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s6k1 inhibitor pf4708671 (ApexBio)

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ApexBio s6k1 inhibitor pf4708671

<t>S6K1</t> phosphorylates PIPKIγ90 at Thr-553 and Ser-555. A, alignment of the Akt/S6K1 consensus sequences from different species. B, transfection of constitutively active Akt1 or S6K1 promoted PIPKIγ90 phosphorylation. FLAG-PIPKIγ90 was co-transfected with an empty vector, Myr-Akt1, and S6K1-F5A-E389-R3A into CHO-K1 cells. FLAG-PIPKIγ90 was immunoprecipitated, and PIPKIγ90 phosphorylation was detected with an anti-RXRXXpS/T motif antibody. a.u., arbitrary unit; *, p < 0.05. C, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in vitro. Recombinant GST-PIPKIγ90501–668, -PIPKIγ90501–668T553A, -PIPKIγ90501–668S555A, and -PIPKIγ90501–668T553A,S555A were phosphorylated with constitutively active S6K1 that was immunoprecipitated from CHO-K1 cells. D, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in CHO-K1 cells. HA-S6K1-F5A-E389-R3A was co-transfected with FLAG-PIPKIγ90, -PIPKIγ90T553A, -PIPKIγ90S555A, and -PIPKIγ90T553A,S555A into CHO-K1 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 versus WT. E, EGF and HGF stimulated PIPKIγ phosphorylation. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved and stimulated with EGF (20 ng/ml), HGF (50 ng/ml), SCF (20 ng/ml), and PDGF (20 ng/ml) for 20 min. FLAG-PIPKIγ90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus control (Ctrl). F, HGF-stimulated PIPKIγ phosphorylation was inhibited by Akt and S6K1 inhibitors. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 μm) or <t>PF4708671</t> (10 μm), and then stimulated with HGF for 20 min. Data are presented as mean ± S.E. of four independent experiments. *, p < 0.05 versus HGF. G, HGF-stimulated PIPKIγ phosphorylation was suppressed by depletion of S6K1. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and then stimulated with HGF (20 ng/ml) for 20 min. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05.

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1) Product Images from "p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion * "

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.742742

S6K1 phosphorylates PIPKIγ90 at Thr-553 and Ser-555. A, alignment of the Akt/S6K1 consensus sequences from different species. B, transfection of constitutively active Akt1 or S6K1 promoted PIPKIγ90 phosphorylation. FLAG-PIPKIγ90 was co-transfected with an empty vector, Myr-Akt1, and S6K1-F5A-E389-R3A into CHO-K1 cells. FLAG-PIPKIγ90 was immunoprecipitated, and PIPKIγ90 phosphorylation was detected with an anti-RXRXXpS/T motif antibody. a.u., arbitrary unit; *, p < 0.05. C, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in vitro. Recombinant GST-PIPKIγ90501–668, -PIPKIγ90501–668T553A, -PIPKIγ90501–668S555A, and -PIPKIγ90501–668T553A,S555A were phosphorylated with constitutively active S6K1 that was immunoprecipitated from CHO-K1 cells. D, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in CHO-K1 cells. HA-S6K1-F5A-E389-R3A was co-transfected with FLAG-PIPKIγ90, -PIPKIγ90T553A, -PIPKIγ90S555A, and -PIPKIγ90T553A,S555A into CHO-K1 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 versus WT. E, EGF and HGF stimulated PIPKIγ phosphorylation. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved and stimulated with EGF (20 ng/ml), HGF (50 ng/ml), SCF (20 ng/ml), and PDGF (20 ng/ml) for 20 min. FLAG-PIPKIγ90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus control (Ctrl). F, HGF-stimulated PIPKIγ phosphorylation was inhibited by Akt and S6K1 inhibitors. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 μm) or PF4708671 (10 μm), and then stimulated with HGF for 20 min. Data are presented as mean ± S.E. of four independent experiments. *, p < 0.05 versus HGF. G, HGF-stimulated PIPKIγ phosphorylation was suppressed by depletion of S6K1. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and then stimulated with HGF (20 ng/ml) for 20 min. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05.

