Nuclear localization of p85s6k: functional requirement for entry into S phase.

Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinity-purified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p70s6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis.     

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.     

Dual requirement for a newly identified phosphorylation site in p70s6k.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.     

The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain.

The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.     

Signal transduction pathways involved in phosphorylation and activation of p70S6K following exposure to UVA irradiation

Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70(S6K) phosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70(S6K). Further, UVA-stimulated p70(S6K) activity and phosphorylation at Thr(389) were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Deltap85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1-/- or Jnk2-/- knockout cells. The p70(S6K) phosphorylation at Ser(411) and Thr(421)/Ser(424) was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Deltap85, or DNM-p38. However, Ser(411), but not Thr(421)/Ser(424) phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1-/- or Jnk2-/- cells. In vitro assays indicated that Ser(411) on immunoprecipitated p70(S6K) proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr(421)/Ser(424) is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70(S6K) co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70(S6K).     

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v-src oncogene

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3'-kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.     

Nitric oxide increases p21(Waf1/Cip1) expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70(S6k)

Nitric oxide (NO) regulates the expression of p21(Waf1/Cip1) in several cell types. The present study examined the role of both the extracellular signal-regulated kinase (ERK) and p70 S6 kinase (p70(S6k)) in the NO-induced increase in p21 expression that occurred in adventitial fibroblasts during the cell cycle. Both ERK and p70(S6k) were phosphorylated in response to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the activation was rapid, transient, and preceded increased p21 expresion under defined conditions where serum was present. Addition of a selective inhibitor of ERK phosphorylation (PD98059) prevented the subsequent phosphorylation of p70(S6k) and the increase in p21 protein. Both cGMP and cAMP activated both ERK and p70(S6k), whereas only selective inhibitors of protein kinase G prevented the activation of the kinases by SNAP. A complex between ERK and p70(S6k) was documented by immunoprecipitation procedures. Rapamycin blocked p70(S6k) phosphorylation induced by NO and also inhibited p53 phosphorylation and p21 expression whereas PD98059 only prevented the NO-induced increase in p21 protein without influencing either p53 activation or p21 mRNA expression. The studies show a unique relationship between NO, ERK, and p70(S6k) and also provide evidence for a novel role of p70(S6k) in the activation of p53.     

The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k.

Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70s6k or whether they reside on parallel pathways which bifurcate upstream of p70s6k. Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70s6k variants to distinguish between these possibilities. The rapamycin-resistant p70s6k, which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70s6k inhibited insulin-induced 4E-BP1 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70s6k constructs, which also blocked insulin-induced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70s6k activation, although only the kinase-dead p70s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70s6k revealed that the kinase-dead p70s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70s6k, with the two pathways sharing a common rapamycin-sensitive activator.     

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.     

Beta-adrenergic agonists regulate Na-K-ATPase via p70S6k

We have recently reported that the beta-adrenergic agonist isoproterenol regulates the alveolar epithelial cell Na-K-ATPase via MAPK/extracellular signal-regulated kinase and rapamycin-sensitive pathways. Here we report that isoproterenol phosphorylated the protein S6 kinase (p70S6k) in alveolar epithelial cells, which was inhibited by both rapamycin and the MEK1/2 inhibitor U-0126. In alveolar epithelial cells transfected with a p70S6k dominant negative construct, isoproterenol did not increase Na-K-ATPase total protein expression, whereas in cells transfected with a rapamycin-resistant mutant, the isoproterenol-mediated increase in Na-K-ATPase was not prevented by rapamycin. Accordingly, we provide here first evidence that isoproterenol regulates Na-K-ATPase via p70S6k in alveolar epithelial cells.     

VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis,p70, and p90 S6 kinase phosphorylation

FVIIa binding to tissue factor (TF) and subsequent signal transduction have now been implicated in a variety of pathophysiological processes, including cytokine production during sepsis, tumor angiogenesis and neoangiogenesis, and leukocyte diapedesis. The molecular details, however, by which FVIIa/TF affects gene expression and cellular physiology, remain obscure. Here we show that FVIIa induces a transient phosphorylation of p70/p85(S6K) and p90(RSK) in BHK cells stably transfected with either full-length TF or with a cytoplasmic domain-truncated TF but not in wild type BHK cells. Phosphorylation of these kinases was also observed in HaCaT cells, expressing endogenous TF. Phosphorylation of p70/p85(S6K) coincided with protein kinase B and GSK-3beta phosphorylation. Activation of p70/p85(S6K) was sensitive to inhibitors of phosphatidylinositol 3-kinase and to rapamycin, whereas phosphorylation of p90(RSK) was sensitive to PD98059. FVIIa stimulation of p70/p85(S6K) and p90(RSK) correlated with phosphorylation of the eukaryotic initiation factor eIF-4E, up-regulation of protein levels of eEF1alpha and eEF2, and enhanced [(35)S]methionine incorporation. These effects were not influenced by inhibitors of thrombin or FXa generation and were strictly dependent on the presence of the extracellular domain of TF, but they did not require the intracellular portion of TF. We propose that a TF cytoplasmic domain-independent stimulation of protein synthesis via activation of S6 kinase contributes to FVIIa effects in pathophysiology.     

PF-4708671 Activates AMPK Independently of p70S6K1 Inhibition

The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.     

Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons

The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNalpha and IFNbeta induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3'-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3'-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNalpha or IFNbeta. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3'-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85alpha and p85beta subunits of the PI 3'-kinase (p85alpha-/-beta-/-). Treatment of sensitive cell lines with IFNalpha or IFNbeta also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3'-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3'-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3'-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses.     

S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway

Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.     

IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K,PDK-1, and p70S6 kinase

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr(421)/Ser(424), Thr(389), and Thr(229) that paralleled p70S6 kinase activation. p70S6 kinase(Thr(421)/Ser(424)) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr(389)) and p70S6 kinase(Thr(229)) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser(308)), Akt(Ser(473)), and mammalian target of rapamycin(Ser(2448)) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.