S6K (S6 Kinase)
The ribosomal S6 protein kinases (S6Ks) represent a superfamily of proteins which were originally discovered to phosphorylate ribosomal protein S6. Proteins known as p90 ribosomal S6 kinases (RSKs) have been first identified as able to phosphorylate ribosomal protein S6 (Jones et al. 1988). A short time later, S6K1 protein was discovered gaining a major role in S6 phosphorylation in somatic cells, while RSKs gained less significance in this process (Banerjee et al. 1990). It is important to note that RSKs and S6Ks belong to different subfamilies of serine/threonine kinases but received similar names because S6 protein is a common substrate for both. After 10 years since S6K1 has been cloned, an S6K1 homologous protein was discovered, which was named S6K2 (Saitoh et al. 1998).
Since it was confirmed a direct correlation between S6K1 and mTOR, when it was shown that S6K1 is phosphorylated at T389 by mTOR through kinase assays in vitro (Burnett et al. 1998a), many studies have been performed to discover the functions of the mTOR/S6K1 signaling axis. For instance, it has been determined that this axis mainly acts on the control of protein biosynthesis and cell growth and proliferation (Magnuson et al. 2011).
As observed, many studies have been focused on understanding the S6K1 functions, given that p70-S6K1 is the canonical isoform of S6Ks. Due to the high degree of similarity between S6K1 and S6K2, it was believed that both isoforms had overlapping functions and for a long time S6K2 was neglected. However, it has been shown that S6K2 may present a different regulation and may be involved in different cellular processes (Pardo et al. 2006; Pavan et al. 2016).
The S6K Family
S6K1 and S6K2 share high identity in their kinase domains (84%), with less similarities in their N-terminal (43%) and C-terminal (59%) (Magnuson et al. 2011; Saitoh et al. 1998). S6Ks exhibit some conserved sites of serine and threonine in their kinase domains, linker domain, and C-terminal, which are essential for their activation by phosphorylation (Fig. 2; Weng et al. 1998). Interestingly, there are some differences between S6K1 and S6K2 in their architectures, specifically at the N- and C-terminal, indicating that S6Ks may be related to different cellular compartments or have different molecular targets. For example, a PDZ-binding domain at C-terminal in S6K1 is important for its interaction with Neurabin and, consequently, recruitment to the actin cytoskeleton (Burnett et al. 1998b). On the other hand, S6K2 contains a proline-rich domain in its C-terminal, consisting of five consecutive prolines, which may support the interaction with SH3 (Src homology 3) and/or WW domain-containing proteins (Macias et al. 2002).
Although S6K1 and S6K2 present known structural differences, scarce information is attributed to the S6K2 isoforms. A recent proteomic analysis based on p70-S6K1 and p54-S6K2 interactomes has shown that S6Ks have different interacting partners, strongly suggesting that some proteins may be new targets for the different S6K isoforms (Pavan et al. 2016). Interactions will be described in a later section, showing different functions of S6K proteins and their involvement in several cellular processes.
A shorter S6K1 isoform termed p31-S6K1 is required for cellular transformation and is generated by alternative splicing, which is regulated by the splicing factor SF2/ASF. Functions of p31-S6K1 are still unclear, but it is known that its kinase domain is severely truncated and it is overexpressed in breast cancer (Fig. 2; Karni et al. 2007).
S6Ks Phosphorylation and Activation
Other Posttranslational Modification
S6K1 and S6K2 are also acetylated by p300 acetyltransferase on the K516 residue at the C-terminal domain, inhibiting phosphorylation and activation by mTORC1 on the T389/T388 residue (Fenton et al. 2010). Besides, a study has shown that both S6K1 and S6K2 are ubiquitinated (Wang et al. 2008). The ubiquitin ligase protein ROC1 specifically interacts with S6K1, leading to its degradation (Panasyuk et al. 2008).
