PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression

<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"</p><p>http://www.biomedcentral.com/1471-2199/8/28</p><p>BMC Molecular Biology 2007;8():28-28.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858702.</p><p></p> (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file

KSRP associates with AUF1p45 and hnRNPA1 in cytoplasmic extracts of aT3-1 cells

<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"</p><p>http://www.biomedcentral.com/1471-2199/8/28</p><p>BMC Molecular Biology 2007;8():28-28.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858702.</p><p></p> (A) S100 extracts from αT3-1 cells were subjected to gel filtration chromatography on a Superose 6 column. Aliquots of the eluted fractions were analyzed by Western blotting using the indicated antibodies. (B) RNase A-treated S100 extracts from αT3-1 cells were immunoprecipitated with preimmune (lane 2) or anti-KSRP (lane 3) sera and analyzed by immunoblotting with either anti-AUF1 (top) or anti-HnRNPA1 (bottom) antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1, while the asterisk marks a anti-AUF1 cross-reacting band. (C) GST-pulldown of either endogenous AUF1p45 (top) or endogenous hnRNPA1 (bottom) from S100 extracts of αT3-1 cells using either control GST or GST-KSRP. Proteins were analyzed by immunoblotting using the indicated antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1

mRNA Encoding β-Catenin Is Labile and Is Stabilized by LiCl and Wnt-3A

<div><p>(A) Expression of β-catenin and β2-MG (control transcript), monitored by RT-PCR, in control and in LiCl-treated (20 mM for 6 h) αT3-1 cells.</p> <p>(B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(C) Quantitative RT-PCR analysis of both CAT and CAT–β-catenin transcripts in either CAT or CAT–β-catenin transiently transfected αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(D) Semiquantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either control or LiCl-treated αT3-1 cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either control-treated αT3-1, or Wnt-3A–treated (10 ng/ml, 6 h) αT3-1 cells.</p> <p>(F) Semiquantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either control or Wnt-3A–treated αT3-1 cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The amount of each transcript was quantitated and represented as described in (D).</p></div

Insulin Stabilizes β-Catenin mRNA and Increases Its Expression

<div><p>(A) HIRc-B cells were treated for 1 h with DMSO (control), insulin (10⁻⁶ M) plus DMSO, or LY (25 μM for 16 h) plus insulin. Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ-³²P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography.</p> <p>(B) Expression of β-catenin and β2-MG, monitored by RT-PCR, in control HIRc-B cells and in HIRc-B treated with either insulin or LY plus insulin.</p> <p>(C) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in control HIRc-B cells, in HIRc-B treated with either insulin or LY plus insulin. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(D) Immunoblot analysis of either S100 or nuclear extracts from either control- or insulin-treated HIRc-B cells with anti–β-catenin, β-actin, and HDAC2 antibodies. The amount of each band was quantitated by densitometry and insulin was found to increase β-catenin expression by 3.2- and 2.1-fold over the control in S100 and nuclear extracts, respectively.</p> <p>(E) HIRc-B cells were maintained for 16 h in DMEM containing 0.1% FCS; then either PBS (control) or insulin (10⁻⁶ M) was added for 1 h. Cultures were then treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared, and the levels of β-catenin quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. β-Actin immunoblotting was used to verify the equal protein loading.</p></div

AKT Activation Affects KSRP-Exosome Interaction

<div><p>(A) The interaction between ³²P-labeled ARE β-catenin RNA and recombinant purified KSRP (increasing amounts from 30 to 300 nM) subjected to kinase reactions in the absence or in the presence of AKT2 (as indicated) was evaluated by UV-crosslinking.</p> <p>(B) The interaction between ³²P-labeled ARE β-catenin RNA and recombinant purified KSRP (50 nM) subjected to AKT2 phosphorylation in the presence of either GST (50 nM) or GST–14-3-3 (50 nM, as indicated) was evaluated by UV-crosslinking.</p> <p>(C) Coimmunoprecipitation of FLAG-KSRP and either endogenous exosome components (hRrp4p and hMtr4p, as indicated) or endogenous deadenylase PARN in 293T cells transiently transfected with FLAG-KSRP and either pCMV empty vector (mock-293T) or pCMV-myrAKT1 (293T-myrAKT1). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using the indicated antibodies.</p> <p>(D) GST pull-down of endogenous hRrp4p and hMtr4p from total extracts of αT3-1 cells using GST–KH1-4 subjected to kinase reaction either in the absence or in the presence of AKT2. Proteins were analyzed by immunoblotting using the indicated antibodies. Asterisks indicate antibody cross-reactivity with GST-fusion proteins.</p> <p>(E) GST pull-down of endogenous hRrp4p and hMtr4p from total extracts of αT3-1–myrAKT1 cells using either control GST, GST–KH1-4, or GST–KH1-4 preincubated with 50 nM difopein (as indicated). Proteins were analyzed by immunoblotting using either anti-hRrp4p or anti-hMtr4p antibodies.</p> <p>(F) In vitro RNA degradation assays using S100s from either mock αT3-1 or αT3-1–myrAKT1 cells. Internally ³²P-labeled, capped, and polyadenylated β-catenin and GAPDH RNA substrates were incubated with S100s for the indicated times, and their decay was analyzed as described in Materials and Methods. Arrows point to either poly(A)⁺ or poly(A)⁻ β-catenin RNA species.</p></div

