Phytohormones participate in an S6 kinase signal transduction pathway in Arabidopsis

Addition of fresh medium to stationary cells of Arabidopsis suspension culture induces increased phosphorylation of the S6 ribosomal protein and activation of its cognate kinase, AtS6k. Analysis of the activation response revealed that medium constituents required for S6 kinase activation were the phytohormones 1-naphthylacetic acid (auxin) and kinetin. Pretreatment of cells with anti-auxin or PI3-kinase drugs inhibited this response. Consistent with these findings, LY294002, a PI3-kinase inhibitor, efficiently suppressed phytohormone-induced S6 phosphorylation and translational up-regulation of ribosomal protein S6 and S18A mRNAs without affecting global translation. These data indicate that (1) activation of AtS6k is regulated by phytohormones, at least in part, via a lipid kinase-dependent pathway, that (2) the translational regulation of ribosomal proteins appears to be conserved throughout the plant and animal kingdom, and that (3) these events are hallmarks of a growth-related signal transduction pathway novel in plants.     

A Fas-associated death domain protein/caspase-8-signaling axis promotes S-phase entry and maintains S6 kinase activity in T cells responding to IL-2

Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity.     

Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and linker regulatory domains but differs from S6K1 in the N- and C-terminal regions and is differently localized primarily to the nucleus because of a C-terminal nuclear localization signal unique to S6K2. We have recently demonstrated that S6K2 is regulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effectors in vivo. However, deletion of the C-terminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs that confer this differential regulation. We demonstrate that the inhibitory effects of the S6K2 C-terminal domain are only partly attributable to the nuclear localization signal but that three C-terminal proline-directed potential mitogen-activated protein kinase phosphorylation sites are critical mediators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme which is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylation site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation sites appear to be essential for relief of S6K2 inhibition but less critical for activation of S6K1. These data suggest a mechanism by which weak PI3-K agonists can regulate S6 phosphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.     

A Heat-Sensitive Arabidopsis thaliana Kinase Substitutes for Human p70s6k Function In Vivo

In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70^s6k has been implicated in the selective translational upregulation of 5′TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25°C but not at 37°C. When Arabidopsis suspension culture cells are shifted from 25 to 37°C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70^s6k; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70^s6k and identify a new signalling pathway in plants.     

环境变化和时序衰老过程中酵母蛋白激酶Sch9磷酸化的调控

出芽酵母(Saccharomyces cerevisiae)蛋白激酶Sch9与哺乳动物蛋白激酶S6K1同源.S6K1是哺乳动物雷帕霉素靶蛋白(mTOR)和磷脂酰肌醇3激酶(PI3K)的底物,且与很多人类疾病相关,包括肥胖症、糖尿病和癌症.Sch9和S6K1都对不同营养条件和环境胁迫条件下的细胞生长调控很重要.Sch9激活环内的磷酸化位点570位苏氨酸残基也被称为PDK1位点,而737位苏氨酸位点也被称为PDK2位点,这两个位点的磷酸化对Sch9的活性非常重要.蛋白激酶Pkh1/2磷酸化Sch9的PDK1位点,而雷帕霉素靶蛋白复合体1(TORC1)磷酸化PDK2位点.为了深入了解Sch9在细胞中的功能,阐明不同环境条件下及时序衰老过程中Sch9的PDK1和PDK2位点磷酸化的调控就显得尤为重要.利用特异性识别570位苏氨酸残基磷酸化的Sch9蛋白和特异性识别737位苏氨酸残基磷酸化的Sch9蛋白的两种抗体,对不同环境条件下和时序衰老过程中Sch9的两个位点的磷酸化调控进行了研究.研究结果揭示了Sch9的两个磷酸化位点在营养感受、胁迫应答、热量限制和时序衰老过程中的调控方式.揭示Sch9的PDK1位点磷酸化的调控与热量限制延长出芽酵母时序寿命密切相关.     

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Plant casein kinases coordinate cell cycle by regulating the stability of a cyclin-dependent kinase inhibitor through promoting interaction with E3 ubiquitin ligases and proteasomal degradation by phosphorylation.     

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.     

