302 research outputs found

    PKC-Mediated USP Phosphorylation at Ser35 Modulates 20-Hydroxyecdysone Signaling in <i>Drosophila</i>

    No full text
    The nuclear receptor complex of the steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer composed of EcR and USP. Our previous studies in <i>Drosophila</i> suggest that PKC modulates 20E signaling by phosphorylating EcR-USP. However, the exact phosphorylation sites in EcR and USP have not been identified. Using LC–MS/MS analysis, we first identified Ser35 of USP as a PKC phosphorylation site. Mutation of USP Ser35 to Ala35 in S2 cells not only eliminated USP phosphorylation, but also attenuated the 20E-induced luciferase activity, mimicking the treatment with a PKC-specific inhibitor chelerythrine chloride in Kc cells. In the larval salivary glands (SG), inhibition of PKC activity with the binary GAL4/UAS system reduced USP phosphorylation and down-regulated the 20E primary-response genes, <i>E75B</i> and <i>Br-C</i>, and RNAi knockdown of <i>Rack1</i> had stronger inhibitory effects than overexpression of <i>PKCi</i>. Moreover, RNAi knockdown of four PKC isozyme genes expressed in the SG exhibited a variety of inhibitory effects on USP phosphorylation and expression of <i>E75B</i> and <i>Br-C</i>, with the strongest inhibitory effects occurring when <i>aPKC</i> was knocked down by RNAi. Taken together, we conclude that PKC-mediated USP phosphorylation at Ser35 modulates 20E signaling in <i>Drosophila</i>

    PKC-Mediated USP Phosphorylation at Ser35 Modulates 20-Hydroxyecdysone Signaling in <i>Drosophila</i>

    No full text
    The nuclear receptor complex of the steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer composed of EcR and USP. Our previous studies in <i>Drosophila</i> suggest that PKC modulates 20E signaling by phosphorylating EcR-USP. However, the exact phosphorylation sites in EcR and USP have not been identified. Using LC–MS/MS analysis, we first identified Ser35 of USP as a PKC phosphorylation site. Mutation of USP Ser35 to Ala35 in S2 cells not only eliminated USP phosphorylation, but also attenuated the 20E-induced luciferase activity, mimicking the treatment with a PKC-specific inhibitor chelerythrine chloride in Kc cells. In the larval salivary glands (SG), inhibition of PKC activity with the binary GAL4/UAS system reduced USP phosphorylation and down-regulated the 20E primary-response genes, <i>E75B</i> and <i>Br-C</i>, and RNAi knockdown of <i>Rack1</i> had stronger inhibitory effects than overexpression of <i>PKCi</i>. Moreover, RNAi knockdown of four PKC isozyme genes expressed in the SG exhibited a variety of inhibitory effects on USP phosphorylation and expression of <i>E75B</i> and <i>Br-C</i>, with the strongest inhibitory effects occurring when <i>aPKC</i> was knocked down by RNAi. Taken together, we conclude that PKC-mediated USP phosphorylation at Ser35 modulates 20E signaling in <i>Drosophila</i>

    PKC-Mediated USP Phosphorylation at Ser35 Modulates 20-Hydroxyecdysone Signaling in <i>Drosophila</i>

    No full text
    The nuclear receptor complex of the steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer composed of EcR and USP. Our previous studies in <i>Drosophila</i> suggest that PKC modulates 20E signaling by phosphorylating EcR-USP. However, the exact phosphorylation sites in EcR and USP have not been identified. Using LC–MS/MS analysis, we first identified Ser35 of USP as a PKC phosphorylation site. Mutation of USP Ser35 to Ala35 in S2 cells not only eliminated USP phosphorylation, but also attenuated the 20E-induced luciferase activity, mimicking the treatment with a PKC-specific inhibitor chelerythrine chloride in Kc cells. In the larval salivary glands (SG), inhibition of PKC activity with the binary GAL4/UAS system reduced USP phosphorylation and down-regulated the 20E primary-response genes, <i>E75B</i> and <i>Br-C</i>, and RNAi knockdown of <i>Rack1</i> had stronger inhibitory effects than overexpression of <i>PKCi</i>. Moreover, RNAi knockdown of four PKC isozyme genes expressed in the SG exhibited a variety of inhibitory effects on USP phosphorylation and expression of <i>E75B</i> and <i>Br-C</i>, with the strongest inhibitory effects occurring when <i>aPKC</i> was knocked down by RNAi. Taken together, we conclude that PKC-mediated USP phosphorylation at Ser35 modulates 20E signaling in <i>Drosophila</i>

    Experimental Set-up.

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    <p>Experimental Set-up.</p

    Quantitative measurement of thresholds for both dominant (DH) and non-dominant (NDH) hands before and after BreEStim and EStim.

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    <p>Quantitative measurement of thresholds for both dominant (DH) and non-dominant (NDH) hands before and after BreEStim and EStim.</p
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