90-kDa ribosomal S6 kinase is a direct target for the nuclear fibroblast growth factor receptor 1 (FGFR1): role in FGFR1 signaling

Fibroblast growth factor receptor 1 (FGFR1) is a transmembrane protein capable of transducing stimulation by secreted FGFs. In addition, newly synthesized FGFR1 enters the nucleus in response to cellular stimulation and during development. Nuclear FGFR1 can transactivate CRE (cAMP responsive element), activate CRE-binding protein (CREB)-binding protein (CBP) and gene activities causing cellular growth and differentiation. Here, a yeast two-hybrid assay was performed to identify FGFR1-binding proteins and the mechanism of nuclear FGFR1 action. Ten FGFR1-binding proteins were identified. Among the proteins detected with the intracellular FGFR1 domain was a 90-kDa ribosomal S6 kinase (RSK1), a regulator of CREB, CBP, and histone phosphorylation. FGFR1 bound to the N-terminal region of RSK1. The FGFR1-RSK1 interaction was confirmed by co-immunoprecipitation and colocalization in the nucleus and cytoplasm of mammalian cells. Predominantly nuclear FGFR1-RSK1 interaction was observed in the rat brain during neurogenesis and in cAMP-stimulated cultured neural cells. In TE671 cells, transfected FGFR1 colocalized and coimmunoprecipitated, almost exclusively, with nuclear RSK1. Nuclear RSK1 kinase activity and RSK1 activation of CREB were enhanced by transfected FGFR1. In contrast, kinase-deleted FGFR1 (TK-), which did not bind to RSK1 failed to stimulate nuclear RSK1 activity or RSK1 activation of CREB. Kinase inactive FGFR1 (K514A) bound effectively to nuclear RSK1, but it failed to stimulate RSK1. Thus, active FGFR1 kinase regulates the functions of nuclear RSK1. The interaction of nuclear FGFR1 with pluripotent RSK1 offers a new mechanism through which FGFR1 may control fundamental cellular processes.     

Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase

Here we demonstrate that mammalian target of rapamycin (mTOR) is phosphorylated in a rapamycin-sensitive manner. We show that S6 kinase 1 (S6K1), but not Akt, directly phosphorylates mTOR in cell-free in vitro system and in cells. Expression of a constitutively active, rapamycin- and wortmannin-resistant S6K1 leads to constitutive phosphorylation of mTOR, whereas knock-down of S6K1 using small inhibitory RNA greatly reduces mTOR phosphorylation despite elevated Akt activity. Importantly, phosphorylation of mTOR by S6K1 occurs at threonine 2446/serine 2448. This region has been shown previously to be part of a regulatory repressor domain. These sites are also constitutively phosphorylated in the breast cancer cell line MCF7 carrying an amplification of the S6K1 gene, but not in a less tumorigenic cell line, MCF10a. Many models for Akt signaling to mTOR have been presented, suggesting direct phosphorylation by Akt. These models must be reconsidered in light of the present findings.     

SHP-2 regulates cell growth by controlling the mTOR/S6 kinase 1 pathway

Cell growth (accumulation in cell mass) ensues through the promotion of macromolecular biosynthesis. S 6 ribosomal kinase 1 (S6K1), which is activated by the mammalian target of rapamycin, is critical for cell growth. The early events that control S6K1 signaling remain unclear. Here we show that SHP-2 suppresses S6K1 activity under conditions of growth factor deprivation. We show that under conditions of growth factor deprivation, S6K1 activity was increased in fibroblasts lacking functional SHP-2 and in cells where knock down of SHP-2 expression was established by small interference RNA. Consistent with these findings, fibroblasts lacking functional SHP-2 exhibited increased cell size as compared with wild type cells. Growth factor deprivation reduces cellular energy, and the energy-sensing 5'-AMP-activated protein kinase (AMPK) negatively regulates S6K1. We found that SHP-2 promoted AMPK activity under conditions of growth factor deprivation (low energy), suggesting that SHP-2 negatively regulates S6K1 via an AMPK-dependent pathway. These results implicate SHP-2 as an early mediator in the S6K1 signaling pathway to limit cell growth in low energy states.     

A nuclear transport signal in mammalian target of rapamycin is critical for its cytoplasmic signaling to S6 kinase 1

