Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) Transgenic mice expressing a doxycycline-inducible cyclin D1 WT and cyclin D1 KE mutant cDNA targeted to the mammary gland by MMTV-RtTA were treated with doxycycline for 14 days and immunohistochemical analysis was conducted for phosphorylated targets of Akt1. (B–D) Representative immunohistochemistry (IHC) results are shown, and quantitative data are indicated as mean ± SEM at the right of the panels. IHC was conducted for (B) cyclin D1 cDNA (FLAG), (C) phospho-Akt1 Ser473, and (D) Akt1. (E) The cyclin D1 fl/fl mice were intercrossed with ROSA26-ER-Cre, and the mice treated with tamoxifen for 5 days. Immunohistochemical staining of the mammary epithelium was conducted. (F–H) Immunohistochemical staining of the mammary gland for cyclin D1 and Akt1 or to downstream Akt signaling substrates. The data are indicated as semiquantitative data, as mean ± SEM for the relative abundance of proteins.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Transgenic Assay, Expressing, Mutagenesis, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) Western blot analysis of MCF-7 cells transduced with an expression vector encoding cyclin D1 with antibodies as indicated to cyclin D1, Akt1, Akt1(Ser473), and the Akt1 signaling pathway and its substrate TSC2 (Ser939). The comparison of FLAG-tagged cyclin D1 (high molecular weight, labeled “Ex” for exogenous) and endogenous (labeled “End”) cyclin D1 indicates a 2-fold increase in cyclin D1 abundance. The data are representative of n = 3 separate experiments. (B and C) (B) Cyclin D1 −/− cells transduced with a retroviral vector for (B) cyclin D1 or (C) cyclin D1 WT and cyclin D1 KE with antibodies as indicated. (D and E) In (D), cyclin D1 immune-precipitation kinase assays were conducted using GST fusion proteins as substrates including pRB, and Akt1. Left panel: proteins on an SDS-PAGE stained with Coomassie. Right panel: γ 32 p IP-kinase assay reactions, with relative incorporation into substrates indicated in (E) as mean ± SEM for n = 3 separate experiments. (F) Representative mass spectrometry spectrum to map the Akt1 Ser473 phosphorylation status in vivo . The liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrum of the singly phosphorylated doubly charged peptide RPHFPQFpSYSASGTA representing S473 in the modified Akt1 sequence. The neutral loss of phosphate confirms the phosphorylation status, and sites are localized to S10 (S473 full length) in the peptide based on the b-ion series (N-terminal fragments) starting at b7 and the y-ion series starting at y10 that contain a phosphate group. (G) Cyclin D1 immune-precipitation kinase assays were conducted using GST fusion proteins including Akt1-WT and Akt1 Ser473 mutation. Left panel: proteins on an SDS-PAGE stained with Coomassie. Right panel: γ 32 P IP-kinase assay reactions.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Western Blot, Transduction, Expressing, Plasmid Preparation, Molecular Weight, Labeling, SDS Page, Staining, IP-Kinase Assay, Mass Spectrometry, In Vivo, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Modification, Sequencing, Mutagenesis

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A–F) The PLA for endogenous Akt1 with WT cyclin D1 or cyclin D1 mutants is indicated by red dots, and nucleus was stained by DAPI. Cyclin D1 expression plasmids were introduced by transduction of cyclin D1 −/− 3T3 cells. 3D reconstruction of the z stack is indicated for representative examples of multiplicate experiments. (G) Schematic representation of cyclin D1 expression plasmids used. (H) Model of the murine cyclin D1-CDK4/CDK6-Akt1 (C-terminal peptide) complex. The model indicates cyclin D1 and CDK4/CDK6 in blue and yellow cartoons, respectively. The Akt1 c-terminal peptide bound at the active site of CDK4/CDK6 is indicated in green stick. The ATP and Mg 2+ ions are indicated in blue sticks and red spheres, respectively.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Staining, Expressing, Transduction

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) The fluorescent intensity (FI) of LS456 in cyclin D1 −/− 3T3 cells transduced with either vector, cyclin D1 WT , or cyclin D1 KE . The FI of LS456 decreased in the 800-nm channel and increased in the 700-nm channel in response to insulin, indicated as fluorescent images in cells. (B) Quantitative analysis of the FI in cyclin D1 WT versus cyclin D1 KE or vector control. Fluorescent images in cells are superimposed on differential interference contrast images. Scale bars, 10 μm. Data are presented as mean ± SEM for n = 8 separate cells. (C) Schematic representation of c- fos promoter luciferase reporter genes including point mutations of the SRE and TCF site. (D and E) c- fos -LUC promoter activity in MCF-7 cells (D) transfected with expression vectors encoding either cyclin D1 WT or cyclin D1 KE or (E) transfected with c- fos -LUC reporter mutants. (F–H) Cyclin D1 −/− 3T3 cells were co-transfected with expression vector encoding c- fos -LUC WT or mutant and activated Akt (myr-Akt1) or (F) cyclin D1, (G) membrane-localized cyclin D1, or (H) nuclear-localized cyclin D1.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Transduction, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Mutagenesis

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) Schematic representation of transgenic mice used for gene expression analysis. MMTV-Akt +/− mice were intercrossed with MMTV-ErbB2 transgenics, and the mammary epithelium was used for a source of mRNA. The cyclin D1-induced state was recapitulated through doxycycline induction of MMTV-RtTA-cyclin D1 transgenics treated for 10 days. (B) Pie diagrams representing the number of genes either induced or repressed by Akt or cyclin D1. (C) KEGG pathway analysis used to identify functional pathways regulated by either Akt1 or cyclin D1 and those regulated by both Akt1 and cyclin D1 . (D) The names of pathways identified by KEGG analysis regulated by both cyclin D1 and Akt1 in the mammary gland (migration 1, adherens junction; migration 2, tight junction). (E) Schematic representation of the relative fold enrichment in gene number of the individual KEGG pathways regulated by both cyclin D1 and Akt1.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Transgenic Assay, Expressing, Functional Assay, Migration