S6K1 and E2FB are in mutually antagonistic regulatory links controlling cell growth and proliferation in Arabidopsis

Plant development is dependent on the coordination between growth and cell proliferation. The nutrient sensing TOR kinase and its downstream target, the 40S ribosomal S6 Kinase, are central controllers of cell growth that were also shown to determine cell size by inhibiting the onset of mitosis in yeast and animal cells. We have shown that the Arabidopsis S6 Kinase1 inhibits cell proliferation through the RBR-E2FB complex. S6K1 interacts with RBR via its N-terminal RBR binding motif, promotes its nuclear localization and consequent RBR-dependent repression of cell cycle genes through E2FB. Here we show that S6K1 and E2FB are in a mutually antagonistic relationship both in their protein abundance and in their activity. We propose that this double inhibitory regulatory connection between S6K1 and E2FB forms a regulatory switch that might be important to determine whether cells divide or grow.     

Tyrosine phosphorylation in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.¹ Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)-Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr-831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants expressing BRI1(Y831F)-Flag may have applications for agriculture.Key words: brassinosteroids, LRR-RLK, autophosphorylation, tyrosine phosphorylation, signal transduction     

Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes: <Emphasis Type="Italic">AtEXP10</Emphasis> from <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">PnEXPA1</Emphasis> from poplar

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.     

The role of phosphatidylinositol-3 kinase in vanadate-promoted S phase entry

Phosphatidylinositil-3 kinase (PI3K) is a heterodimer of catalytic and regulatory subunits. It is involved in various signaling pathways and key functions of the cells. The present study investigated the role of PI3K in vanadate-induced alteration in cell cycle regulation in C141 mouse epidermal cells. Vanadate caused a time- and dose-dependent increase in PI3K activity and phosphorylation of p70 S6 kinase (p70S6K) at Thr421/Ser424 and Thr389 sites. The phosphorylation at these sites was inhibited by PI3K inhibitor, LY294002, and p70S6K mutation. Vanadate promoted S phase entry and this promotion was inhibited by LY294002 and rapmycin, a p70S6K inhibitor. Vanadate-induced enhancement in S phase entry was also inhibited in transfection with dominant negative p70S6K mutant cells. The results obtained show that vanadate is able to increase PI3K activity through phosphorylation. PI3K activated p70S6K, which phosphated protein S6, and promoted S phase entry.     

Tumour suppressors hamartin and tuberin: intracellular signalling

Tumour suppressors hamartin and tuberin, encoded by tuberous sclerosis complex 1(TSC1) and TSC2 genes, respectively, are critical regulators of cell growth and proliferation. Mutations in TSC1 and TSC2 genes are the cause of an autosomal dominant disorder known as tuberous sclerosis complex (TSC). Another genetic disorder, lymphangioleiomyomatosis (LAM), is also associated with mutations in the TSC2 gene. Hamartin and tuberin control cell growth by negatively regulating S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), potentially through their upstream modulator mammalian target of rapamycin (mTOR). Growth factors and insulin promote Akt/PKB-dependent phosphorylation of tuberin, which in turn, releases S6K1 from negative regulation by tuberin and results in the activation of S6K1. Although much has been written regarding the molecular genetics of TSC and LAM, which is associated with either the loss of or mutation in the TSC1 and TSC2 genes, few reviews have addressed the intracellular signalling pathways regulated by hamartin and tuberin. The current review will fill the gap in our understanding of their role in cellular signalling networks, and by improving this understanding, an integrated picture regarding the normal function of tuberin and hamartin is beginning to emerge.     

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.     

Kip-related protein 3 is required for control of endoreduplication in the shoot apical meristem and leaves of Arabidopsis

The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.     

Silica induces cell cycle changes through PI-3K/AP-1 pathway in human embryo lung fibroblast cells

Exposure to silica is associated with progressive pulmonary inflammation and fibrosis. Our previous study had demonstrated silica exposure could cause cell cycle alternation and activator protein-1 (AP-1) activation. This study showed that silica exposure induced phosphorylation of p70S6 kinase (p70S6K) and Akt in human embryo lung fibroblasts (HELFs). These changes were blocked by overexpression of dominant-negative mutants of phosphatidylinositol-3 kinase (Δp85) or Akt (DN-Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70S6K inhibitor, could inhibit silica-induced cell cycle alteration, AP-1 activation, and phosphorylation of p70S6K, but had no effect on Akt phosphorylation. This suggested that phosphatidylinositol-3 kinase (PI-3K)/AP-1 pathway was likely responsible for cell cycle changes. Furthermore, we observed the effect of the pathway on cell cycle regulatory proteins. Our results indicated that inactivation of PI-3K, Akt, or p70S6K could inhibit silica-induced overexpression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) and decreased expression of E2F-4. Taken together, silica could induce cell cycle changes through PI-3K/ AP-1 pathway in HELFs.