Figure Legend Snippet: S6K1 phosphorylates PIPKIγ90 at Thr-553 and Ser-555. A, alignment of the Akt/S6K1 consensus sequences from different species. B, transfection of constitutively active Akt1 or S6K1 promoted PIPKIγ90 phosphorylation. FLAG-PIPKIγ90 was co-transfected with an empty vector, Myr-Akt1, and S6K1-F5A-E389-R3A into CHO-K1 cells. FLAG-PIPKIγ90 was immunoprecipitated, and PIPKIγ90 phosphorylation was detected with an anti-RXRXXpS/T motif antibody. a.u., arbitrary unit; *, p < 0.05. C, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in vitro. Recombinant GST-PIPKIγ90501–668, -PIPKIγ90501–668T553A, -PIPKIγ90501–668S555A, and -PIPKIγ90501–668T553A,S555A were phosphorylated with constitutively active S6K1 that was immunoprecipitated from CHO-K1 cells. D, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in CHO-K1 cells. HA-S6K1-F5A-E389-R3A was co-transfected with FLAG-PIPKIγ90, -PIPKIγ90T553A, -PIPKIγ90S555A, and -PIPKIγ90T553A,S555A into CHO-K1 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 versus WT. E, EGF and HGF stimulated PIPKIγ phosphorylation. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved and stimulated with EGF (20 ng/ml), HGF (50 ng/ml), SCF (20 ng/ml), and PDGF (20 ng/ml) for 20 min. FLAG-PIPKIγ90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus control (Ctrl). F, HGF-stimulated PIPKIγ phosphorylation was inhibited by Akt and S6K1 inhibitors. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 μm) or PF4708671 (10 μm), and then stimulated with HGF for 20 min. Data are presented as mean ± S.E. of four independent experiments. *, p < 0.05 versus HGF. G, HGF-stimulated PIPKIγ phosphorylation was suppressed by depletion of S6K1. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and then stimulated with HGF (20 ng/ml) for 20 min. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05.

Techniques Used: Transfection, Phospho-proteomics, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Stable Transfection, Expressing, Control

S6K1-mediated PIPKIγ90 phosphorylation is essential for the invasion. A, PIPKIγ90 and PIPKIγ90T553E,S555E restored the invasive capacity of PIPKIγ-depleted cells but PIPKIγT553A,S555A did not. PIPKIγ-depleted MDA-MB-231 cells were infected with retroviruses that express codon-modified ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or PIPKIγ90T553E,S555E and then selected with neomycin. Cells that express shRNA control (Ctrl) were used as controls. w/o, without. B, quantification of the experiment in A. White column, without HGF; gray column, 20 ng/ml HGF. Data are presented as mean ± S.E., n = 3. *, p < 0.05; **, p < 0.01 versus shRNA A1. C, inhibition of the invasion of MDA-MB-231 cells in the absence (white columns) and presence (gray columns) of HGF by the S6K1 inhibitor DG2. Data are mean ± S.E. of three independent experiments. **, p < 0.01. D, S6K1 and Akt activation in MDA-MB-231 cells expressing a control shRNA or S6K1 shRNAs. E, depletion of S6K1 by shRNA inhibited the invasion of MDA-MB-231 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001. F, S6K1 and ribosomal protein S6 phosphorylation in MDA-MB-231 cells expressing a control shRNA or Akt1 shRNAs. G, Akt1 knockdown did not significantly affect the invasion of MDA-MB-231 cells. White columns, without HGF; pink columns, with HGF. The data are expressed as mean ± S.E. of three independent experiments. H, effects of the S6K1 inhibitor DG2 on the invasion of PIPKI-depleted MDA-MB-231 cells that express ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E. Cell invasion was performed in the presence of DG2 (black columns, 10 μm) or vehicle (white columns) with HGF (20 ng/ml) in the lower chambers. Data are mean ± S.E. of three independent experiments. *, p < 0.05; ***, p < 0.001.

Figure Legend Snippet: S6K1-mediated PIPKIγ90 phosphorylation is essential for the invasion. A, PIPKIγ90 and PIPKIγ90T553E,S555E restored the invasive capacity of PIPKIγ-depleted cells but PIPKIγT553A,S555A did not. PIPKIγ-depleted MDA-MB-231 cells were infected with retroviruses that express codon-modified ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or PIPKIγ90T553E,S555E and then selected with neomycin. Cells that express shRNA control (Ctrl) were used as controls. w/o, without. B, quantification of the experiment in A. White column, without HGF; gray column, 20 ng/ml HGF. Data are presented as mean ± S.E., n = 3. *, p < 0.05; **, p < 0.01 versus shRNA A1. C, inhibition of the invasion of MDA-MB-231 cells in the absence (white columns) and presence (gray columns) of HGF by the S6K1 inhibitor DG2. Data are mean ± S.E. of three independent experiments. **, p < 0.01. D, S6K1 and Akt activation in MDA-MB-231 cells expressing a control shRNA or S6K1 shRNAs. E, depletion of S6K1 by shRNA inhibited the invasion of MDA-MB-231 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001. F, S6K1 and ribosomal protein S6 phosphorylation in MDA-MB-231 cells expressing a control shRNA or Akt1 shRNAs. G, Akt1 knockdown did not significantly affect the invasion of MDA-MB-231 cells. White columns, without HGF; pink columns, with HGF. The data are expressed as mean ± S.E. of three independent experiments. H, effects of the S6K1 inhibitor DG2 on the invasion of PIPKI-depleted MDA-MB-231 cells that express ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E. Cell invasion was performed in the presence of DG2 (black columns, 10 μm) or vehicle (white columns) with HGF (20 ng/ml) in the lower chambers. Data are mean ± S.E. of three independent experiments. *, p < 0.05; ***, p < 0.001.

