Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.     

Triad 1 induces apoptosis by p53 activation

Triad 1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1) is an E3 ligase that induces apoptosis and clonogenic inhibition in myeloid cells through Gfi-1 stabilization. Here we demonstrate that Triad 1 induces apoptosis in several cancer cell lines including MCF7, A549, U2OS, and HCT 116 p53^+/+ cells via its RING ligase activity. Interestingly, in these cancer cells, Triad 1-induced apoptosis is not mediated by Gfi-1 stabilization but is instead p53-dependent. Moreover, Triad 1 promotes transactivation of p53. These results suggest that Triad 1 can induce apoptosis through its ligase activity via p53 activation.     

STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65

We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

Direct phosphorylation of NF-kappaB1 p105 by the IkappaB kinase complex on serine 927 is essential for signal-induced p105 proteolysis

The p105 precursor protein of NF-kappaB1 acts as an NF-kappaB inhibitory protein, retaining associated Rel subunits in the cytoplasm of unstimulated cells. Tumor necrosis factor alpha (TNFalpha) and interleukin-1alpha (IL-1alpha) stimulate p105 degradation, releasing associated Rel subunits to translocate into the nucleus. By using knockout embryonic fibroblasts, it was first established that the IkappaB kinase (IKK) complex is essential for these pro-inflammatory cytokines to trigger efficiently p105 degradation. The p105 PEST domain contains a motif (Asp-Ser(927)-Gly-Val-Glu-Thr), related to the IKK target sequence in IkappaBalpha, which is conserved between human, mouse, rat, and chicken p105. Analysis of a panel of human p105 mutants in which serine/threonine residues within and adjacent to this motif were individually changed to alanine established that only serine 927 is essential for p105 proteolysis triggered by IKK2 overexpression. This residue is also required for TNFalpha and IL-1alpha to stimulate p105 degradation. By using a specific anti-phosphopeptide antibody, it was confirmed that IKK2 overexpression induces serine 927 phosphorylation of co-transfected p105 and that endogenous p105 is also rapidly phosphorylated on this residue after TNFalpha or IL-1alpha stimulation. In vitro kinase assays with purified proteins demonstrated that both IKK1 and IKK2 can directly phosphorylate p105 on serine 927. Together these experiments indicate that the IKK complex regulates the signal-induced proteolysis of NF-kappaB1 p105 by direct phosphorylation of serine 927 in its PEST domain.     

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.     

Ordered multisite phosphorylation of Xenopus ribosomal protein S6 by S6 kinase II.

Phosphorylated ribosomal proteins were isolated from Xenopus 40 S ribosomal subunits by reversed-phase high performance liquid chromatography (HPLC) to enable direct analysis of the phosphorylation sites in ribosomal protein S6. Xenopus S6 closely resembled mammalian S6 with respect to the following properties: (i) reversed-phase HPLC elution behavior, (ii) amino-terminal sequence (96% identity in the first 37 residues), and (iii) an identical sequence within the region of its phosphorylation sites. Whereas S6 was the only ribosomal protein phosphorylated in vitro by Xenopus S6 kinase II, ribosomes phosphorylated in vivo were found to be associated with an additional phosphoprotein having an amino-terminal sequence identical to that of the ubiquitin carboxyl-terminal extension protein CEP 80. S6 kinase II phosphorylated at least four sites (serines 1-3 and 5) in the sequence Arg-Arg-Leu-Ser(1)-Ser(2)-Leu-Arg-Ala-Ser(3)-Thr-Ser(4)-Lys-Ser(5)-, which correspond to the residues known to be phosphorylated in the carboxyl-terminal region of mammalian S6. The in vivo S6 phosphorylation sites in maturing Xenopus oocytes were shown to be located within the same cluster of serine residues, although individual sites were not identified. Kinetic analysis of S6 kinase II-catalyzed phosphorylation events indicated a simple sequential mechanism of multisite phosphorylation initiating at either serine 2 (preferred) or serine 1, with the rates of phosphorylation of individual sites occurring in the order serine 2 greater than serine 1 greater than serine 3 greater than serine 5.     

Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK)

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na(+)/H(+) exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na(+)/H(+) exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.     

The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C

Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo.     

Acute alcohol exposure exerts anti-inflammatory effects by inhibiting IkappaB kinase activity and p65 phosphorylation in human monocytes

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.