A study has shown that S6K1 and S6K2 have different subcellular localizations, since it has been evidenced that GFP-tagged p70-S6K1 is spread throughout the cell, while p54-S6K2 presents an intense localization in the nucleus and nucleolus (Pavan et al. 2016). Thus, these findings agree with the presence of a nuclear localization signal at the C-terminal domain of p54-S6K2, which is not present in p70-S6K1. Although p85-S6K1 presents a nuclear localization signal at its N-terminal domain, a study has shown that it is mainly cytoplasmic (Rosner and Hengstschläger 2011).
S6K Targets and Their Related Biological Processes
Several targets of S6Ks have been described and will be presented bellow along with their biological processes involved. Their relationships with S6Ks are summarized in Fig. 1.
Chaperonin containing TCP-1 (CCTβ) is a large multi-subunit complex that mediates protein folding in eukaryotic cells, participating in the folding of newly synthesized polypeptides, like actin, tubulin, and regulators of cell cycle. A study has identified CCTβ as a downstream target for p90-S6K (RSK) and p70-S6K1. p90- and p70-S6K phosphorylate the CCTβ subunit on S260, indicating that its phosphorylation is an important contributor to cell division (Abe et al. 2009).
Apoptosis is a molecular process of programmed cell death, critical for development, tissue homeostasis, and protection against pathogens. The Bcl-2 family regulates cell death and is regulated by cytokines or other death-survival signals at different levels. Studies have demonstrated the involvement of S6K1 in cell survival by blocking apoptosis through phosphorylation of Bcl-2-associated death promoter (BAD) (Harada et al. 2001). Besides, S6K1 binds and phosphorylates murine double minute 2 (Mdm2) (Lai et al. 2010), preventing its translocation to the nucleus, where Mdm2 leads to the ubiquitination of the nuclear tumor suppressor protein p53. As a consequence, the increase of p53 protein levels leads to cell cycle arrest and/or apoptosis. Furthermore, S6K1 can phosphorylate and inhibit GSK3, generating a signal for cell surviving (Park et al. 2002).
Phosphorylation of the ribosomal S6 protein, target of S6K1 and S6K2, is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery (Park et al. 2002). It has been demonstrated that Cdc42, an important protein in cell growth, can stimulate mRNA splicing via S6K1 and CBC, the nuclear cap-binding complex. S6K1, together with PI3 kinase, can phosphorylate the 80-kDa subunit of the CBC, CBP80, at residues that are growth factor dependent and rapamycin sensitive (Wilson et al. 2000). The role of S6Ks in the splicing process was also investigated for the nuclear protein SKAR (S6K1 Aly/REF-like target), identified as another target of S6K1 and a linker between pre-mRNA splicing and the mTOR pathway (Richardson et al. 2004).
Transforming growth factor-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, being essential for the production of tumor necrosis factor-α TNF-α. TAK1 also plays a key regulatory role in several cytokine-mediated signal transduction cascades, including Interleukin-1 (IL-1) and the downstream signaling of Toll-like receptors (TLRs). It has been shown that S6K1 negatively regulates TLR-mediated signals by inhibiting TAK1 activity (Kim et al. 2014). S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activities. Conversely, cells with S6K1 knockout or knockdown display enhanced production of inflammatory cytokines.
S6K1 has emerged as a critical signaling component in the development of insulin resistance through phosphorylation and inhibition of insulin receptor substrate 1 (IRS-1), acting as a negative feedback loop. A study has shown that S6K1 can directly phosphorylate IRS-1 on S1101 in vitro at its C-terminal domain, inhibiting its downstream activity. The phosphorylation of IRS-1 S1101 is increased in the liver of obese db/db and wild type on a high-fat diet, but not in S6K1 knockout mice maintained on the same diet, indicating that nutrient and hormonal-dependent activation of S6K1 causes insulin resistance (Tremblay et al. 2007).