Techniques Used: Phospho-proteomics, Infection, Modification, shRNA, Control, Inhibition, Activation Assay, Expressing, Knockdown

S6K1-mediated PIPKIγ phosphorylation is crucial for matrix degradation. A, effect of PIPKIγT553A,S555A and PIPKIγT553E,S555E on gelatin degradation in PIPKIγ-depleted cells. PIPKIγ-depleted MDA-MB-231 cells that express FLAG-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E were resuspended in DMEM containing 1% FBS and HGF, plated on Alexa 488 gelatin-coated glass-bottom dishes, and cultured for 10 h. Scale bar = 20 μm. B, quantification of the experiment in A. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus shRNA control (Ctrl). AU, arbitrary unit. C, inhibition of invadopodium formation in MDA-MB-231 cells by the S6K1 inhibitor DG2. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Figure Legend Snippet: S6K1-mediated PIPKIγ phosphorylation is crucial for matrix degradation. A, effect of PIPKIγT553A,S555A and PIPKIγT553E,S555E on gelatin degradation in PIPKIγ-depleted cells. PIPKIγ-depleted MDA-MB-231 cells that express FLAG-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E were resuspended in DMEM containing 1% FBS and HGF, plated on Alexa 488 gelatin-coated glass-bottom dishes, and cultured for 10 h. Scale bar = 20 μm. B, quantification of the experiment in A. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus shRNA control (Ctrl). AU, arbitrary unit. C, inhibition of invadopodium formation in MDA-MB-231 cells by the S6K1 inhibitor DG2. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Techniques Used: Phospho-proteomics, Cell Culture, shRNA, Control, Inhibition

S6K1 activation correlates with breast cancer metastasis in human clinical specimens. A, human breast cancer primary tumors and the matched metastatic tumors of lymph node tissue were stained with anti-phospho-S6 ribosome protein antibody. B, the intensities of phospho-S6 staining were scored from 0–4, with 4 as the strongest. AU, arbitrary unit.

Figure Legend Snippet: S6K1 activation correlates with breast cancer metastasis in human clinical specimens. A, human breast cancer primary tumors and the matched metastatic tumors of lymph node tissue were stained with anti-phospho-S6 ribosome protein antibody. B, the intensities of phospho-S6 staining were scored from 0–4, with 4 as the strongest. AU, arbitrary unit.

Techniques Used: Activation Assay, Staining

S6K1-mediated phosphorylation regulates PIPKIγ degradation. A, the steady-state levels of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells that were transiently transfected with FLAG-PIPKIγWT, -PIPKIγT553A,S555A and -PIPKIγT553E,S555E, respectively, and treated with DMSO or carfilzomib (1 μm). B, substitution of Thr-553 and Ser-555 with Ala, but not Glu, inhibited degradation of PIPKIγ. CHO-K1 cells expressing BirA were transfected with Avi-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E and labeled with biotin. The levels of PIPKIγ were detected by Western blotting using Dylight 680-streptavidin. C, time course of degradation of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells. Data represent mean ± S.E. of three experiments. **, p < 0.01; ***, p < 0.001. D, CHO-K1 cells were transiently transfected with Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E and plated on fibronectin. The cells were irradiated for 2 min by a 408-nm laser to convert the Dendra2 fusion protein into red Dendra2 fusion protein. The intensities of the red fluorescence were recorded using time-lapse imaging. Scale bar = 20 μm. E, quantification of the degradation of Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E. Data are presented as mean ± S.E. of four independent experiments. F, the S6K1 inhibitor DG2 or PF4708671 stabilizes PIPKIγ90WT. CHO-K1 cells were transfected with Dendra2-PIPKIγ90WT. 24 h post-transfection, cells were treated with DG2 (10 μm) or PF4708671 (10 μm) for 30 min and then irradiated for 2 min using a 408-nm laser. Data are presented as mean ± S.E. of three experiments. G, the S6K1 inhibitor DG2 (10 μm) had little effect on the degradation of Dendra2-PIPKIγ90T553A,S555E. Data are presented as mean ± S.E. of three experiments.