The mammalian target of rapamycin (mTOR) regulates nutrient-dependent cell growth and proliferation through cytoplasmic targets, such as S6 kinase 1 (S6K1). Consistent with its main function in the cytoplasm, mTOR is predominantly cytoplasmic. However, previously we have found that mTOR shuttles between the nucleus and cytoplasm, and we have proposed that the nucleocytoplasmic shuttling of mTOR is required for the maximal activation of S6K1. The intrinsic signals directing mTOR nuclear transport and the underlying mechanisms are unknown. In this study we initially set out to identify nuclear export signals in mTOR. A systematic scan of the mTOR sequence revealed 16 peptides conforming to the canonical leucine-rich nuclear export signal, of which 3 were found by reporter assays to contain leptomycin B-sensitive and leucine-dependent nuclear export activity. Unexpectedly, mTOR proteins with those conserved leucines mutated to alanines were unable to enter the nucleus. Further investigation revealed that the L982A/L984A and L1287A/L1289A mutations likely induced a global structural change in mTOR, whereas the L545A/L547A mutation directly impaired the nuclear import of the protein, potentially regulated by a nucleocytoplasmic shuttling signal. The loss of nuclear import was accompanied by the significantly reduced ability of the L545A/L547A mutant to activate S6K1 in cells. Most importantly, when nuclear import was restored in the L545A/L547A mutant by the addition of an exogenous nuclear import signal, signaling to S6K1 was rescued. Taken together, our observations suggest the existence of a nuclear shuttling signal in mTOR and provide definitive evidence for the requirement of mTOR nuclear import in its cytoplasmic signaling to S6K1.     

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. Here, we have characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. Our Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, we demonstrated that ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, our genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation.     

The mammalian target of rapamycin-p70 ribosomal S6 kinase but not phosphatidylinositol 3-kinase-Akt signaling is responsible for fibroblast growth factor-9-induced cell proliferation

Fibroblast growth factor-9 (FGF9) is a potent mitogen that stimulates normal and cancer cell proliferation though the signaling mechanism is not fully understood. In this study, we aimed to unravel the signaling cascades mediate FGF9 actions in human uterine endometrial stromal cell. Our results demonstrate that the mitogenic effect of FGF9 is transduced via two parallel but additive signaling pathways involving mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase. Activation of mTOR by FGF9 induces p70 ribosomal S6 kinase (S6K1) phosphorylation, cyclin expression, and cell proliferation, which are independent of phosphatidylinositol 3-kinase and Akt. Coimmunoprecipitation analysis demonstrates that mTOR physically associates with S6K1 upon FGF9 treatment, whereas ablation of mTOR activity using RNA interference or pharmacological inhibitor blocks S6K1 phosphorylation and cell proliferation induced by FGF9. Further study demonstrates that activation of mTOR is regulated by a phospholipase Cgamma-controlled calcium signaling pathway. These studies provide evidence to demonstrate, for the first time, that a novel signaling cascade involving phospholipase Cgamma, calcium, mTOR, and S6K1 is activated by FGF9 in a receptor-specific manner.     

ZRF1 is a novel S6 kinase substrate that drives the senescence programme

The inactivation of S6 kinases mimics several aspects of caloric restriction, including small body size, increased insulin sensitivity and longevity. However, the impact of S6 kinase activity on cellular senescence remains to be established. Here, we show that the constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) by tuberous sclerosis complex (TSC) mutations induces a premature senescence programme in fibroblasts that relies on S6 kinases. To determine novel molecular targets linking S6 kinase activation to the control of senescence, we set up a chemical genetic screen, leading to the identification of the nuclear epigenetic factor ZRF1 (also known as DNAJC2, MIDA1, Mpp11). S6 kinases phosphorylate ZRF1 on Ser47 in cultured cells and in mammalian tissues in vivo. Knock-down of ZRF1 or expression of a phosphorylation mutant is sufficient to blunt the S6 kinase-dependent senescence programme. This is traced by a sharp alteration in p16 levels, the cell cycle inhibitor and a master regulator of senescence. Our findings reveal a mechanism by which nutrient sensing pathways impact on cell senescence through the activation of mTORC1-S6 kinases and the phosphorylation of ZRF1.     

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid-signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2-1, regulated by the 3'-phosphoinositide-dependent kinase-1 (AtPDK1). AGC2-1 specifically interacts with AtPDK1 through a conserved C-terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2-1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2-1 in an AtPDK1-dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2-1 is abundant in fast-growing organs and dividing cells, and activated during re-entry of cells into the cell cycle after sugar starvation-induced G1-phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1-regulated AGC2-1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP-AGC2-1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2-1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2-1 in root hair growth and development.     

Cdc42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex

Cdc42 is a low molecular weight GTP-binding protein that plays a key regulatory role in a variety of cellular activities. The importance of the coordination of different cell functions by Cdc42 is underscored by the fact that a constitutively active Cdc42 mutant induces cellular transformation. In this study, we describe a novel function for Cdc42: its ability to stimulate pre-messenger RNA splicing. This activity is dependent on cysteine 37 in the effector loop of Cdc42 but is not dependent on cell growth. A likely candidate protein for mediating the Cdc42 effects on pre-mRNA splicing is the nuclear RNA cap-binding complex (CBC), which plays a key role in an early step of cap-dependent RNA splicing. Activation of the CBC by Cdc42 can be inhibited by rapamycin. Additionally, phosphatidylinositol 3-kinase and the Cdc42 effector, pp70 S6 kinase, stimulate the RNA cap-binding activity of the CBC. S6 kinase may directly target the CBC in vivo as it can phosphorylate the 80-kDa subunit of the CBC, CBP80, at residues that are subject to a growth factor-dependent and rapamycin-sensitive phosphorylation in vivo. Together these data suggest the involvement of a Cdc42-S6 kinase pathway in the regulation of RNA splicing, mediated by an increase in capped RNA binding by the CBC, as well as raise the possibility that the effects of Cdc42 on cell growth may be due in part to its regulation of RNA processing.     

