Journal: The Journal of Biological Chemistry

Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers

doi: 10.1016/j.jbc.2021.100325

Figure Lengend Snippet: Gβγ translocation from the PM to the GA in DU145, PC3, and HEK293 cells. A , Gγ9 translocation from the PM to the GA. The cells were cultured on 6-well dishes and transfected with YFP-Gγ9, Gβ1, Gαi1, and pmTurquoise2-Golgi (500 ng each). After starvation for 48 h, the cells were stimulated with SDF1α at 1 μg/ml. The images shown are obtained after stimulation for 10 s. B , quantification of the translocation of different Gγ subunits in complex with Gβ1 in response to SDF1α stimulation. The cells were transfected with individual YFP-Gγ, Gβ1, Gαi1, and pmTurquoise2-Golgi and stimulated as in A . The YFP signal at the GA before and after SDF1α stimulation was measured as shown in Fig. S1A . The increase in the YFP signal at the GA after SDF1α stimulation was expressed as the translocation of Gβγ to the GA. ∗ p < 0.05 versus Gγ3. C , relative expression of YFP-Gγ9 at the PM, nucleus (nuc), and GA before and after SDF1α stimulation for 10 s in a representative DU145 cell based on the line scan analysis. D , the half time (t 1/2 ) of Gγ translocation from the PM to the GA after SDF1α stimulation. The images shown in A and C are representatives of five to eight experiments. The quantitative data are presented as means ± SD (n = 5–8). The scale bars represent 10 μm. GA, Golgi apparatus; PM, plasma membrane.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam.

Techniques: Translocation Assay, Cell Culture, Transfection, Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers

doi: 10.1016/j.jbc.2021.100325

Figure Lengend Snippet: Gγ9 subunit is required for ERK1/2 activation by SDF1α. A , SDF1α dose dependently activated ERK1/2. The cells were cultured on 6-well dishes. After starvation for 48 h, the cells were stimulated with different concentrations of SDF1α for 5 min. B , quantitative data shown in A . C , effect of PTX, gallein, and AMD3100 on ERK1/2 activation by SDF1α. The cells were incubated with PTX (100 ng/ml for 16 h), gallein (10 μM for 30 min), and AMD3100 (100 μM for 1 h) before SDF1α stimulation (200 ng/ml for 5 min). D , effect of Golgi-targeting GRK2ct (Golgi-GRK2ct) on ERK1/2 activation by SDF1α. The cells were transfected with Golgi-GRK2ct or its mutant GRK2ctR587Q and then stimulated with SDF1α. E , expression of endogenous Gγ3 and Gγ9 in CRISPR-Cas9–mediated Gγ9 ( left panel ) or Gγ3 ( right panel ) knockout cells by Western blotting using Gγ-specific antibodies. F , ERK1/2 activation in Gγ3 and Gγ9 knockout cells in response to SDF1α stimulation. G , rescue of ERK1/2 activation in response to SDF1α stimulation by transient expression of sgRNA-resistant Gγ9 (γ9res) in Gγ9 knockout PC3 cells. The quantitative data are presented as means ± SD (n = 3). The Western blots shown in each panel are representatives of at least three experiments. PTX, pertussis toxin.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam.

Techniques: Activation Assay, Cell Culture, Incubation, Transfection, Mutagenesis, Expressing, CRISPR, Knock-Out, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers

doi: 10.1016/j.jbc.2021.100325

Figure Lengend Snippet: Inducible expression of Gβγ at the GA directly activates ERK1/2. A , inducible expression of Gγ2, Gγ3, and Gγ9 at the GA, but not at the PM, activated ERK1/2. The cells were cultured on 6-well dishes, transfected with individual FRB-Gγ and Gβ1, together with Golgi-Gγ or PM-Gγ (500 ng each), and then induced with rapamycin at 1 μM for 30 min. SDF1α simulation (200 ng/ml for 5 min) was used as a control. B , time courses of ERK1/2 activation by inducible expression of Gγ9 at the GA. C , quantitative data shown in B . D , effect of Golgi disruptors on Gγ9-mediated ERK1/2 activation. The cells transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP were treated with BFA (3 μM), ilimaquinone (10 μM), monensin (5 μM), nigericin (2 μM), nocodazole (10 μM), and swainsonine (5 μM) for 40 min before incubation with rapamycin for 30 min to induce Gβγ translocation. E , dose-dependent action of BFA on Golgi-Gγ9-induced ERK1/2 activation. The cells were transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP and then treated with BFA at different concentrations (0–4 μM) for 40 min before incubation with rapamycin for 30 min to induce Gβγ translocation. F , quantitative data shown in E . G , effect of Gβ, Gα, and GRK2ct on ERK1/2 activation by Golgi-Gγ9. The cells were transfected with FRB-Gγ9 and Golgi-FKBP with or without cotransfection with Gβ1, different Gα subunits, Golgi-GRK2ct, or GRK2ctR587Q (500 ng each) and then incubated with rapamycin for 30 min. ERK1/2 activation by SDF1α (200 ng/ml for 5 min) was used as a control. H , Gβγ translocation in the presence of Gα subunits. PC3 cells were transfected with venus-Gβ1, mCherry-Gαi1, FRB-Gγ9, and Golgi-FKBP (500 ng each) and then treated with rapamycin at 1 μM for 30 min. The quantitative data are presented as means ± SD (n = 3). The Western blots and images shown are representatives of at least three experiments. The scale bar represents 10 μm. BFA, brefeldin A; FRB, FKBP-rapamycin binding; GA, Golgi apparatus; PM, plasma membrane.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam.