Figure Legend Snippet: S6K1-mediated phosphorylation regulates PIPKIγ degradation. A, the steady-state levels of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells that were transiently transfected with FLAG-PIPKIγWT, -PIPKIγT553A,S555A and -PIPKIγT553E,S555E, respectively, and treated with DMSO or carfilzomib (1 μm). B, substitution of Thr-553 and Ser-555 with Ala, but not Glu, inhibited degradation of PIPKIγ. CHO-K1 cells expressing BirA were transfected with Avi-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E and labeled with biotin. The levels of PIPKIγ were detected by Western blotting using Dylight 680-streptavidin. C, time course of degradation of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells. Data represent mean ± S.E. of three experiments. **, p < 0.01; ***, p < 0.001. D, CHO-K1 cells were transiently transfected with Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E and plated on fibronectin. The cells were irradiated for 2 min by a 408-nm laser to convert the Dendra2 fusion protein into red Dendra2 fusion protein. The intensities of the red fluorescence were recorded using time-lapse imaging. Scale bar = 20 μm. E, quantification of the degradation of Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E. Data are presented as mean ± S.E. of four independent experiments. F, the S6K1 inhibitor DG2 or PF4708671 stabilizes PIPKIγ90WT. CHO-K1 cells were transfected with Dendra2-PIPKIγ90WT. 24 h post-transfection, cells were treated with DG2 (10 μm) or PF4708671 (10 μm) for 30 min and then irradiated for 2 min using a 408-nm laser. Data are presented as mean ± S.E. of three experiments. G, the S6K1 inhibitor DG2 (10 μm) had little effect on the degradation of Dendra2-PIPKIγ90T553A,S555E. Data are presented as mean ± S.E. of three experiments.

Techniques Used: Phospho-proteomics, Transfection, Expressing, Labeling, Western Blot, Irradiation, Fluorescence, Imaging

PIPKIγ90 degradation is required for cancer cell-mediated matrix degradation. A, ubiquitination of PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E. Avi-ubiquitin (Avi-Ub) was co-transfected with ZZ-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E into CHO-K1 cells expressing BirA. The cells were labeled with biotin, and the ZZ-tagged proteins were immunoprecipitated with IgG-agarose. The ubiquitination was detected using Dylight 680-streptavidin. Data are representative of two independent experiments. B, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or S6K1 shRNAs, treated with DMSO or carfilzomib (Carf, 5 μm). Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05. Ctrl, control. C, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or Akt1 shRNA, treated with DMSO or bortezomib/carfilzomib (B+C, 1 μm each). Data are presented as mean ± S.E. of three independent experiments. D, the expression levels of PIPKIγ in MDA-MB-231 cells expressing a control shRNA or PIPKIγ shRNA A1 and the PIPKIγ-depleted cells that stably express ZZ-PIPKIγ and -PIPKIγK97R. E, PIPKIγWT restored gelatin degradation in PIPKIγ-depleted MDA-MB-231 cells but PIPKIγK97R, a ubiquitination-deficient mutant, did not. Scale bar = 20 μm. F, quantification of the experiment in E. Data are mean ± S.E. of three independent experiments. *, p < 0.05. AU, arbitrary unit.

Figure Legend Snippet: PIPKIγ90 degradation is required for cancer cell-mediated matrix degradation. A, ubiquitination of PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E. Avi-ubiquitin (Avi-Ub) was co-transfected with ZZ-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E into CHO-K1 cells expressing BirA. The cells were labeled with biotin, and the ZZ-tagged proteins were immunoprecipitated with IgG-agarose. The ubiquitination was detected using Dylight 680-streptavidin. Data are representative of two independent experiments. B, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or S6K1 shRNAs, treated with DMSO or carfilzomib (Carf, 5 μm). Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05. Ctrl, control. C, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or Akt1 shRNA, treated with DMSO or bortezomib/carfilzomib (B+C, 1 μm each). Data are presented as mean ± S.E. of three independent experiments. D, the expression levels of PIPKIγ in MDA-MB-231 cells expressing a control shRNA or PIPKIγ shRNA A1 and the PIPKIγ-depleted cells that stably express ZZ-PIPKIγ and -PIPKIγK97R. E, PIPKIγWT restored gelatin degradation in PIPKIγ-depleted MDA-MB-231 cells but PIPKIγK97R, a ubiquitination-deficient mutant, did not. Scale bar = 20 μm. F, quantification of the experiment in E. Data are mean ± S.E. of three independent experiments. *, p < 0.05. AU, arbitrary unit.

Techniques Used: Ubiquitin Proteomics, Transfection, Expressing, Labeling, Immunoprecipitation, Plasmid Preparation, Control, shRNA, Stable Transfection, Mutagenesis