Atf1 is a target of the mitogen-activated protein kinase Pmk1 and regulates cell integrity in fission yeast

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.     

Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site

A critical step in S6 kinase 1 (S6K1) activation is Thr(229) phosphorylation in the activation loop by the phosphoinositide-dependent protein kinase (PDK1). Thr(229) phosphorylation requires prior phosphorylation of the Ser/Thr-Pro sites in the autoinhibitory domain and Thr(389) in the linker domain, consistent with PDK1 more effectively catalyzing Thr(229) phosphorylation in a variant harboring acidic residues in these positions (S6K1-E389D(3)E). S6K1-E389D(3)E has high basal activity and exhibits partial resistance to rapamycin and wortmannin, and its activity can be further augmented by mitogens, effects presumably mediated by Thr(229) phosphorylation. However, PDK1-induced Thr(229) phosphorylation is reported to be constitutive rather than phosphatidylinositide 3,4,5-trisphosphate-dependent, suggesting that S6K1-E389D(3)E activity is mediated through a distinct site. Here we use phosphospecific antibodies to show that Thr(229) is fully phosphorylated in S6K1-E389D(3)E in the absence of mitogens and that regulation of S6K1-E389D(3)E activity by mitogens, rapamycin, or wortmannin parallels Ser(371) phosphorylation. Consistent with this observation, a dominant interfering allele of the mammalian target of rapamycin, mTOR, inhibits mitogen-induced Ser(371) phosphorylation and activation of S6K1-E389D(3)E, whereas wild type mTOR stimulates both responses. Moreover, in vitro mTOR directly phosphorylates Ser(371), and this event modulates Thr(389) phosphorylation by mTOR, compatible with earlier in vivo findings.     

Glycogen synthase kinase (GSK)-3 and mammalian target of rapamycin complex 1 (mTORC1) cooperate to regulate protein S6 kinase 1 (S6K1)

Comment on: Shin S, et al. Proc Natl Acad Sci USA 2011; 108:1204–13     

Hsp70 is a new target of Sgt1--an interaction modulated by S100A6

Here, two temperature sensitive promoters, P2 and P7, isolated from Bacillus subtilis, were characterized. The production of beta-galactosidase driven by these promoters was much higher at 45 degrees C than that at 37 degrees C both in Escherichia coli and B. subtilis and that the P2 promoter showed higher expression strength in B. subtilis at 45 degrees C. Thereby, an efficient temperature-inducible expression system was constructed by using P2 promoter in B. subtilis. Thus, we isolated and characterized a newly temperature inducible promoter and exploited it as a potential expression element in B. subtilis.     

Protein kinase Ctheta is a specific target for inhibition of the HIV type 1 replication in CD4+ T lymphocytes

Integration of HIV-1 genome in CD4(+) T cells produces latent reservoirs with long half-life that impedes the eradication of the infection. Control of viral replication is essential to reduce the size of latent reservoirs, mainly during primary infection when HIV-1 infects CD4(+) T cells massively. The addition of immunosuppressive agents to highly active antiretroviral therapy during primary infection would suppress HIV-1 replication by limiting T cell activation, but these agents show potential risk for causing lymphoproliferative disorders. Selective inhibition of PKC, crucial for T cell function, would limit T cell activation and HIV-1 replication without causing general immunosuppression due to PKC being mostly expressed in T cells. Accordingly, the effect of rottlerin, a dose-dependent PKC inhibitor, on HIV-1 replication was analyzed in T cells. Rottlerin was able to reduce HIV-1 replication more than 20-fold in MT-2 (IC(50) = 5.2 μM) and Jurkat (IC(50) = 2.2 μM) cells and more than 4-fold in peripheral blood lymphocytes (IC(50) = 4.4 μM). Selective inhibition of PKC, but not PKCδ or -ζ, was observed at <6.0 μM, decreasing the phosphorylation at residue Thr(538) on the kinase catalytic domain activation loop and avoiding PKC translocation to the lipid rafts. Consequently, the main effector at the end of PKC pathway, NF-κB, was repressed. Rottlerin also caused a significant inhibition of HIV-1 integration. Recently, several specific PKC inhibitors have been designed for the treatment of autoimmune diseases. Using these inhibitors in combination with highly active antiretroviral therapy during primary infection could be helpful to avoid massive viral infection and replication from infected CD4(+) T cells, reducing the reservoir size at early stages of the infection.