Autoregulation of the Mechanistic Target of Rapamycin (mTOR) Complex 2 Integrity Is Controlled by an ATP-dependent Mechanism

Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mm and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.     

Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation

The mammalian target of rapamycin, mTOR, is a Ser/Thr kinase that promotes cell growth and proliferation by activating ribosomal protein S6 kinase 1 (S6K1). We previously identified a conserved TOR signaling (TOS) motif in the N terminus of S6K1 that is required for its mTOR-dependent activation. Furthermore, our data suggested that the TOS motif suppresses an inhibitory function associated with the C terminus of S6K1. Here, we have characterized the mTOR-regulated inhibitory region within the C terminus. We have identified a conserved C-terminal "RSPRR" sequence that is responsible for an mTOR-dependent suppression of S6K1 activation. Deletion or mutations within this RSPRR motif partially rescue the kinase activity of the S6K1 TOS motif mutant (S6K1-F5A), and this rescued activity is rapamycin resistant. Furthermore, we have shown that the RSPRR motif significantly suppresses S6K1 phosphorylation at two phosphorylation sites (Thr-389 and Thr-229) that are crucial for S6K1 activation. Importantly, introducing both the Thr-389 phosphomimetic and RSPRR motif mutations into the catalytically inactive S6K1 mutant S6K1-F5A completely rescues its activity and renders it fully rapamycin resistant. These data show that the N-terminal TOS motif suppresses an inhibitory function mediated by the C-terminal RSPRR motif. We propose that the RSPRR motif interacts with a negative regulator of S6K1 that is normally suppressed by mTOR.     

Mechanistic Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation Is Governed by Competition between Substrates for Interaction with Raptor

In this study, the interaction of mTORC1 with its downstream targets p70S6K1 and 4E-BP1 was evaluated in both mouse liver and mouse embryonic fibroblasts following combined disruption of the genes encoding 4E-BP1 and 4E-BP2. Phosphorylation of p70S6K1 was dramatically elevated in the livers of mice lacking 4E-BP1 and 4E-BP2 following feeding-induced activation of mTORC1. Immunoprecipitation of mTORC1 suggested that elevated phosphorylation was the result of enhanced interaction of p70S6K1 with raptor. These findings were extended to a cell culture system wherein loss of 4E-BP1 and 4E-BP2 resulted in elevated interaction of p70S6K1 with IGF1-induced activation of mTORC1 in conjunction with an enhanced rate of p70S6K1 phosphorylation at Thr-389. Furthermore, cotransfecting HA-p70S6K1 with 4E-BP1, but not 4E-BP1(F114A), reduced recovery of mTORC1 in HA-p70S6K1 immunoprecipitates. Together, these findings support the conclusion that, in the absence of 4E-BP proteins, mTORC1-mediated phosphorylation of p70S6K1 is elevated by a reduction in competition between the two substrates for interaction with raptor.     

Cyclin-dependent Kinase 5 (Cdk5)-dependent Phosphorylation of p70 Ribosomal S6 Kinase 1 (S6K) Is Required for Dendritic Spine Morphogenesis

The maturation and maintenance of dendritic spines depends on neuronal activity and protein synthesis. One potential mechanism involves mammalian target of rapamycin, which promotes protein synthesis through phosphorylation of eIF4E-binding protein and p70 ribosomal S6 kinase 1 (S6K). Upon extracellular stimulation, mammalian target of rapamycin phosphorylates S6K at Thr-389. S6K also undergoes phosphorylation at other sites, including four serine residues in the autoinhibitory domain. Despite extensive biochemical studies, the importance of phosphorylation in the autoinhibitory domain in S6K function remains unresolved, and its role has not been explored in the cellular context. Here we demonstrated that S6K in neuron was phosphorylated at Ser-411 within the autoinhibitory domain by cyclin-dependent kinase 5. Ser-411 phosphorylation was regulated by neuronal activity and brain-derived neurotrophic factor (BDNF). Knockdown of S6K in hippocampal neurons by RNAi led to loss of dendritic spines, an effect that mimics neuronal activity blockade by tetrodotoxin. Notably, coexpression of wild type S6K, but not the phospho-deficient S411A mutant, could rescue the spine defects. These findings reveal the importance of cyclin-dependent kinase 5-mediated phosphorylation of S6K at Ser-411 in spine morphogenesis driven by BDNF and neuronal activity.     

