Figure 1. S6 kinase signaling pathway
S6 kinase overview
S6 kinase (ribosomal protein S6 kinase)
S6K1 and S6K2 are members of the AGC kinase family. AGC kinases are known as the serine–threonine kinases. AGC kinases have some structural similarities. They play an important role in regulating some cellular activities including growth, survival and metabolism. In addition to S6Ks, some well-known proteins like PKA, PKG, PKC and Akt are also members of the AGC kinase family. Many data in precious studies show that the mTOR/S6K signaling pathway make a great contribution to many common diseases like cancer, diabetes and obesity, which shows its importance. Although S6K1 and S6K2 have many similarities and S6K1 is much better studied, there are some significant differences in terms of their functions both in vitro and in vivo found in new studies. In the kinase domain, they have about 83% of identity, while in they show lower similarity in N terminus and C-terminal domain.
Figure 2. S6K isoforms, domain structure and phosphorylation sites
S6Kα2 is one of isoforms of S6K1. It is a well-described protein with 502 amino acids which is around 70kD. However, there is another isoform named S6Kα1 with an additional 23 N-terminal amino acids and it is around 85 kD. Even if the smaller isoform p70-S6K1 predominantly stay at the cytosol, it is considered that p85-S6K1 is likely to locate at the nucleus. This assumption is based on the presence of an NLS (nuclear localization sequence) within the N terminal extension of p85-S6K1. In addition to S6K1, S6K2 also has several isoforms. The smallest one named p54-S6K2 or S6Kβ2 has 482 amino acids. Another 56 kDa isoform which is also called S6Kβ1) contains an extension in N terminal domain with extra 13 amino acids. Both of larger isoforms of S6K1 and S6K2 contain a nuclear localization sequence region in the N-terminal domain. However, S6K2 also contains another nuclear localization sequence region in the C-terminal domain, which is different from S6K1. There is an even smaller variant of S6K1 named p31-S6K1. Although it is a small protein with fewer amino acids, it is essential for cellular transformation induced by the splicing factor SF2/ASF (splicing factor 2/alternative splicing factor). Its small size come from the absence of most of the kinase domain. Both of S6K1 and S6K2 protein are composed of some critical regulatory domains including an acidic N terminal region which contains the TOS (TOR signaling) motif, the kinase domain with T-loop and a basic C terminal region with an autoinhibitory pseudo-substrate domain. In addition, there is a linker region that contains the TM and HM sites. Especially, the C-terminal domain makes S6Ks unique and different from other AGC family members. At the same time, the C terminal region also have many phosphorylation sites.
S6 kinase signaling pathway
S6K signaling cascade
Thr229 and Thr389 are two important sites for S6K1 activation. The phosphorylation of these two sites shows strong positive co-operativity their temporal relationship to each other. There are two well-described models for the stepwise activation of S6K1 which is regulated by the ordered multi-site phosphorylation. Model 1 is the conventional one which shows PDK1- mediated phosphorylation of Thr229 occurs later than mTORC1- mediated phosphorylation of Thr389. In this model, Thr389 is a docking site for PDK1 phosphorylation and then Thr229 on the activation loop is further phosphorylated, which is similar to PDK1-mediated activation of RSK2. On the other hand, the alternative Model 2 shows that PDK1-mediated phosphorylation of Thr229 occurs prior to mTORC1-mediated phosphorylation of Thr389.
In loss of function studies, an S6K1-T389A mutant shows the level of growth-factor-stimulated Thr229 phosphorylation decrease. In vitro, S6K1-T389E-D3E is phosphorylated by PDK-1 with a higher level than that of wild-type. T389E or D3E plays an important role in the HM site and C-terminal phosphorylation during the process of S6K1 phosphorylation which is mediated by PDK-1. As for Model 2, an S6K1-T229A mutant shows the level of growth-factor-stimulated Thr389osphorylation decrease. Recent data suggests that phosphorylation of S6K1 on Thr389 mediated by mTOR in vitro is prior to the phosphorylation on Thr229 by PDK1. Because of the support for alternate Model 2, TM site phosphorylation is considered to represent an early event that occurs prior to the phosphorylation of HM and T-loop site.