KSRP Phosphorylation by AKT Promotes Its Interaction with 14-3-3 and Affects Its mRNA-Destabilizing Function

<div><p>(A) In vitro RNA degradation assays using S100s from either mock αT3-1 (lanes 1–4) or αT3-1–shKSRP cells (lanes 5–28) preincubated with BSA (lanes 1–8), GST (lanes 17–20), KSRP (30 nM, lanes 9–12), AKT2-phosphorylated KSRP (30 nM, lanes 13–16), KH1-4 (S193A) (30 nM, lanes 21–24), or AKT2-phosphorylated KH1-4(S193A) (30 nM, lanes 25–28), respectively. Internally ³²P-labeled and capped RNA substrates were added, and their decay was monitored as described above.</p> <p>(B) Coimmunoprecipitation of FLAG-KSRP and endogenous 14-3-3 in 293T cells transiently transfected with FLAG-KSRP and either pCMV empty vector (mock 293T) or pCMV-myrAKT1 (293T-myrAKT1). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using anti–14-3-3 antibody.</p> <p>(C) GST pull-down of endogenous 14-3-3 from total extracts of either mock αT3-1 (lanes 1–3) or αT3-1–myrAKT1 (lanes 4–11) cells using either control GST, GST–KH1-4, or the additional KSRP deletion mutants fused with GST (as indicated, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050005#pbio-0050005-g004" target="_blank">Figure 4</a>B for a schematic representation of KSRP deletion mutants). Proteins were analyzed by immunoblotting using anti–14-3-3 antibody.</p> <p>(D) Coimmunoprecipitation of either FLAG-KSRP or FLAG-KSRP(S193A) and endogenous 14-3-3 in 293T cells transiently transfected with pCMV-myrAKT1 (293T-myrAKT1) and either FLAG-KSRP or FLAG-KSRP(S193A). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using anti–14-3-3 antibody.</p> <p>(E) In vitro RNA degradation assays using S100s from αT3-1–shKSRP cells pre-incubated with either GST (lanes 1–4), KH1-4 (30 nM, lanes 5–8), AKT2-phosphorylated KH1-4 (30 nM, lanes 9–12), or AKT2-phosphorylated KH1-4 (30 nM) preincubated with difopein (50 nM) (lanes 13–16), respectively. Internally ³²P-labeled and capped RNA substrates were added, and their decay monitored was as described above.</p> <p>(F) GST pull-down of endogenous 14-3-3 from total extracts of αT3-1-myrAKT1 cells using either control GST, GST–KH1-4, or GST–KH1-4 preincubated with 50 nM difopein (as indicated). Proteins were analyzed by immunoblotting using anti–14-3-3 antibody.</p></div

PI3K-AKT Signaling Stabilizes β-Catenin mRNA and Increases Its Expression

<div><p>(A) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of LY, control), DMSO plus LiCl, or LY (25 μM for 16 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of triciribine, control), DMSO plus LiCl (20 mM for 6 h), or triciribine (1 μM for 1 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(C) In vitro RNA degradation assays using S100s from either LiCl-treated mock αT3-1 or LiCl-treated αT3-1 cells expressing the AKT dominant negative mutant AKT1(K179M) (αT3-1–AKTDN). Internally ³²P-labeled and capped RNA substrates were incubated with S100s for the indicated times and their decay analyzed as described in Materials and Methods.</p> <p>(D) Either mock αT3-1 or αT3-1–myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ-³²P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography.</p> <p>(E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either mock αT3-1 or αT3-1–myrAKT1 cells.</p> <p>(F) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either mock αT3-1 or αT3-1–myrAKT1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(G) Immunoblot analysis of either S100 or nuclear extracts from the indicated cell lines with anti- β-catenin, α-tubulin, and HDAC2 antibodies.</p></div

KSRP Is Required for β-Catenin mRNA Degradation

<div><p>(A) Immunoblot analysis of total extracts from either mock αT3-1 (empty pSUPER-Puro vector-transfected) or αT3-1-shKSRP (pSUPER-Puro-shKSRP–transfected) cells using affinity-purified anti-KSRP and anti–α-tubulin antibodies.</p> <p>(B) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either mock αT3-1 or αT3-1–shKSRP cells.</p> <p>(C) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either mock αT3-1 or αT3-1–shKSRP cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p> <p>(D) Immunoblot analysis of total extracts from the indicated cell lines with anti–β-catenin and α-tubulin antibodies.</p> <p>(E) Either mock αT3-1 or αT3-1–shKSRP cells were treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared and the levels of β-catenin quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. α-Tubulin immunoblotting was used to verify the equal protein loading.</p> <p>(F) Either mock αT3-1 or αT3-1–shKSRP cells were transiently transfected with either TOP-FLASH or c-myc-LUC reporter vectors, cultured for 2 d, and collected, and luciferase activity was measured. The values shown are averages (±SEM) of four independent experiments performed in duplicate.</p></div