As a constituent of insulin pathway, the heterotrimeric AMP-activated protein kinase (AMPK) is a known energy sensor that plays a key role in regulating energy metabolism. In response to the reduction of intracellular ATP levels, AMPK inhibits protein, carbohydrate, and lipid biosynthesis, as well as cell growth and proliferation. A study has shown that S6K1 is an inhibitory AMPK kinase, forming a complex with the α2AMPK catalytic subunit and phosphorylating it on S491 (Dagon et al. 2012). This phosphorylation is critical for leptin action on food intake and body weight. S6K1 is also able to phosphorylate and activate PFK-2 (6-phosphofructo-2-kinase), which regulates glycolysis by catalyzing formation of fructose-2,6-bisphosphate, an allosteric activator of PFK-1 of the glycolytic pathway (Deprez 1997).
In the energy metabolism, the production of ketone bodies as an alternative energy source is critical to maintain energy homeostasis during fasting, and its regulation is accomplished by transcriptional networks, such as the peroxisome proliferator-activated receptor (PPAR) family. A study demonstrated that S6K2 can suppress PPARα activity, a ligant-activated transcription factor, impairing the recruitment of nuclear receptor corepressor 1 (NCoR1) to the cell’s nucleus. The S6K2 knockout mice exhibit increased activity of PPARα and consequently increased production of ketone bodies. Nonetheless, the ob/ob mice present increased S6K2 phosphorylation activity and NCoR1 primarily located to the nucleus, leading to decreased production of ketone bodies (Kim et al. 2012).
The anabolic effects of the mTOR/S6K1 pathway are also related to the production of nucleotides, which are necessary for ribosome biogenesis and cell growth. Indeed, S6K1 is able to phosphorylate carbamoyl-phosphate synthase, aspartate carbamoyltransferase, and dihydroorotase (CAD) on S1859, an enzyme that participates in the early stages of pyrimidine synthesis pathway (Ben-Sahra et al. 2013).
Akt, a protein that regulates many processes including metabolism, proliferation, cell survival, growth, and angiogenesis, is upstream to the mTOR/S6K pathway (Fig. 1; Magnuson et al. 2011). It has been reported that S6K2 presents a positive feedback regulation on Akt (Sridharan and Basu 2011). Another protein, B-Raf, involved in the transduction of mitogenic signals from the cell membrane to the nucleus, forms a complex with S6K2 and protein kinase C epsilon (PKCɛ), mediating the activation of S6K2, but not S6K1, by FGF-2 (Pardo et al. 2006). Finally, it has been shown that activation of Erk is able to activate mTOR by inhibiting the TSC1/2 complex (Ma et al. 2005).
One of the involvements of S6Ks in cell proliferation can be highlighted by the interaction of S6K2 with hnRNPF, a protein involved in the splicing process. It has been reported that S6K2, mTOR, and hnRNPF form a complex that is predominantly nuclear and regulates cell proliferation (Goh et al. 2010).
It is largely reported that S6Ks participate in protein synthesis by their interaction with the ribosomal protein S6, considered the main target of S6Ks (Tavares et al. 2015). S6Ks also interact with programmed cell death protein 4 (PDCD4), a protein that inhibits translation initiation and plays a role in apoptosis. The interaction seems to inhibit PDCD4 leading to its ubiquitination and thus increased translation (Carayol et al. 2008). Besides, it has been described that both S6Ks phosphorylate eukaryotic translation initiation factor 4B (eIF4B), a protein required for the binding of the 40S ribosome subunit to the mRNA 5′ cap. eIF4B phosphorylation by S6Ks increases its interaction with eukaryotic translation initiation factor 3 (eIF3), promoting protein synthesis (Jastrzebski et al. 2007; Raught et al. 2004).
S6K1 also binds and inhibits fragile X mental retardation protein (FMRP), a protein that participates in the development of synapses between neurons and associates to several mRNAs, repressing their translation (Narayanan et al. 2008). Finally, S6K1 inhibits eukaryotic elongation factor 2 kinase (eEF2K), a threonine kinase that diminishes protein synthesis (Wang et al. 2001).
