Identification of S6K2 as a centrosome-located kinase

Ribosomal S6 kinase 2 (S6K2) acts downstream of the mammalian target of rapamycin (mTOR). Here, we show that some S6K2 localize at the centrosome throughout the cell cycle. S6K2 is found in the pericentriolar area of the centrosome. S6K2 centrosomal localization is unaffected by serum withdrawal or treatment with rapamycin, wortmannin, U0126, or phorbol-12-myristate-13-acetate (PMA). Unlike S6K2, S6 kinase 1 (S6K1) does not localize at the centrosome, suggesting the two kinases may also have nonoverlapping functions. Our data suggest that centrosomal S6K2 may have a role in the phosphoinositide-3-kinase (PI3K)/Akt/mTOR signaling pathway that has also been detected in the centrosome.     

Specific interaction between S6K1 and CoA synthase: a potential link between the mTOR/S6K pathway, CoA biosynthesis and energy metabolism

Ribosomal protein S6 kinase (S6K) is a key regulator of cell size and growth. It is regulated via phosphoinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR) signaling pathways. We demonstrate for the first time that CoA synthase associates specifically with S6K1. The association was observed between native and transiently overexpressed proteins in vivo, as well as by BIAcore analysis in vitro. The sites of interaction were mapped to the C-terminal regions of both CoA synthase and S6K1. In vitro studies indicated that the interaction does not affect their enzymatic activities and that CoA synthase is not a substrate for S6 kinase. This study uncovers a potential link between mTor/S6K signaling pathway and energy metabolism through CoA and its thioester derivatives, but its physiological relevance should be further elucidated.     

Cross-talk between Sirtuin and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in the Regulation of S6 Kinase 1 (S6K1) Phosphorylation

p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges on the mechanisms underlying mTORC1-dependent S6K1 activation.     

A phospho-proteomic screen identifies novel S6K1 and mTORC1 substrates revealing additional complexity in the signaling network regulating cell growth

S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70^S6K1 isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85^S6K1 isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85^S6K1 substrates. Four novel putative p85^S6K1 substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.     

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.     

mTOR/S6K1信号通路与胰岛素抵抗的关系及有氧运动对其影响的研究

目的:通过研究高脂饮食和有氧运动对胰岛素抵抗(IR)小鼠骨骼肌雷帕霉素靶蛋白/核糖体S6激酶1(mTOR/S6K1)通路的影响,试图为运动防治IR提供理论依据。方法:8周C57BL/6小鼠随机分为正常饮食组和高脂饮食组,每组各20只,高脂饮食组喂养8周后建立IR模型。随后将正常饮食组再次随机分为正常饮食安静组(NC)和正常饮食运动组(NE);高脂饮食组也随机分为高脂饮食安静组(HC)和高脂饮食运动组(HE)。各运动组进行为期6周、75%VO2max强度跑台训练,每天1次,每次60min,每周5次。实验结束后采用OGTT检测葡萄糖耐量,组织学检测胰岛形态变化,ELISA法检测血清空腹胰岛素水平,Northern blot、Western blot检测骨骼肌中mTOR和S6K1 mRNA和蛋白及其磷酸化蛋白pS6K1-Thr389的表达。结果:与NC组相比,HC组小鼠体重、空腹血清胰岛素值和胰岛β细胞团面积百分比均呈显著增加,且OGTT曲线显示糖耐量明显受损,然而6周有氧运动后以上各指标呈显著性降低,葡萄糖耐量也得到明显改善;且骨骼肌中mTOR、S6K1、pS6K1-Thr389 mRNA和蛋白表达均明显降低。结论:mTOR/S6K1信号通路与高脂饮食诱导IR的发生密切相关,有氧运动明显增加了机体组织对胰岛素的敏感性,推测有氧运动可能通过抑制mTOR/S6K1信号通路,增加IR小鼠骨骼肌的能量代谢从而改善IR。     

Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver

Leucine stimulates protein synthesis by modulating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that promotion of the branched-chain amino acid (BCAA) catabolism might influence the leucine-induced protein synthesis. Clofibric acid (an active metabolite of clofibrate) is known to promote the BCAA catabolism by activation of branched-chain alpha-keto acid dehydrogenase complex (BCKDC), the rate-limiting enzyme of the BCAA catabolism. In the present study, we examined the phosphorylation state of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1) in liver of rats with or without activation of the BCKDC by clofibrate treatment. Clofibrate-treated rats were prepared by oral administration of clofibrate 5 h before sacrifice. In order to stimulate phosphorylation of components in the mTOR signaling pathway, rats were orally administered with leucine 1 h before sacrifice. Clofibrate treatment almost fully activated hepatic BCKDC and significantly decreased the plasma leucine concentration in rats without leucine administration, resulting in decreased mTOR and 4E-BP1 phosphorylation. Similarly, in rats administered with leucine, clofibrate treatment attenuated the predicted increase in plasma leucine concentration as well as the phosphorylation of mTOR, 4E-BP1, and S6K1. These results suggest that BCAA catabolism enhanced by clofibrate treatment has significant influences on the leucine-induced activation of translation initiation processes.     

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.     

Identification of a Dual Inhibitor of Janus Kinase 2 (JAK2) and p70 Ribosomal S6 Kinase1 (S6K1) Pathways

Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach.     

Recombinant production, purification and biochemical characterization of domain 6 of LEKTI: a temporary Kazal-type-related serine proteinase inhibitor

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor which is of pathophysiological relevance for skin diseases and atopy. Domains 2 and 15 of LEKTI contain six cysteine residues and match the Kazal-type inhibitor motif almost exactly. The other 13 domains seem to be Kazal-type derived but lack the cysteines in positions 3 and 6 usually conserved within this family of inhibitors. Here, we report the recombinant production and comprehensive biochemical characterization of the 7.7 kDa LEKTI domain 6 (LD-6). Testing a selected number of different serine proteinases, we show that both native and recombinant LD-6 exhibit a significant but temporary inhibitory activity on trypsin. Furthermore, the relation of LEKTI domain 6 to Kazal-type inhibitors is confirmed by determining its disulfide bond pattern (1-4/2-3) and its P(1) site located after the second Cys residue of LD-6. The established strategy for the recombinant production of LEKTI domain 6 will enable further investigation of its mode of action and its physiological role.     

Phylogenetic relationships of Cyprinidae (Teleostei: Cypriniformes) inferred from the partial <Emphasis Type="Italic">S6K1</Emphasis> gene sequences and implication of indel sites in intron 1

The family Cyprinidae is widely distributed in East Asia, and has the important phylogenetic significance in the fish evolution. In this study, the 5′ end partial sequences (containing exon 1, exon 2 and indel 1) of S6K1 gene were obtained from 30 representative species in Cyprinidae and outgroup using PCR amplification and sequencing. The phylogenetic relationships of Cyprinidae were reconstructed with neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. Myxocyprinus asiaticus (Catostomidae) was assigned to the outgroup taxon. Similar phylogenetic relationships within the family Cyprinidae were achieved with the four analyses. Leuciscini and Barbini were monophyletic lineages respectively with the high nodal supports. Leuciscini comprises Hypophthalmichthyinae, Xenocyprinae, Cultrinae, Gobioninae, Acheilognathinae and East Asian species of Leuciscinae and Danioninae. Monophyly of East Asian clade was supported with high nodal support. Barbini comprises Schizothoracinae, Barbinae, Cyprininae and Labeoninae. The monophyletic lineage consisting of Danio rerio, D. myersi, and Rasbora trilineata was basal in the tree. In addition, the large fragment indels in intron 1 were analyzed to improve the understanding of Cyprinidae relationships. The results showed that the large fragment indels were correlated with the relations among species. Some conserved regions in intron 1 were thought to be involved in the functional regulation. However, no correlation was found between sequence variations and species characteristic size.     

Role of p70S6K1-mediated Phosphorylation of eIF4B and PDCD4 Proteins in the Regulation of Protein Synthesis

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5′-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.     

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.