Image Search Results

Average population spike amplitude and EPSP slope following local delivery of pharmacological agents into the dentate gyrus. ( A ) Average population spike amplitude and EPSP slope ( B ) following local delivery of the PI3-kinase inhibitor, wortmannin. Saline baseline was recorded for 15 min. The saline-filled micropipette was replaced for a wortmannin-filled micropipette and baseline was recorded for 20 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( C ) Average population spike amplitude and EPSP slope ( D ) following delivery of the reversible PI3-kinase inhibitor, LY294002. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette with the LY294002-filled micropipette, no test pulses were delivered for 15 min; baseline was then recorded for 20 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( E ) Average population spike amplitude and EPSP slope ( F ) following delivery of the MEK inhibitor, U0126. Baseline responses were recorded for 20 min via the Hamilton micropipette before injection, then injection of U0126 was administered within a 5-min time window. No test pulses were delivered for 35 min following injection; baseline was then recorded for 10 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( G ) Average population spike amplitude and EPSP slope ( H ) following local delivery of the mTOR inhibitor, rapamycin. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette for a rapamycin-filled micropipette, no test pulses were delivered for 20 min; baseline was then recorded for 25 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( I ) Average population spike amplitude and EPSP slope ( J ) following local delivery of the RSK inhibitor, SL0101-1. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette for an SL0101-1-filled micropipette, no test pulses were delivered for 15 min; baseline was then recorded for 25 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( K ) Average population spike amplitude and EPSP slope ( L ) following local delivery of the S6K1 inhibitor, PF4708671. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette for a PF4708671-filled micropipette, no test pulses were delivered for 15 min; baseline was then recorded for 20 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow).

Journal: Learning & Memory

Article Title: Synaptically driven phosphorylation of ribosomal protein S6 is differentially regulated at active synapses versus dendrites and cell bodies by MAPK and PI3K/mTOR signaling pathways

doi: 10.1101/lm.044974.117

Figure Lengend Snippet: Average population spike amplitude and EPSP slope following local delivery of pharmacological agents into the dentate gyrus. ( A ) Average population spike amplitude and EPSP slope ( B ) following local delivery of the PI3-kinase inhibitor, wortmannin. Saline baseline was recorded for 15 min. The saline-filled micropipette was replaced for a wortmannin-filled micropipette and baseline was recorded for 20 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( C ) Average population spike amplitude and EPSP slope ( D ) following delivery of the reversible PI3-kinase inhibitor, LY294002. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette with the LY294002-filled micropipette, no test pulses were delivered for 15 min; baseline was then recorded for 20 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( E ) Average population spike amplitude and EPSP slope ( F ) following delivery of the MEK inhibitor, U0126. Baseline responses were recorded for 20 min via the Hamilton micropipette before injection, then injection of U0126 was administered within a 5-min time window. No test pulses were delivered for 35 min following injection; baseline was then recorded for 10 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( G ) Average population spike amplitude and EPSP slope ( H ) following local delivery of the mTOR inhibitor, rapamycin. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette for a rapamycin-filled micropipette, no test pulses were delivered for 20 min; baseline was then recorded for 25 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( I ) Average population spike amplitude and EPSP slope ( J ) following local delivery of the RSK inhibitor, SL0101-1. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette for an SL0101-1-filled micropipette, no test pulses were delivered for 15 min; baseline was then recorded for 25 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow). ( K ) Average population spike amplitude and EPSP slope ( L ) following local delivery of the S6K1 inhibitor, PF4708671. Saline baseline was recorded for 20 min. Following replacement of the saline-filled micropipette for a PF4708671-filled micropipette, no test pulses were delivered for 15 min; baseline was then recorded for 20 min, followed by delivery of 30 trains of HFS (time of HFS delivery denoted by arrow).

Article Snippet: S6K1 inhibitor, PF4708671, (100 µM (39.0 µg/mL) in 10% DMSO in saline; Tocris, Cat. 4032).

Techniques: Saline, Injection

Local infusion of the S6K1 inhibitor, PF4708671, partially attenuates phosphorylation of rpS6. ( A ) Immunostaining for p-ser235/236 ipsilateral to stimulation in a section distant from the infusion area. ( B ) High-magnification image of A . ( C ) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the infusion area, ipsilateral sections within the infusion area, and contralateral, nonstimulated sections. ( D ) Statistical assessment at four sites along the dentate gyrus, GCL, IML, MML, OML (ser235/236, N = 7), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction ( F (6,72) = 7.50, P < 0.0001), a significant main effect of treatment ( F (2,72) = 139.31, P < 0.0001), and a significant main effect of region ( F (3,72) = 72.31, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed that distant sections were statistically different than contralateral sections in all regions and sections within the infusion area were significantly different than contralateral sections in the GCL, IML, and MML. Notably, distant sections were also statistically different than sections within the infusion area in the IML and MML (two-way ANOVA, P < 0.05). ( E ) Immunostaining of p-ser235/236 ipsilateral to the stimulation in a section within the infusion area in the presence of PF4708671 (arrow denotes electrode track). ( F ) High-magnification image of E . Note local infusion of PF4708671 attenuates phosphorylation of rpS6 throughout the molecular layer of the dentate gyrus. The distinct band of rpS6 phosphorylation remained discernable. ( G ) Immunostaining of p-ser235/236 contralateral to the stimulation. ( H ) High-magnification image of G. ( I ) Immunostaining of p-ser240/244 in a section distant from the infusion area. ( J ) High-magnification image of I . ( K ) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the infusion area, ipsilateral sections within the infusion area, and contralateral sections. ( L ) Statistical assessment at four sites along the dentate gyrus, GCL, IML, MML, OML (ser240/244, N = 7), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction ( F (6,72) = 5.29, P = 0.0001), a significant main effect of treatment ( F (2,72) = 147.93, P < 0.0001), and a significant main effect of region ( F (3,72) = 180.77, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed that distant sections were statistically different than sections within the infusion area and contralateral sections in all regions. Notably, sections within the infusion area were also statistically different than contralateral sections in the GCL, IML, and MML (two-way ANOVA, P < 0.05). ( M ) Immunostaining of p-ser240/244 ipsilateral to stimulation in a section within the infusion area in the presence of PF4708671 (arrow denotes electrode track). ( N ) High-magnification image of M . ( O ) Immunostaining of p-ser240/244 contralateral to the stimulation. ( P ) High-magnification image of O . Scale bars: A , 200 µm; B , 20 µm.