Techniques: Expressing, Cell Culture, Transfection, Control, Activation Assay, Incubation, Translocation Assay, Cotransfection, Western Blot, Binding Assay, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers

doi: 10.1016/j.jbc.2021.100325

Figure Lengend Snippet: Effect of pharmacological inhibition of Gβγ downstream effectors on ERK1/2 activation by SDF1α and Golgi-Gγ9. A , the cells were incubated with LY294002 (50 μM), wortmannin (10 μM), HS-173 (0.1 μM), TGX-221 (0.5 μM), AS-604850 (2.5 μM), and GSK2292767 (0.5 μM) for 6 h ( left panel ); U73122 (10 μM), U73433 (10 μM), CRT0066101 (5 μM) for 1 h, or Go6976 (1 μM) for 30 min ( right panel ) before stimulation with SDF1α at 200 ng/ml for 5 min. B , dose-dependent effect of AS-604850 treatment for 6 h on ERK1/2 activation by SDF1α. C , quantitative data shown in B . D , the cells were transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP (500 ng each) and then incubated with HS-173, TGX-221, AS-604850, and GSK2292767 as in A ; AZD5363 (1 μM) for 3 h; U0126 (10 μM), PD98059 (50 μM), or GW5074 (10 μM) for 14 h before incubation with rapamycin for 30 min. The quantitative data are presented as means ± SD (n = 3). The Western blots shown in each panel are representatives of at least three experiments.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam.

Techniques: Inhibition, Activation Assay, Incubation, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers

doi: 10.1016/j.jbc.2021.100325

Figure Lengend Snippet: Depletion of p110γ and p101 abolishes ERK1/2 activation by SDF1α and Golgi-γ9. A , expression of p110γ in control and p110γ knockout cells. B , ERK1/2 activation by SDF1α at 200 ng/ml for 5 min in control and p110γ knockout cells. C , Golgi-γ9-induced ERK1/2 activation in p110γ knockout cells. The cells were transfected with FRB-γ9, Gβ1, and Golgi-FKBP (500 ng each) and then incubated with rapamycin for 30 min. D , ERK1/2 activation by SDF1α at 200 ng/ml for 5 min in p87 and p101 knockdown PC3 cells. E , Golgi-γ9–induced ERK1/2 activation in p87 and p101 knockdown PC3 cells. F , effect of Gγ3, Gγ9, and p110γ knockout on ERK1/2 activation by UK14304 at 1 μM for 5 min. G , effect of Gγ3, Gγ9, and p110γ knockout on ERK1/2 activation by EGF at 50 ng/ml for 5 min ( upper panel ), and insulin-like growth factor 1 at 200 ng/ml for 1 h ( lower panel ) in PC3 cells. The Western blots shown in each panel are representatives of at least three experiments.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam.

Techniques: Activation Assay, Expressing, Control, Knock-Out, Transfection, Incubation, Knockdown, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers

doi: 10.1016/j.jbc.2021.100325

Figure Lengend Snippet: Effect of Gγ9 and PI3Kγ knockout on PC3 migration and invasion in vitro and metastasis in vivo . A , inhibition of PC3 migration by Gγ9 and p110γ knockout after SDF1α stimulation at 1 μg/ml for 48 h as measured in transwell assays. EGF stimulation (50 ng/ml for 48 h) in control cells was used as a positive control. B , enhancement of PC3 migration by Golgi-Gγ9. PC3 cells were transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP (500 ng each) and treated with rapamycin at 1 μM with or without U0126 (10 μM) and PD98059 (50 μM), GW5074 (10 μM), or AS-604850 (2.5 μM) for 48 h. C , inhibition of PC3 invasion by Gγ9 and p110γ knockout after SDF1α stimulation at 1 μg/ml for 48 h. FBS (10%) stimulation in control cells was used as a positive control. D , PC3 invasion induced by Golgi-Gγ9. PC3 cells were transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP (500 ng each) and treated with rapamycin with or without U0126 and PD98059, GW5074, or AS-604850 as in B . E , the tumor growth in nude mice inoculated with PC3 cells by intracardiac injection at 21, 28, and 35 days (n = 10 in each group). One mouse died in the control group at 29 days. F , quantitative data of tumor growth as measured by bioluminescence imaging of whole animals. G , a model depicting the function of Gβγ translocation from the PM to the GA in activation of the ERK1/2 pathway via the PI3Kγ heterodimer p110γ-p101 (see text for details). The quantitative data are presented as means ± SD (n = 3 in A – D ; n = 9–10 in F ). ∗ and ∗∗, p < 0.05 versus basal and Ctrl, respectively, in A – D . ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.001 in F . The scale bars represent 5 cm. EGF, epidermal growth factor; FBS, fetal bovine serum.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam.

Techniques: Knock-Out, Migration, In Vitro, In Vivo, Inhibition, Control, Positive Control, Transfection, Injection, Imaging, Translocation Assay, Activation Assay