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, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Stable Transfection, Expressing

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: Infection, Modification, shRNA, Inhibition, Activation Assay, Expressing

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: Cell Culture, shRNA, 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: 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: Transfection, Expressing, Labeling, Immunoprecipitation, Plasmid Preparation, shRNA, Stable Transfection, Mutagenesis