream targets, S6K1 and 4E-BP (Fingar
et al., 2002; Ohanna et al., 2005). Indeed, S6K has been implicated as an
important positive regulator of cell and body size. Thus, most dS6K null
Drosophila exhibit embryonic lethality, with the few surviving adults having
a severely reduced body size, due to a decrease in cell size rather than a
decrease in cell number (Montagne et al., 1999). S6K1
/
mice are signif-
icantly smaller at birth, due to a proportional decrease in the size of all organs
(Shima et al., 1998). A smaller cell size in these mice was reported for
pancreatic bcells (Pende et al., 2000) and myoblasts (Ohanna et al.,
2005). In contrast, the birth weight of S6K2
/
mice, as well as the size
of their myoblasts, is similar to those of wild-type mice (Pende et al.,
2000; Ohanna et al., 2005). In accordance with the phenotypes of each of
these mutant mice, the embryonic and postnatal growth, as well as the
size of myoblasts of the double knockout mice, S6K1
/
/S6K2
/
, are
similar to those of S6K1
/
mice (Ohanna et al., 2005; Pende et al.,
2000). The fact that mammalian cell size is predominantly determined by
S6K1 and not S6K2 posed a question regarding the effector(s) of S6K1
involved in this mode of regulation.
Of the known multiple substrates of S6K1, it is rpS6 phosphorylation
that is directly involved in the control of cell size. Thus, a wide variety
of cell types derived from rpS6
P/
mice are significantly smaller than their
wild-type counterparts. These include pancreatic bcells, interleukin-7-
dependent cells derived from fetal livers, MEFs (Granot et al., 2009;
Ruvinsky et al., 2005), muscle myotubes (Ruvinsky et al., 2009). It ap-
pears, however, that the small cell phenotype is not ubiquitous, as acinar
cells in the pancreas display a similar size regardless of the absence of
S6K1 (Pende et al., 2000) or phosphorylatable serine residues in rpS6
Ribosomal Protein S6 Phosphorylation 57
(Ruvinsky et al., 2005). Notably, even though the birth weight of
rpS6
P/
mice is similar to that of their wild-type littermates, the knock
mice start to display relative retarded weight gain at the age of 6 weeks
(Ruvinsky et al., 2005, 2009).
Several studies have demonstrated that cell cycle progression and cell
growth are separable and are therefore distinct processes, at least in some
mammalian cells (Conlon et al., 2004; Fingar and Blenis, 2004). The
apparent small size of rpS6
P/
MEFs are accompanied by accelerated divi-
sion (Ruvinsky et al., 2005), yet several lines of evidence lend support to the
notion that the small cell size phenotype reflects impaired growth, rather
than being a by-product of enhanced cell division: (1) rpS6
P/
MEFs
remained smaller than their wild-type counterparts, even when progression
through the cell cycle was arrested by aphidicolin, an inhibitor of DNA po-
lymerase- ; and;(2) The size of immortalized rpS6
P/
MEFs is increased to
the extent that it equalize with that of rpS6
Pþ/þ
MEFs. Nevertheless, this
increase was not accompanied by lengthening of the doubling time, as
would be expected if the size was inversely proportional to the division
rate (Ruvinsky et al., 2005).
Interestingly, rapamycin treatment decreased the size of rpS6
Pþ/þ
MEFs, whereas the size of rpS6
P/
MEFs remained unchanged
(Ruvinsky et al., 2005). The rapamycin-resistance displayed by the latter
is reminiscent of that exhibited by S6K1
/
myoblasts (Ohanna et al.,
2005), implying that cells already displaying a small size phenotype, due
to deficiency of S6K1 or of phosphorylatable serine residues in rpS6, are
not further affected by rapamycin. Furthermore, it seems that rpS6 phos-
phorylation is a critical effector of mTORC1 in regulation of cell growth
and that its absence is equivalent to inhibition of mTORC1. Notably, the
small size of S6K1
/
myoblasts is apparent, even though their rpS6 is still
phosphorylated, most probably by S6K2 (Ohanna et al., 2005). It is likely,
therefore, that this small cell size phenotype reflects a reduced activity of
yet another S6K1-specific effector(s), which is involved in this mode of
regulation, such as SKAR (Richardson et al., 2004). Alternatively, if
S6K2 is inactive during muscle differentiation in early embryo, then it is
possible that S6K1 deficiency is indeed equivalent to the lack of phosphor-
ylatable serine in rpS6. If the latter is the case, then it should be assumed
that once the growth of a specific cell lineage is blocked by a temporary
deficiency of rpS6 phosphorylation, as a result of S6K1 deficiency, the
small size is maintained thereafter, regardless of a later phosphorylation
of rpS6 by a different kinase (S6K2, for example).