Journal: Learning & Memory

Article Title: Synaptically driven phosphorylation of ribosomal protein S6 is differentially regulated at active synapses versus dendrites and cell bodies by MAPK and PI3K/mTOR signaling pathways

doi: 10.1101/lm.044974.117

Figure Lengend Snippet: Local infusion of the S6K1 inhibitor, PF4708671, partially attenuates phosphorylation of rpS6. ( A ) Immunostaining for p-ser235/236 ipsilateral to stimulation in a section distant from the infusion area. ( B ) High-magnification image of A . ( C ) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the infusion area, ipsilateral sections within the infusion area, and contralateral, nonstimulated sections. ( D ) Statistical assessment at four sites along the dentate gyrus, GCL, IML, MML, OML (ser235/236, N = 7), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction ( F (6,72) = 7.50, P < 0.0001), a significant main effect of treatment ( F (2,72) = 139.31, P < 0.0001), and a significant main effect of region ( F (3,72) = 72.31, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed that distant sections were statistically different than contralateral sections in all regions and sections within the infusion area were significantly different than contralateral sections in the GCL, IML, and MML. Notably, distant sections were also statistically different than sections within the infusion area in the IML and MML (two-way ANOVA, P < 0.05). ( E ) Immunostaining of p-ser235/236 ipsilateral to the stimulation in a section within the infusion area in the presence of PF4708671 (arrow denotes electrode track). ( F ) High-magnification image of E . Note local infusion of PF4708671 attenuates phosphorylation of rpS6 throughout the molecular layer of the dentate gyrus. The distinct band of rpS6 phosphorylation remained discernable. ( G ) Immunostaining of p-ser235/236 contralateral to the stimulation. ( H ) High-magnification image of G. ( I ) Immunostaining of p-ser240/244 in a section distant from the infusion area. ( J ) High-magnification image of I . ( K ) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the infusion area, ipsilateral sections within the infusion area, and contralateral sections. ( L ) Statistical assessment at four sites along the dentate gyrus, GCL, IML, MML, OML (ser240/244, N = 7), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction ( F (6,72) = 5.29, P = 0.0001), a significant main effect of treatment ( F (2,72) = 147.93, P < 0.0001), and a significant main effect of region ( F (3,72) = 180.77, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed that distant sections were statistically different than sections within the infusion area and contralateral sections in all regions. Notably, sections within the infusion area were also statistically different than contralateral sections in the GCL, IML, and MML (two-way ANOVA, P < 0.05). ( M ) Immunostaining of p-ser240/244 ipsilateral to stimulation in a section within the infusion area in the presence of PF4708671 (arrow denotes electrode track). ( N ) High-magnification image of M . ( O ) Immunostaining of p-ser240/244 contralateral to the stimulation. ( P ) High-magnification image of O . Scale bars: A , 200 µm; B , 20 µm.

Article Snippet: S6K1 inhibitor, PF4708671, (100 µM (39.0 µg/mL) in 10% DMSO in saline; Tocris, Cat. 4032).

Techniques: Immunostaining

Summary of PI3-kinase/mTOR- and MAPK/ERK-dependent signaling to rpS6 phosphorylation following synaptic stimulation. Somatodendritic activation of rpS6 phosphorylation at ser240/244 via PI3-kinase/mTOR-dependent kinases, i.e., S6K1, is the primary mechanism for modifying ribosomes throughout the postsynaptic cell ( left side). Synapse-specific phosphorylation of rpS6 at ser235/236 via MAPK/ERK-dependent kinases, that is, RSK, provides a mechanism for selective modification of ribosomes at activated synapses ( right side).