S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2

S6K1 (p70S6K) is a serine kinase downstream from Akt in the insulin signaling pathway that is involved in negative feedback regulation of insulin action. S6K1 is also activated by TNF-alpha, a pro-inflammatory cytokine. However, its role remains to be characterized. In the current study, we elucidated a mechanism for S6K1 to mediate TNF-alpha-induced insulin resistance in adipocytes and hepatocytes. S6K1 was phosphorylated at Thr-389 in response to TNF-alpha. This led to phosphorylation of IRS-1 by S6K1 at multiple serine residues including Ser-270, Ser-307, Ser-636, and Ser-1101 in human IRS-1 (Ser-265, Ser-302, Ser-632, and Ser-1097, in rodent IRS-1). Direct phosphorylation of these sites by S6K1 was observed in an in vitro kinase assay using purified IRS-1 and S6K1. Phosphorylation of all these serines was increased in the adipose tissue of obese mice. RNAi knockdown demonstrated an important role for S6K1 in mediating TNF-alpha-induced IRS-1 inhibition that led to impaired insulin-stimulated glucose uptake in adipocytes. A point mutant of IRS-1 (S270A) impaired association of IRS-1 with S6K1 resulting in diminished phosphorylation of IRS-1 at three other S6K1 phosphorylation sites (Ser-307, Ser-636, and Ser-1101). Expression of a dominant negative S6K1 mutant prevented TNF-induced Ser-270 phosphorylation and IRS-1 protein degradation. Moreover, in IKK2 (but not IKK1)-null cells, TNF-alpha treatment did not result in Thr-389 phosphorylation of S6K1. We present a new mechanism for TNF-alpha to induce insulin resistance that involves activation of S6K by an IKK2-dependent pathway. S6K directly phosphorylates IRS-1 on multiple serine residues to inhibit insulin signaling.     

Characterization of phosphatidylinositol 3-kinase-dependent phosphorylation of the hydrophobic motif site Thr(389) in p70 S6 kinase 1

Phosphorylation of the highly conserved hydrophobic motif site in AGC kinases is necessary for phosphotransferase activity. Phosphorylation of this motif (FLGFT389Y) in p70 S6 kinase (S6K1) is both rapamycin- and wortmannin-sensitive, suggesting a role for both mammalian target of rapamycin- and phosphatidylinositol 3-kinase-dependent pathways. We report here that co-expression of phosphoinositide-dependent kinase-1 (PDK1) and the phosphatidylinositol 3-kinase-regulated atypical protein kinase Czeta cooperate to increase both phosphorylation of the hydrophobic motif site Thr(389), as well as the activation loop site Thr(229). Interestingly, although PDK1 alone can promote an increase in Thr(389) phosphorylation in both wild type S6K1 and a kinase-inactive mutant of S6K1, the cooperative effect between PDK1 and protein kinase Czeta required S6K1 activity. Furthermore, Akt, another phosphatidylinositol 3-kinase effector and regulator of S6K1, also increased Thr(389) phosphorylation in a S6K1 activity-dependent manner. Consistent with this, epidermal growth factor-induced Thr(389) phosphorylation in wild type S6K1 persisted for up to 120 min, whereas kinase-inactive mutants of S6K1 displayed only a reduced and transient increase in Thr(389) phosphorylation. We conclude that S6K1 activity is required for maximal Thr(389) phosphorylation by mitogens and by multiple phosphatidylinositol 3-kinase-dependent inputs including PDK1, PKCzeta, and Akt, and we propose that autophosphorylation is an important regulatory mechanism for phosphorylation of the hydrophobic motif Thr(389) site in S6K1.     