58 Oded Meyuhas
5.3 Normal Muscle Function
rpS6
P/
mice suffer from muscle weakness as demonstrated by a variety of
physical performance tests (Ruvinsky et al., 2009). This physical inferiority
appears to result from two defects: (1) a decrease in total muscle mass that
reflects impaired growth, rather than aberrant differentiation of myofibers,
as well as a diminished abundance of contractile proteins; and (2) a reduced
content of ATP and phosphocreatine, two readily available energy sources.
However, the apparent energy deficiency in this genotype does not result
from a lower mitochondrial mass or compromised activity of enzymes of
the oxidative phosphorylation, nor does it reflect a decline in insulin-depen-
dent glucose uptake, or diminution in storage of glycogen or triacylglycerol
in the muscle (Ruvinsky et al., 2009). These observations have established
rpS6 phosphorylation as a determinant of muscle strength, through its role
in regulation of myofiber growth and energy content.
5.4 Hypertrophic Responses
The apparent role of rpS6 phosphorylation as a determinant of cell size raised
the possibility that this modification might also be critical during induced
cellular hypertrophy. One such response is the compensatory renal hyper-
trophy, which results from reduction in the number of functioning neph-
rons, as in the case in unilateral nephrectomy (Preisig, 1999). This
response is similarly blunted by either rapamycin treatment or in S6K1-
deficient mice (Chen et al., 2009), indicating the role of the mTORC1/
S6K1 axis in this protective mechanism. Interestingly, among its multiple
substrates, S6K1 transduces its growth signal primarily through rpS6 phos-
phorylation, as is evident by the greatly impaired compensatory renal hyper-
trophy in rpS6
P/
mice (Xu et al., 2015). Surprisingly, induced
hypertrophy of muscle in adolescent rats following progressive resistance
exercise was shown to be associated with reduced phosphorylation of
rpS6 at Ser235/236 (Hellyer et al., 2012). Conceivably, once maximal
growth is attained following repeated bouts of exercise, signaling to rpS6
phosphorylation is silenced.
5.5 Cell Proliferation
The puzzling observations of a similar birth weight of rpS6
P/
and
rpS6
Pþ/þ
mice, despite a smaller size of rpS6
P/
embryonic cells (MEFs
and interleukin-7-dependent cells), have been reconciled by the findings
that rpS6
P/
newborns contain a higher DNA content, which reflects a
Ribosomal Protein S6 Phosphorylation 59
higher cell number (Ruvinsky et al., 2005). It is conceivable, therefore, that
a faster proliferation compensates for the smaller size of embryonic rpS6
P/
cells. Indeed, this possibility is further supported by the apparent shorter
population doubling time of rpS6
P/
MEFs, as well as the faster protein
and nucleic acids accumulation in these cells. This accelerated cell division
primarily reflects a shortening of the G1 phase in rpS6
P/
MEFs (Ruvinsky
et al., 2005). Notably, the deficiency of both S6K1 and S6K2, unlike the
mutation in all phosphorylatable serine residues in rpS6
P/
, had no effect
on the doubling time of MEFs or primary myoblasts (Ohanna et al., 2005;
Pende et al., 2004). This difference might reflect the fact that rpS6 in
S6K1
/
/S6K2
/
is still phosphorylated at Ser235/236.
5.6 Clearance of Apoptotic Cells
Professional and amateur phagocytes rapidly clear apoptotic cells in a process
known as efferocytosis (Korns et al., 2011). It has previously been shown
that the F-box protein Pallbearer (PALL) participates in a complex func-
tioning as an E3-ubiquitin ligase. This complex promotes efficient apoptotic
cell clearance in Drosophila (Silva et al., 2007). A study with Drosophila
Schneider S2 cell line has unveiled the role of PALL in proteasomal degra-
dation of rpS6 preferentially in its phosphorylated form (Xiao et al., 2015).
Evidently, rpS6 appears to act as a negative regulator of efferocytosis, since its
knockdown enhances, whereas its overexpression decreases the engulfment
of apoptotic cells. Finally, the PALL-dependent degradation of rpS6 leads to
upregulation and activation of RAC2 GTPase that is followed by actin
remodeling to promote efferocytosis (Xiao et al., 2015). Interestingly,
rpS6 phosphorylation has been implicated in TRAIL (tumor necrosis
factor-related apoptosis-inducing ligand)-induced apoptosis in mammalian
cells. Thus, rpS6
P/
MEFs were more sensitive to TRAIL than wild-
type MEFs. Yet, they were as sensitive as wild-type cells to the topoisomer-
ase inhibitor, etoposide (Jeon et al., 2008).