Journal: Learning & Memory

Article Title: Synaptically driven phosphorylation of ribosomal protein S6 is differentially regulated at active synapses versus dendrites and cell bodies by MAPK and PI3K/mTOR signaling pathways

doi: 10.1101/lm.044974.117

Figure Lengend Snippet: Summary of PI3-kinase/mTOR- and MAPK/ERK-dependent signaling to rpS6 phosphorylation following synaptic stimulation. Somatodendritic activation of rpS6 phosphorylation at ser240/244 via PI3-kinase/mTOR-dependent kinases, i.e., S6K1, is the primary mechanism for modifying ribosomes throughout the postsynaptic cell ( left side). Synapse-specific phosphorylation of rpS6 at ser235/236 via MAPK/ERK-dependent kinases, that is, RSK, provides a mechanism for selective modification of ribosomes at activated synapses ( right side).

Article Snippet: S6K1 inhibitor, PF4708671, (100 µM (39.0 µg/mL) in 10% DMSO in saline; Tocris, Cat. 4032).

Techniques: Activation Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1 phosphorylates PIPKIγ90 at Thr-553 and Ser-555. A, alignment of the Akt/S6K1 consensus sequences from different species. B, transfection of constitutively active Akt1 or S6K1 promoted PIPKIγ90 phosphorylation. FLAG-PIPKIγ90 was co-transfected with an empty vector, Myr-Akt1, and S6K1-F5A-E389-R3A into CHO-K1 cells. FLAG-PIPKIγ90 was immunoprecipitated, and PIPKIγ90 phosphorylation was detected with an anti-RXRXXpS/T motif antibody. a.u., arbitrary unit; *, p < 0.05. C, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in vitro. Recombinant GST-PIPKIγ90501–668, -PIPKIγ90501–668T553A, -PIPKIγ90501–668S555A, and -PIPKIγ90501–668T553A,S555A were phosphorylated with constitutively active S6K1 that was immunoprecipitated from CHO-K1 cells. D, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in CHO-K1 cells. HA-S6K1-F5A-E389-R3A was co-transfected with FLAG-PIPKIγ90, -PIPKIγ90T553A, -PIPKIγ90S555A, and -PIPKIγ90T553A,S555A into CHO-K1 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 versus WT. E, EGF and HGF stimulated PIPKIγ phosphorylation. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved and stimulated with EGF (20 ng/ml), HGF (50 ng/ml), SCF (20 ng/ml), and PDGF (20 ng/ml) for 20 min. FLAG-PIPKIγ90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus control (Ctrl). F, HGF-stimulated PIPKIγ phosphorylation was inhibited by Akt and S6K1 inhibitors. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 μm) or PF4708671 (10 μm), and then stimulated with HGF for 20 min. Data are presented as mean ± S.E. of four independent experiments. *, p < 0.05 versus HGF. G, HGF-stimulated PIPKIγ phosphorylation was suppressed by depletion of S6K1. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and then stimulated with HGF (20 ng/ml) for 20 min. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Transfection, Phospho-proteomics, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Stable Transfection, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1-mediated PIPKIγ90 phosphorylation is essential for the invasion. A, PIPKIγ90 and PIPKIγ90T553E,S555E restored the invasive capacity of PIPKIγ-depleted cells but PIPKIγT553A,S555A did not. PIPKIγ-depleted MDA-MB-231 cells were infected with retroviruses that express codon-modified ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or PIPKIγ90T553E,S555E and then selected with neomycin. Cells that express shRNA control (Ctrl) were used as controls. w/o, without. B, quantification of the experiment in A. White column, without HGF; gray column, 20 ng/ml HGF. Data are presented as mean ± S.E., n = 3. *, p < 0.05; **, p < 0.01 versus shRNA A1. C, inhibition of the invasion of MDA-MB-231 cells in the absence (white columns) and presence (gray columns) of HGF by the S6K1 inhibitor DG2. Data are mean ± S.E. of three independent experiments. **, p < 0.01. D, S6K1 and Akt activation in MDA-MB-231 cells expressing a control shRNA or S6K1 shRNAs. E, depletion of S6K1 by shRNA inhibited the invasion of MDA-MB-231 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001. F, S6K1 and ribosomal protein S6 phosphorylation in MDA-MB-231 cells expressing a control shRNA or Akt1 shRNAs. G, Akt1 knockdown did not significantly affect the invasion of MDA-MB-231 cells. White columns, without HGF; pink columns, with HGF. The data are expressed as mean ± S.E. of three independent experiments. H, effects of the S6K1 inhibitor DG2 on the invasion of PIPKI-depleted MDA-MB-231 cells that express ZZ-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E. Cell invasion was performed in the presence of DG2 (black columns, 10 μm) or vehicle (white columns) with HGF (20 ng/ml) in the lower chambers. Data are mean ± S.E. of three independent experiments. *, p < 0.05; ***, p < 0.001.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Phospho-proteomics, Infection, Modification, shRNA, Control, Inhibition, Activation Assay, Expressing, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1-mediated PIPKIγ phosphorylation is crucial for matrix degradation. A, effect of PIPKIγT553A,S555A and PIPKIγT553E,S555E on gelatin degradation in PIPKIγ-depleted cells. PIPKIγ-depleted MDA-MB-231 cells that express FLAG-PIPKIγ90, -PIPKIγ90T553A,S555A, or -PIPKIγ90T553E,S555E were resuspended in DMEM containing 1% FBS and HGF, plated on Alexa 488 gelatin-coated glass-bottom dishes, and cultured for 10 h. Scale bar = 20 μm. B, quantification of the experiment in A. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus shRNA control (Ctrl). AU, arbitrary unit. C, inhibition of invadopodium formation in MDA-MB-231 cells by the S6K1 inhibitor DG2. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Phospho-proteomics, Cell Culture, shRNA, Control, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1 activation correlates with breast cancer metastasis in human clinical specimens. A, human breast cancer primary tumors and the matched metastatic tumors of lymph node tissue were stained with anti-phospho-S6 ribosome protein antibody. B, the intensities of phospho-S6 staining were scored from 0–4, with 4 as the strongest. AU, arbitrary unit.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Activation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1-mediated phosphorylation regulates PIPKIγ degradation. A, the steady-state levels of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells that were transiently transfected with FLAG-PIPKIγWT, -PIPKIγT553A,S555A and -PIPKIγT553E,S555E, respectively, and treated with DMSO or carfilzomib (1 μm). B, substitution of Thr-553 and Ser-555 with Ala, but not Glu, inhibited degradation of PIPKIγ. CHO-K1 cells expressing BirA were transfected with Avi-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E and labeled with biotin. The levels of PIPKIγ were detected by Western blotting using Dylight 680-streptavidin. C, time course of degradation of PIPKIγWT, PIPKIγT553A,S555A, and PIPKIγT553E,S555E in CHO-K1 cells. Data represent mean ± S.E. of three experiments. **, p < 0.01; ***, p < 0.001. D, CHO-K1 cells were transiently transfected with Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E and plated on fibronectin. The cells were irradiated for 2 min by a 408-nm laser to convert the Dendra2 fusion protein into red Dendra2 fusion protein. The intensities of the red fluorescence were recorded using time-lapse imaging. Scale bar = 20 μm. E, quantification of the degradation of Dendra2-PIPKIγ90WT, -PIPKIγ90T553A,S555A, and -PIPKIγ90T553E,S555E. Data are presented as mean ± S.E. of four independent experiments. F, the S6K1 inhibitor DG2 or PF4708671 stabilizes PIPKIγ90WT. CHO-K1 cells were transfected with Dendra2-PIPKIγ90WT. 24 h post-transfection, cells were treated with DG2 (10 μm) or PF4708671 (10 μm) for 30 min and then irradiated for 2 min using a 408-nm laser. Data are presented as mean ± S.E. of three experiments. G, the S6K1 inhibitor DG2 (10 μm) had little effect on the degradation of Dendra2-PIPKIγ90T553A,S555E. Data are presented as mean ± S.E. of three experiments.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Phospho-proteomics, Transfection, Expressing, Labeling, Western Blot, Irradiation, Fluorescence, Imaging