Figure 3. Stepwise activation of S6K1 via multi-site phosphorylation
For a long time, rpS6 is studied as the only substrate of S6K. However, there are more new other proteins shown to be substrates of S6K. There are more than 9 substrates have been discovered so far, but their activity and regulation are not all well described. Actually, there are some criteria to settle a protein as the substrate of S6K. First of all, it is important to have the conformity of the phosphorylated serine’s sequence context with the consensus S6K recognition motif. Second, it can be phosphorylated by S6K in vitro. Third, there should be a correlation of the phosphorylation status in vivo with S6K activity. Last but not least, the hypo-phosphorylation on the putative substrate can be conferred through knockout or knockdown of S6K. Current discovered substrates can be divided into different groups based on their function or subcellular location.
At the level of cells, mTORC1, as an essential regulator, plays a role in the translation initiation, which is the rate-limiting step. With the increase of protein synthesis, cell growth is promoted significantly. At the same time, cell division and proliferation are enhanced as well. In these processes, S6K1 phosphorylates its substrates, which is the driving force. In addition to the phosphorylation by S6K1, it can also be mediated by 4EBP1. It has been established that the direct control of translational initiation is via the mTORC1–4EBP1 axis. However, the discovery and identification of substrates and functions of the mTORC1–S6K1 axis are challenging. In addition to rpS6, the first discovered substrate, other substrates are essential for a wide range of biological activities including the transcriptional control of ribosome biogenesis, metabolism, lipid synthesis, adipocyte differentiation, cell survival, DNA damage sensing as well as synaptic plasticity. The mTORC1–S6K1 axis plays a significant role in many feedback loops.
Chronic activation of S6K1 by mTORC1 induces insulin resistance by a kind of special mechanism named the ‘negative-feedback loop’. S6K1 signaling impose a repressing effect on IRS-1 gene expression, which directly phosphorylates IRS-1 on several inhibitory serine residues. At the same time, mTOR phosphorylates IRS-1 (Ser636/Ser639). This phosphorylation of IRS-1 serine initiates IRS-1 degradation via the proteasome. This results in that PI3K departs from the insulin/IGF receptor and then leads to the inhibition of signaling involving downstream PI3K effectors such as Akt and the Ras/MAPK pathway. This negative-feedback loop may provide an explanation for related insulin resistance. In addition to the activation of mTORC1, S6K1 also takes part in other feedback loops. Especially, new studies suggest that mTORC1 can phosphorylate Grb10 to mediate negative feedback to insulin/IGF signaling.
Relationship with diseases
Previous studies have shown the great importance of mTORC1–S6K1 axis in physiology and a wide range of diseases. It plays an essential role in diabetes, obesity, cancer and benign tumor syndromes, organ hypertrophy, neurological disorders like autism spectrum disorders and Alzheimer’s disease as well as aging-related pathology. Many of them are because of the mutations of mTORC1. The mutations exert negative effects on lipid synthesis, cell growth, cell proliferation and cellular metabolism. Particularly, S6K is highly associated with cancer development.
The deregulation of PI3K/Akt and mTOR/S6K signaling pathway where a series of signal transduction mediators and effectors are inhibited or activated has been studied and considered to predispose to carcinogenesis. In several cancers, the expression level and also activity of S6K have increased, which is related to drugs resistance and chemotherapeutic treatment. Data in many studies suggest that S6K2 participates in cell proliferation and survival which are mediated by mTOR activation. At the same time, the inhibition of S6K1 can prevent the migration of cancer cell. This phenomenon is more significant in breast cancer, which shows a great importance in metastasis. Besides the level of protein, alternative splicing of S6K also affects cancer development. Different isoforms of S6K caused by alternative splicing play different roles in it. Data of recent studies have suggested that S6K shows a high expression level in cancer cells. Compared to S6K1, the expression level of S6K2 is remarkably higher in cancer cells. At the same time, S6K2 can also be accumulated in nucleus of cancer cells. This nuclear accumulation is believed to be related to malignant growth signaling of cancer cells as it affects cell growth and proliferation. In addition, S6K2 also takes part in some anti-apoptotic proteins’ transcription includin Bcl-xL and XIAP. With the coordination of FGF-2 (fibroblast growth factor-2), S6K2 is able to form a complex protein with other two factors named B-Raf and PKCε. Therefore, this formation of a signaling protein complex composed with PKCε, B-Raf and S6K2, is able to control transcription of Bcl-xL and XIAP, which promotes cancer cell survival and resistance to chemotherapy.
Figure 4. Downstream effectors of S6 kinase