Limits...
Akt-dependent cytokine production in mast cells.

Kitaura J, Asai K, Maeda-Yamamoto M, Kawakami Y, Kikkawa U, Kawakami T - J. Exp. Med. (2000)

Bottom Line: Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha promoters.Catalytic activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt.Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-kappaB, NF-AT, and AP-1 that contributes to the production of cytokines.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

ABSTRACT
Cross-linking of FcepsilonRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcepsilonRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha promoters. Transcription from the nuclear factor kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IkappaB-alpha, a cytoplasmic protein that binds NF-kappaB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-alpha in FcepsilonRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-kappaB, NF-AT, and AP-1 that contributes to the production of cytokines.

Show MeSH

Related in: MedlinePlus

Activation of Akt in mast cells by growth factor or FcεRI stimulation. (A) BMMCs sensitized overnight with anti-DNP IgE were stimulated with 100 ng/ml DNP-HSA for the indicated amounts of time. Cells were lysed, and lysates were analyzed by SDS-PAGE followed by immunoblotting with antiphospho-Akt antibody that specifically recognizes the phosphorylated Ser-473 residue and its flanking sequence. The same blot was reprobed with anti-Akt antibody (left). Shown is the result representative of at least five independent experiments. Cell lysates were immunoprecipitated with anti-Akt, and immune complexes were subjected to kinase assays using histone H2B as an exogenous substrate (right). The kinase result is representative of three similar experiments. Stim., stimulation. (B) IgE-sensitized BMMCs were pretreated with the indicated concentrations of genistein, wortmannin, or LY294002 for 15 min before antigen (100 ng/ml DNP-HSA) stimulation (Stim.) for 10 min. Cell lysates were analyzed for Ser-473 phosphorylation as above.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193272&req=5

Figure 1: Activation of Akt in mast cells by growth factor or FcεRI stimulation. (A) BMMCs sensitized overnight with anti-DNP IgE were stimulated with 100 ng/ml DNP-HSA for the indicated amounts of time. Cells were lysed, and lysates were analyzed by SDS-PAGE followed by immunoblotting with antiphospho-Akt antibody that specifically recognizes the phosphorylated Ser-473 residue and its flanking sequence. The same blot was reprobed with anti-Akt antibody (left). Shown is the result representative of at least five independent experiments. Cell lysates were immunoprecipitated with anti-Akt, and immune complexes were subjected to kinase assays using histone H2B as an exogenous substrate (right). The kinase result is representative of three similar experiments. Stim., stimulation. (B) IgE-sensitized BMMCs were pretreated with the indicated concentrations of genistein, wortmannin, or LY294002 for 15 min before antigen (100 ng/ml DNP-HSA) stimulation (Stim.) for 10 min. Cell lysates were analyzed for Ser-473 phosphorylation as above.

Mentions: Because Akt is activated by numerous stimuli, we investigated whether FcεRI cross-linking induces Akt activation. In situ Akt activity was monitored before and after cell stimulation by immunoblotting with a phosphospecific antibody to the phosphorylated Ser-473 of Akt. Ser-473 phosphorylation is crucial for Akt activation. Antigen treatment of IgE-primed BMMCs caused a remarkable phosphorylation of Ser-473 at its peak ∼3–10 min after antigen stimulation (Fig. 1 A, left). Enzymatic activation of Akt in a time course similar to that of Ser-473 phosphorylation was shown in in vitro kinase assays on anti-Akt immunoprecipitates using histone H2B as an exogenous substrate (Fig. 1 A, right). Dependence of FcεRI-induced Akt activation on PTK was revealed by pretreatment of BMMCs with genistein, similar to BCR-induced Akt activation 647273. PI3K inhibitors wortmannin and LY294002 also blocked Akt activation very efficiently, confirming the PI3K dependence of Akt activation (Fig. 1 B). These results are consistent with PI3K activation induced by the engagement of FcεRI and other related receptors that induces activation of several PTKs as well 74757677.