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: PIPKIγ90 degradation is required for cancer cell-mediated matrix degradation. A, ubiquitination of PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E. Avi-ubiquitin (Avi-Ub) was co-transfected with ZZ-PIPKIγWT, -PIPKIγT553A,S555A, and -PIPKIγT553E,S555E into CHO-K1 cells expressing BirA. The cells were labeled with biotin, and the ZZ-tagged proteins were immunoprecipitated with IgG-agarose. The ubiquitination was detected using Dylight 680-streptavidin. Data are representative of two independent experiments. B, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or S6K1 shRNAs, treated with DMSO or carfilzomib (Carf, 5 μm). Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05. Ctrl, control. C, the steady-state levels of PIPKIγ in MDA-MB-231 cells that express empty pLKO.1 vector or Akt1 shRNA, treated with DMSO or bortezomib/carfilzomib (B+C, 1 μm each). Data are presented as mean ± S.E. of three independent experiments. D, the expression levels of PIPKIγ in MDA-MB-231 cells expressing a control shRNA or PIPKIγ shRNA A1 and the PIPKIγ-depleted cells that stably express ZZ-PIPKIγ and -PIPKIγK97R. E, PIPKIγWT restored gelatin degradation in PIPKIγ-depleted MDA-MB-231 cells but PIPKIγK97R, a ubiquitination-deficient mutant, did not. Scale bar = 20 μm. F, quantification of the experiment in E. Data are mean ± S.E. of three independent experiments. *, p < 0.05. AU, arbitrary unit.

Article Snippet: The S6K1 inhibitor PF4708671 was from ApexBio (Houston, TX).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Labeling, Immunoprecipitation, Plasmid Preparation, Control, shRNA, Stable Transfection, Mutagenesis

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