Regulation of s6 kinase 1 activation by phosphorylation at ser-411

Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G(1) phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1.     

p70 S6K1 nuclear localization depends on its mTOR-mediated phosphorylation at T389, but not on its kinase activity towards S6

The protein kinase p70 S6K1 is regulated in response to cytokines, nutrients and growth factors, and plays an important role in the development of a variety of human diseases. Mammalian target of rapamycin (mTOR) is known to phosphorylate and thereby activate p70 S6K1. p70 S6K1 phosphorylates different cytoplasmic and nuclear substrates involved in the regulation of protein synthesis, cell cycle, cell growth and survival. Recently, we have shown that mTOR-mediated phosphorylation of p70 S6K1 at T389 also regulates its nucleocytoplasmic localization. Since this phosphorylation is associated with its kinase activity the question whether p70 S6K1 phosphorylation or kinase activity is essential for its proper localization remained elusive. Recently, the chemical compound PF-4708671 has been demonstrated to block p70 S6K1 kinase activity while inducing its phosphorylation at T389. This potential of PF-4708671 to separate p70 S6K1 activity from its T389 phosphorylation allowed us to demonstrate that the proper nucleocytoplasmic localization of this kinase depends on its mTOR-mediated phosphorylation but not on its kinase activity. These findings provide important insights into the regulation of p70 S6K1 and allow a more detailed understanding of subcellular enzyme localization processes.     

Intestinal cell kinase (ICK) promotes activation of mTOR complex 1 (mTORC1) through phosphorylation of Raptor Thr-908

Intestinal cell kinase (ICK), named after its cloning origin, the intestine, is actually a ubiquitously expressed and highly conserved serine/threonine protein kinase. Recently we reported that ICK supports cell proliferation and G(1) cell cycle progression. ICK deficiency significantly disrupted the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling events. However, the biological substrates that mediate the downstream signaling effects of ICK in proliferation and the molecular mechanisms by which ICK interacts with mTORC1 are not well defined. Our prior studies also provided biochemical evidence that ICK interacts with the mTOR/Raptor complex in cells and phosphorylates Raptor in vitro. In this report, we investigated whether and how ICK targets Raptor to regulate the activity of mTORC1. Using the ICK substrate consensus sequence [R-P-X-S/T-P/A/T/S], we identified a putative phosphorylation site, RPGT908T, for ICK in human Raptor. By mass spectrometry and a phospho-specific antibody, we showed that Raptor Thr-908 is a novel in vivo phosphorylation site. ICK is able to phosphorylate Raptor Thr-908 both in vitro and in vivo and when Raptor exists in protein complexes with or without mTOR. Although expression of the Raptor T908A mutant did not affect the mTORC1 integrity, it markedly impaired the mTORC1 activation by insulin or by overexpression of the small GTP-binding protein RheB under nutrient starvation. Our findings demonstrate an important role for ICK in modulating the activity of mTORC1 through phosphorylation of Raptor Thr-908 and thus implicate a potential signaling mechanism by which ICK regulates cell proliferation and division.     

Osmotic stress regulates mammalian target of rapamycin (mTOR) complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor protein phosphorylation

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Critical role of T-loop and H-motif phosphorylation in the regulation of S6 kinase 1 by the tuberous sclerosis complex

The tuberous sclerosis gene products Tsc1 and Tsc2 behave as tumor suppressors by restricting cell growth, a function conserved among metazoans. Recent evidence has indicated that hyperactivation of S6 kinase 1 (S6K1) may represent an important biochemical change in the development of tuberous sclerosis-associated lesions. We show here that deletion of either Tsc1 or Tsc2 or expression of the Rheb (Ras homolog enriched in brain) GTPase leads to hyperphosphorylation of S6K1 at a subset of regulatory sites, particularly those of two essential residues functionally conserved among AGC superfamily serine/threonine kinases, i.e. the activation loop (T-loop; Thr-229) and the hydrophobic motif (H-motif; Thr-389). These sites are reciprocally and dose-dependently regulated when S6K1 is coexpressed with the Tsc1-Tsc2 complex. Mutations that render S6K1 mTOR (mammalian target of rapamycin)-resistant also protect S6K1 activity and phosphorylation from down-regulation by Tsc1/2. We demonstrate that two disease-associated mutations in Tsc2 fail to negatively regulate S6K1 activity concomitant with a failure to modify T-loop and H-motif phosphorylation. Finally, we identify one pathological Tsc2 mutation that retains its ability to negatively regulate S6K1, suggesting that, in some cases, tuberous sclerosis may develop independently of S6K1 hyperactivation. These results also highlight the importance of dual control of T-loop and H-motif phosphorylation of S6K1 by the Tsc1-Tsc2 complex.     

FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

Hyperactive mammalian target of rapamycin (mTOR) is associated with cognitive deficits in several neurological disorders including tuberous sclerosis complex (TSC). The phosphorylation of the mRNA-binding protein FMRP reportedly depends on mTOR complex 1 (mTORC1) activity via p70 S6 kinase 1 (S6K1). Because this phosphorylation is thought to regulate the translation of messages important for synaptic plasticity, we explored whether FMRP phosphorylation of the S6K1-dependent residue (S499) is altered in TSC and states of dysregulated TSC-mTORC1 signaling. Surprisingly, we found that FMRP S499 phosphorylation was unchanged in heterozygous and conditional Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Neither up- nor down-regulation of the mTORC1-S6K1 axis in vivo or in vitro had any effect on phospho-FMRP S499 levels. In addition, FMRP S499 phosphorylation was unaltered in S6K1-knockout mice. Collectively, these data strongly suggest that FMRP S499 phosphorylation is independent of mTORC1-S6K1 activity and is not altered in TSC.     

Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells

The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA-deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non-deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473-an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS-1 phosphorylation, which was dose-dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS-1 via an S6K1-independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on beta-casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells.     

Evidence for cell cycle-dependent, rapamycin-resistant phosphorylation of ribosomal protein S6 at S240/244

The ribosomal protein S6 is essential for the formation of the subunits of higher eukaryotic ribosomes, and S6 heterozygosity leads to early embryonal lethality in mice. S6 is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. So far, the S6Kinases, S6K1 and S6K2 are the only proven S6 S240/244 phosphorylating enzymes in mammalian cells. The activity of these S6Kinases is strictly regulated via the mammalian target of rapamycin (mTOR) enzyme complex with raptor, named mTORC1. In time course experiments with the mTORC1 inhibitor rapamycin we here demonstrate rapamycin-resistant phosphorylation of the ribosomal protein S6 at S240/244. Serum-restimulation experiments further demonstrated that this rapamycin-resistant S6 240/244 phosphorylation is induced via serum factors in a cell cycle-dependent manner. Our data allow new insights into the regulation of S6 phosphorylation and provide evidence for the existence of rapamycin-resistant S6 phosphorylating kinase activities.     

RGD-containing peptides activate S6K1 through beta3 integrin in adult cardiac muscle cells

The enzyme p70S6 kinase (S6K1) is critical for cell growth, and we have reported its activation during cardiac hypertrophy. Because cardiac hypertrophy also involves integrin activation, we analyzed whether integrins could contribute to S6K1 activation. Using adult feline cardiomyocytes, here we report that integrin-interacting Arg-Gly-Asp (RGD) peptides activate S6K1 as observed by band shifting, kinase activity and phosphorylation at Thr-389 and Thr-421/Ser-424 of S6K1, and S6 protein phosphorylation. Perturbation of specific integrin function with blocking antibodies and by overexpressing the beta1A cytoplasmic tail revealed that beta3 but not beta1 integrin mediates the RGD-induced S6K1 activation. This activation is focal adhesion complex-independent and is accompanied by the activation of extracellular signal-regulated kinases 1/2 (ERK) and mammalian target of rapamycin (mTOR). Studies using specific inhibitors and dominant negative c-Raf expression in cardiomyocytes indicate that the S6K1 activation involves mTOR, MEK/ERK, and phosphatidylinositol 3-kinase pathways and is independent of protein kinase C and c-Raf. Finally, addition of fluorescent-labeled RGD peptide to cardiomyocytes exhibits its internalization and localization to the endocytic vesicles, and pretreatment of cardiomyocytes with endocytic inhibitors reduced the S6K1 activation. These data suggest that RGD interaction with beta3 integrin and its subsequent endocytosis trigger specific signaling pathway(s) for S6K1 activation in cardiomyocytes and that this process may contribute to hypertrophic growth and remodeling of myocardium.     

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.     

Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K)

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.