Akt-dependent cytokine production in mast cells.

Kitaura J, Asai K, Maeda-Yamamoto M, Kawakami Y, Kikkawa U, Kawakami T - J. Exp. Med. (2000)

Activation of Akt in mast cells by growth factor or FcεRI stimulation. (A) BMMCs sensitized overnight with anti-DNP IgE were stimulated with 100 ng/ml DNP-HSA for the indicated amounts of time. Cells were lysed, and lysates were analyzed by SDS-PAGE followed by immunoblotting with antiphospho-Akt antibody that specifically recognizes the phosphorylated Ser-473 residue and its flanking sequence. The same blot was reprobed with anti-Akt antibody (left). Shown is the result representative of at least five independent experiments. Cell lysates were immunoprecipitated with anti-Akt, and immune complexes were subjected to kinase assays using histone H2B as an exogenous substrate (right). The kinase result is representative of three similar experiments. Stim., stimulation. (B) IgE-sensitized BMMCs were pretreated with the indicated concentrations of genistein, wortmannin, or LY294002 for 15 min before antigen (100 ng/ml DNP-HSA) stimulation (Stim.) for 10 min. Cell lysates were analyzed for Ser-473 phosphorylation as above.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193272&req=5

Figure 1: Activation of Akt in mast cells by growth factor or FcεRI stimulation. (A) BMMCs sensitized overnight with anti-DNP IgE were stimulated with 100 ng/ml DNP-HSA for the indicated amounts of time. Cells were lysed, and lysates were analyzed by SDS-PAGE followed by immunoblotting with antiphospho-Akt antibody that specifically recognizes the phosphorylated Ser-473 residue and its flanking sequence. The same blot was reprobed with anti-Akt antibody (left). Shown is the result representative of at least five independent experiments. Cell lysates were immunoprecipitated with anti-Akt, and immune complexes were subjected to kinase assays using histone H2B as an exogenous substrate (right). The kinase result is representative of three similar experiments. Stim., stimulation. (B) IgE-sensitized BMMCs were pretreated with the indicated concentrations of genistein, wortmannin, or LY294002 for 15 min before antigen (100 ng/ml DNP-HSA) stimulation (Stim.) for 10 min. Cell lysates were analyzed for Ser-473 phosphorylation as above.
Mentions: Because Akt is activated by numerous stimuli, we investigated whether FcεRI cross-linking induces Akt activation. In situ Akt activity was monitored before and after cell stimulation by immunoblotting with a phosphospecific antibody to the phosphorylated Ser-473 of Akt. Ser-473 phosphorylation is crucial for Akt activation. Antigen treatment of IgE-primed BMMCs caused a remarkable phosphorylation of Ser-473 at its peak ∼3–10 min after antigen stimulation (Fig. 1 A, left). Enzymatic activation of Akt in a time course similar to that of Ser-473 phosphorylation was shown in in vitro kinase assays on anti-Akt immunoprecipitates using histone H2B as an exogenous substrate (Fig. 1 A, right). Dependence of FcεRI-induced Akt activation on PTK was revealed by pretreatment of BMMCs with genistein, similar to BCR-induced Akt activation 647273. PI3K inhibitors wortmannin and LY294002 also blocked Akt activation very efficiently, confirming the PI3K dependence of Akt activation (Fig. 1 B). These results are consistent with PI3K activation induced by the engagement of FcεRI and other related receptors that induces activation of several PTKs as well 74757677.

Bottom Line: Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha promoters.Catalytic activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt.Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-kappaB, NF-AT, and AP-1 that contributes to the production of cytokines.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

ABSTRACT
Cross-linking of FcepsilonRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcepsilonRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha promoters. Transcription from the nuclear factor kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IkappaB-alpha, a cytoplasmic protein that binds NF-kappaB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-alpha in FcepsilonRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-kappaB, NF-AT, and AP-1 that contributes to the production of cytokines.

Show MeSH
Related in: MedlinePlus