5.7 Tumorigenicity
The importance of mTORC1 in cancer is well appreciated (Xu et al., 2014),
yet it is believed that the key downstream effector of this pathway in cancer
is 4E-BP (Hsieh et al., 2010). Nevertheless, S6K has also been implicated as
an important player in the development of cancer (Alliouachene et al.,
2008). Not surprisingly, therefore, that rpS6 phosphorylation has attracted
much attention as a diagnostic maker for various types of tumors (see
60 Oded Meyuhas
Section 6 below). However, it is only recently that rpS6 phosphorylation has
proven instructive for neoplastic transformation.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal hu-
man cancers. Oncogenic mutations in Kras are found in more than 95% of
PDACs and appear to drive the formation of precursor lesions (Vincent
et al., 2011). Several studies have shown that pharmacologic inhibition of
mTORC1 can attenuate the growth of pancreatic cancer cell lines and, at
least, a subset of PDAC in vivo (Mirzoeva et al., 2011; Morran et al.,
2014), but the identity of downstream effectors remains largely unknown.
Phosphorylation of rpS6 was increased in pancreatic acinar cells upon
implantation of the chemical carcinogen 7,12-dimethylbenz(a)anthracene
(DMBA) or transgenic expression of mutant Kras. Strikingly, the develop-
ment of pancreatic cancer precursor lesions induced by either DMBA or
mutant Kras was greatly reduced in rpS6
P/
mice. rpS6 phosphoryla-
tion-deficient mice, expressing oncogenic Kras, showed increased nuclear
accumulation of the tumor suppressor, p53. This increase is accompanied
with enhanced staining of the DNA damage markers g-H2AX and 53bp1
(Trp53bp1) in areas of acinar to ductal metaplasia, suggesting that rpS6 phos-
phorylation attenuates Kras-induced DNA damage and p53-mediated tu-
mor suppression (Khalaileh et al., 2013). These results attest to the critical
role of rpS6 phosphorylation in the initiation of pancreatic cancer.
A previously published report has shown that pancreata in about 30% of
mice expressing a constitutively active myr-Akt1 in their bcells underwent
hyperplastic transformation leading to insulinoma formation. However,
deficiency of S6K1, but not of S6K2, fully protected the animals from
this tumorigenesis (Alliouachene et al., 2008). The readily detectable
phosphorylated rpS6 in myr-Akt1 transgenic islets, despite S6K1 deficiency,
might argue against a tumorigenic role of rpS6 phosphorylation. However,
rpS6 phosphorylation deficiency in rpS6
P/
mice led to a complete protec-
tion against the development of myr-Akt1-induced insulinoma (Dreazen
et al., unpublished data). These observations imply that not only S6K1,
but also rpS6 phosphorylation can promote malignant transformation, albeit
through distinct mechanisms.
It should be noted that the role of rpS6 phosphorylation in Akt-mediated
tumorigenesis should not be referred to as a general mechanism, but rather a
tissue-specific phenomenon. Thus, Akt
T
mice, in which a constitutively
active Akt2 is expressed in immature T cells, develop spontaneous thymic
lymphomas, which cannot be prevented in rpS6
P/
;Akt
T
double mutant
mice. It appears, therefore, that rpS6 phosphorylation is dispensable for
Ribosomal Protein S6 Phosphorylation 61
transformation downstream of oncogenic Akt signaling in the thymus
(Hsieh et al., 2010).
5.8 Glucose Homeostasis
It has previously been shown that insulin secretion closely correlates with the
size of bcells (Giordano et al., 1993; Pende et al., 2000). Mice deficient in
S6K1 exhibited impaired glucose homeostasis, due to insufficient insulin
secretion in response to glucose load. The reason for this defect was pro-
posed to be the small size of bcells in S6K1
/
mice (Pende et al.,
2000). This phenotype is recapitulated in rpS6
P/
mice, which show a
twofold reduction in both circulating levels and pancreatic content of insu-
lin, in addition to a higher and prolonged hyperglycemic response after
glucose challenge, compared to wild-type mice (Ruvinsky et al., 2005).
Interestingly, the apparent glucose intolerance in rpS6
P/
and
S6K1
/
mice is reminiscent of impaired glucose tolerance observed in
offsprings of rats that were undernourished during pregnancy, or in adult
human beings after prenatal exposure to famine ((Ravelli et al., 1998) and
references therein). Possibly, malnutrition during pregnancy leads to insuf-
ficient signals through mTOR, an integrator of nutritional signals (Dann
et al., 2007; Proud, 2007), which in turn leads to reduced activation of
S6K1 and hypophosphorylation of rpS6 during a critical stage of pancreatic
development and consequently to impaired pancreatic function in the adult
organism. It should be pointed out, however, that the effect of perinatal
famine on the size of bcells, a hallmark of rpS6
P/
and S6K1
/
mice,
is currently unknown.