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Recombinant, In Situ, shRNA, Plasmid Preparation, Mutagenesis, Software

Journal: Oncogene

Article Title: The PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway

doi: 10.1038/onc.2014.179

Figure Lengend Snippet: ( A ) A conserved substrate consensus site of Akt kinase identified in Eya1. ( B and C ) Immunoprecipitation (IP) using antibodies against either a pan-AKT (AKT) (B), EYA1 (C) or control IgG from BT549 cell lysates, and resultants were immunoblotted (IB) using indicated antibodies. ( D and E ) Co-IP of full-length mouse Akt1 and Flag-tagged Eya1 (Flag/Eya1) fragments from transfected HEK293 cells. Results are summarized in E. *, inconclusive. ( F ) Immunoprecipitates (Flag/Eya1 or Flag/S298A) were incubated with recombinant AKT1 (r-AKT1) in an in vitro kinase assay with γ-[ 32 P]-ATP. Top panel, autoradiography; bottom panel, protein immunoblot using a Flag-specific antibody. ( G ) Immunoprecipitates from the phospho-specific Akt substrate antibody (p-sub) were probed using indicated antibodies. Flag/S298A, an Eya1 serine 298 to alanine mutation; myr-HA/Akt1, myristoylated HA-tagged Akt1, GAPDH was used as loading control.

Article Snippet: Eya1 and S298A mutant proteins and recombinant active AKT1 (R&D Systems) were mixed and incubated in kinase reaction buffer (5 mM MOPS pH 7.2, 2.5 mM beta-glycerophosphate, 5 mM MgCl2, 1mM EGTA, 0.4 mM EDTA, 0.4% glycerol, 5 μM ATP and 5 μCi gamma-[32P]-ATP) at room temperature for 30 min.

Techniques: Immunoprecipitation, Control, Co-Immunoprecipitation Assay, Transfection, Incubation, Recombinant, In Vitro, Kinase Assay, Autoradiography, Western Blot, Mutagenesis

Journal: Oncogene

Article Title: The PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway

doi: 10.1038/onc.2014.179

Figure Lengend Snippet: ( A ) HEK293 cells were transiently transfected with the Flag/Eya1 and/or HA/Akt1 expression constructs and analyzed by indirect immunocytochemistry using a Flag-antibody (green, 1), HA- antibody (Red, 2) and counter-stained with DAPI (blue). 3, a merged image of 1 and 2. ( B–C ) Combinations of Akt1, Flag/Eya1 and Myc/Six1 were co-expressed in HEK293 cells. co-IP and IB were done using indicated antibodies. ( D ) A transient transcription reporter assay using a Six1/Eya1-dependent reporter (SE1/luc), which was co-expressed with combinations of indicated expression constructs. Relative fold changes were calculated using basal and Renilla controls (mean ± SEM, n=5). ( E ) Schematic diagram of regulation of Eya1 activities. Resting state Eya1 has low transcription activity (low, in a dash box). Akt1 increases Eya1 transcription (HIGH) in part through the conserved S298 phosphorylation (p) site.

Article Snippet: Eya1 and S298A mutant proteins and recombinant active AKT1 (R&D Systems) were mixed and incubated in kinase reaction buffer (5 mM MOPS pH 7.2, 2.5 mM beta-glycerophosphate, 5 mM MgCl2, 1mM EGTA, 0.4 mM EDTA, 0.4% glycerol, 5 μM ATP and 5 μCi gamma-[32P]-ATP) at room temperature for 30 min.

Techniques: Transfection, Expressing, Construct, Immunocytochemistry, Staining, Co-Immunoprecipitation Assay, Reporter Assay, Activity Assay, Phospho-proteomics

Journal: Oncogene

Article Title: The PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway

doi: 10.1038/onc.2014.179

Figure Lengend Snippet: ( A–C ) Inhibition of PI3K/Akt signaling with an inhibitor LY294002 (25 µM, 2 h, A and B) or AKT siRNA oligo sets (C, targeting all three AKT1/2/3 isoforms, Dharmacon) enhanced endogenous EYA1 (A) and Flag/Eya1 (B and C) SUMOylation levels. IP and IB were performed using indicated antibodies. ( D ) S298A mutant had enhanced SUMOylation level. ( E and F ) Constitutively active myr-HA/Akt1 suppressed SUMOylation of wild type Eya1 (E) but not S298A mutant (F). Flag/Eya1, Flag/S298A and HA/Sumo1 were transiently expressed in HEK293 cells with gradient amount of myr-HA/Akt1. ( G ) Akt1 induced Eya1 transcription activity via inhibition of Eya1 SUMOylation. The Six1/Eya1-dependent reporter (SE1/luc) was co-transfected with indicated constructs and the luciferase reporter activity was measure at 48 hours. Relative fold changes were calculated using basal and Renilla controls (mean ± SEM, n=3). ns, not significant; KS/RA, K43R/K164R/S298A triple mutation.

Article Snippet: Eya1 and S298A mutant proteins and recombinant active AKT1 (R&D Systems) were mixed and incubated in kinase reaction buffer (5 mM MOPS pH 7.2, 2.5 mM beta-glycerophosphate, 5 mM MgCl2, 1mM EGTA, 0.4 mM EDTA, 0.4% glycerol, 5 μM ATP and 5 μCi gamma-[32P]-ATP) at room temperature for 30 min.

Techniques: Inhibition, Mutagenesis, Activity Assay, Transfection, Construct, Luciferase