S6K1 involvement in transcription is based on its interaction with transcription factors. The relationships between S6K1 and cAMP-responsive element modulator (CREM), a transcriptional regulator that binds to the cAMP response element (CRE), and S6K1 and ERα (estrogen receptor α), a nuclear hormone receptor involved in the regulation of eukaryotic gene expression, have been described (Degroot et al. 1994; Yamnik et al. 2009). S6K1 also interacts with the signaling adaptor STING, forming a complex that is necessary for the activation of the transcription factor IRF3, a protein involved in immunity (Wang et al. 2016).
S6K2 also has interacting partners involved in transcription. Yin Yang 1 (YY1), a multifunctional transcription factor that exhibits positive and negative control on a large number of genes, binds to S6K2 forming a complex that is mediated by the mTOR pathway (Ismail et al. 2013). Besides, S6K2 interacts with the transcription factor RAR-related orphan receptor gamma (RORγ), which is important for Th17 cells differentiation (McGuire et al. 2014). Finally, S6K2 is able to phosphorylate histone H3 on T45, modulating transcription (Ismail et al. 2014).
It has been reported that S6K1 interacts directly with F-actin (Ip et al. 2011), participating in the cytoskeleton organization. S6K1 also activates Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (CDC42 small effector protein 1), two GTPases from the Rho family involved in the regulation of cytoskeleton reorganization, playing a role in the migration of ovarian cancer cells (Ip et al. 2011).
S6Ks in Diseases
Studies have demonstrated the participation of the mTOR/S6K1 axis in the aging process and age-associated disorders. The mTOR pathway inhibition by rapamycin has been demonstrated to increase the life span of mice (Roizen 2010). Additionally, increased S6K1 activity has been associated with endothelial cells senescence due to increased superoxide production by mitochondria, uncoupling endothelial nitric oxide synthase (eNOS), and reducing nitric oxide (NO) levels (Rajapakse et al. 2011). NO is known to impair the aging process of those cells.
In the central nervous system, multiple lines of evidence point to a potential role of S6Ks in synaptic plasticity and consequently learning and memory. A study indicates the involvement of both S6Ks in those processes, showing deficiencies in synaptic plasticity of S6K1 or S6K2 knockout mice (Antion et al. 2008). In addition, another study has demonstrated an important role of S6K1 in the phosphorylation of FMRP, a protein involved in the fragile X mental retardation syndrome (Narayanan et al. 2008).
The relation between S6Ks, mTOR, and cancer has been widely investigated (Tavares et al. 2015). A study has shown that the overexpression of both S6Ks increases viability, migration, and chemotherapy resistance of prostate cancer cells (Amaral et al. 2016). It has been also demonstrated that 4E-BP1 and S6K2 play an important role in breast cancer development (Karlsson et al. 2013). Besides, as already mentioned, it is known that the short isoform p31-S6K1 is overexpressed in breast cancer cells (Karni et al. 2007). In 2010, PF-4708671, an specific cell-permeable inhibitor of S6K1, but not of S6K2, has been reported, suggesting that it may contribute to the treatment of cancer and others human diseases associated with S6Ks (Pearce et al. 2010).
Over the past 30 years, several studies have contributed to a better understanding of the mTOR/S6Ks signaling pathway. However, many questions remain unclear, such as how do post translational modifications regulate S6Ks activity? Despite the high similarity between S6K isoforms, S6K1 still remains the most studied isoform, while literature has limited information about S6K2. Understanding how these isoforms act in different biological processes and what are their protein targets may clarify the specific involvement of S6K1 or S6K2 in human diseases, such as cancer, diabetes, obesity, and neurological disorders. Nonetheless, the development of specific inhibitors, as PF-4708671 for S6K1 and a still unknown inhibitor for S6K2, may guide us to a better understanding of this protein family and open the field for therapeutic applications targeting S6Ks.
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