It should be noted that S6K1
/
mice, unlike rpS6
P/
mice, display an
in utero developmental defect manifested in smaller birth size (Shima et al.,
1998), and that the disruption of both S6K1 and S6K2 leads to decreased
viability due to perinatal lethality (Pende et al., 2004). Clearly, these pheno-
types attest to the involvement of S6K targets other than rpS6, in normal in
utero development.
5.9 rpS6 Phosphorylation as Diagnostic Marker
The usage of the phosphorylation state of Ser235/236 in rpS6 as a biomarker
for activation of the PI3K/mTORC1/S6K pathway in tissue samples from
tumor biopsies (Li et al., 2014; Pinto et al., 2013; Robb et al., 2006, 2007)
or transplants (Lepin et al., 2006; Li et al., 2015) has been repeatedly pro-
posed in recent years. However, these sites can also be phosphorylated by
RSK (Roux et al., 2007), and therefore, their phosphorylation cannot be
62 Oded Meyuhas
used as an indication for therapeutic strategy involving blockage of PI3K/
mTORC1/S6K signaling. Indeed, it has recently been shown that phos-
phorylation of Ser235/236 might be upregulated in tumors with activation
of the Ras/Raf/ERK pathway, rather than activation of the PI3K/
mTORC1/S6K pathway (Chow et al., 2006; Ma et al., 2007). Hence,
differential diagnosis of the activated pathway should depend on the use
of specific biomarkers, such as phospho-rpS6(Ser240/244) for the PI3K/
mTORC1/S6K pathway and phospho-ERK or phospho-RSK for the
Ras/Raf/MEK pathway. Indeed, monitoring the phosphorylation of rpS6
at Ser240/244, rather than Ser235/236, has recently been reported for
several tumors (Chaisuparat et al., 2013; Kim et al., 2013) and epidermal
hyperproliferation conditions (Ruf et al., 2014).
6. CONCLUDING REMARKS AND FUTURE
PERSPECTIVES
Many of the phenotypic manifestations of mice deficient in S6K1 are
recapitulated in the rpS6 knockin mice. Thus both these mutants exhibit: (1)
small bcell size phenotype that is accompanied by hypoinsulinemia and
impaired glucose homeostasis (Pende et al., 2000; Ruvinsky et al., 2005);
(2) small myoblasts and reduced muscle mass (Ohanna et al., 2005; Ruvinsky
et al., 2009); and (3) blunted compensatory renal hypertrophy following
contralateral nephrectomy (Chen et al., 2009; Xu et al., 2015). Clearly,
based on these observations it is tempting to argue that this phenotypic sim-
ilarity simply reflects the fact that rpS6 phosphorylation is a critical S6K1
effector. However, this explanation is inconsistent with the observation
that rpS6 is still fully phosphorylated in S6K1-deficient mouse, due to the
compensatory activity of S6K2 (Alliouachene et al., 2008; Ohanna et al.,
2005; Shima et al., 1998). It appears, therefore, that despite the similarity
in their phenotypic manifestations, the impaired functions are caused by
distinct mechanisms operating in phosphorylation-deficient and S6K1
knockout mice.
Despite a major progress in understanding the physiological roles of rpS6
phosphorylation, the mechanism underlying its highly diverse effects is
poorly understood, if at all. Several explanations can be proposed to account
for this diversity: (1) The phosphorylation of rpS6 within, or outside, the
ribosome affects the translation efficiency of specific mRNAs encoding pro-
teins participating in various processes; (2) rpS6 might be one of the many
bifunctional ribosomal proteins, that can carry out extraribosomal tasks often
Ribosomal Protein S6 Phosphorylation 63
unrelated to the mechanics of protein synthesis (Warner and McIntosh,
2009); (3) Phosphorylated rpS6 might not affect protein synthesis, but
instead interacts with cellular protein(s), which consequently become active
or inactive, and thus affects the cell physiology. This notion is further
supported by reports on the coimmunoprecipitation of rpS6 with several
extraribosomal proteins, suggesting an in vivo interaction, either directly
or indirectly with these proteins (Kim et al., 2006, 2014; Schumacher
et al., 2006). Not surprisingly, therefore, rpS6
P/
mice show altered
transcription, rather than translation, of the ribosome biogenesis program
in hepatocytes (Chauvin et al., 2014). Clearly, resolving any of these mech-
anistic issues will have to wait for further studies in the coming years.
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