Journal: International journal of molecular sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Figure 1. BAG5 interacts with Akt. (A) Schematic representation of Akt and BAG5 structures indicating Bait and Prey and their corresponding interacting domains. (B) BAG5 and Akt interact in the yeast two-hybrid system. Akt-KDD (dominant negative mutant of the catalytic domain of Akt) was used as bait to screen a human fetal brain cDNA library in a yeast two-hybrid system. A full-length clone of BAG5 was identified as a novel Akt-interactor protein. The specificity of the interaction between BAG5 and Akt was determined using p53 and pTD1 (T antigen) as negative control bait and prey, respectively, whereas the known interaction between p53 and T antigen served as positive control of the system. All yeast grew on media lacking leucine and tryptophan (-LT, which selects for the presence of the plasmids, left), but only those in which interactions occurred grew in media lacking histidine, leucine, and tryptophan and were positive for the activity of α-galactosidase (-HLT + Xα-Gal, right). (C,D) Transfected Akt and BAG5 interact in mammalian cells. HEK293T cells were cotransfected with HA-Akt and Myc-BAG5 (C) or with HA-Akt and GST-BAG5 (D), as indicated. Total cell lysates (TCL) were either immunoprecipitated using anti-HA antibodies ((C), IP:HA) or pulled down using glutathione beads ((D), PD:GST). BAG5 that coimmunoprecipitated with Akt was detected with Myc-specific antibodies (C), whereas Akt that interacted with pulled-down BAG5

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Dominant Negative Mutation, cDNA Library Assay, Negative Control, Positive Control, Activity Assay, Transfection, Immunoprecipitation

Journal: International journal of molecular sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Figure 2. BAG5 promotes Akt ubiquitination and degradation. (A) BAG5 controls Akt ubiquitination. HeLa cells were transfected with HA-Ubiquitin and Myc-BAG5. Thirty-six hours after transfection, cells were serum-starved overnight and then left untreated or treated with 20 µM of MG132 for 6 h. Akt was immunoprecipitated from total cell lysates, and the presence of ubiquitinated Akt was detected as a high molecular smear in the immunoblots using anti-HA antibodies. (B) Akt protein levels are regulated by changes in the expression of BAG5. Increasing amounts of Myc- BAG5 induce a decrease in Akt protein expression, regardless of the effect of MG132, without altering the expression of Erk detected in the same total cell lysates. (C) Protein ubiquitination in total cell lysates of HeLa cells transfected with BAG5 and treated with MG132, as indicated in (A). (D) BAG5 accelerates Akt degradation under Akt destabilizing conditions (ADC). HeLa cells transiently transfected with HA-Akt and Myc-BAG5 were treated with 5 µM of cycloheximide (CHX) and 1 µM of 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) in serum-free media, a condition known to destabilize Akt, during the indicated times. The expression of Akt, phospho-Akt (T-450),

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: International journal of molecular sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Figure 3. Hsp70 mediates Akt degradation promoted by BAG5. (A) Hypothetical model showing that BAG5 contributes to Akt regulation by Hsp70. BAG5 structure is shown as predicted by AlphaFold. (B) BAG5 interacts with Akt and Hsp70. HEK293T cells were transiently transfected with HA-Akt either in the presence or absence of GST-BAG5 alone or with EGFP-Hsp70, as indicated. GST-BAG5 was isolated by pull-down (PD:GST) from total cell lysates (TCL) using glutathione beds. The interaction of HA-Akt and EGFP-Hsp70 proteins with BAG5 was detected in the pull-down, whereas their expression was confirmed in total cell lysates (TCL), using anti-HA or anti-EGFP antibodies, respectively. (C) Hsp70 promotes Akt degradation. HeLa cells transiently transfected with HA-Akt, and increasing amounts of EGFP-Hsp70 were analyzed by Western blot of total cell lysates for the expression of Akt (anti-HA and anti-Akt), Hsp70 (anti-EGFP), and S6, 48 h post-transfection. Anti-S6 immunoblot served as a loading control. (D) Graph illustrates the densitometric analysis of HA-Akt expression in the presence or absence of Hsp70 and the effect of BAG5 knockdown on the effect of Hsp70. Bars represent the mean value ± SEM of three independent experiments, **, p < 0.01. (E) Hsp70 involves BAG5 to promote Akt degradation. HeLa cells were transiently transfected with shRNA-BAG5 for 24 h; then, cells were transfected again with HA-Akt and EGFP-Hsp70 for 48 h more to achieve 72 h of shRNA BAG5 effect. Akt, Hsp70, BAG5, Erk, and S6 levels were analyzed by immunoblot using antibodies against HA or EGFP for transfected Akt and Hsp70, respectively, or antibodies that recognize endogenous BAG5, Akt, Erk, or S6 as indicated. Erk and S6 immunoblots served as loading controls. The numbers below the top panels (B,C,E) indicate the normalized densitometric value with respect to the condition indicated at 1.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Transfection, Isolation, Expressing, Western Blot, Control, Knockdown, shRNA

Journal: International journal of molecular sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Figure 4. Profile of Deubiquitinases and E3 ligases linked to BAG5 expression in BRCA, UCEC, LUSC, and OV TCGA cancer patients. (A) BAG5 mRNA expression in BRCA, UCEC, LUSC, and OV TCGA studies in diploid and BAG5 amplified samples. Mean ± S.E.M values. ***, p < 0.001, ****, p < 0.0001. Unpaired t test with Welch’s correction. (B) Heatmap for the top ten DUBs correlated genes with BAG5 in BRCA, UCEC, LUSC, and OV cancer studies. (C) Heatmap for the top ten E3 ligases correlated genes with BAG5 in BRCA, UCEC, LUSC, and OV cancer studies. (D) Akt1 protein expression according to diploid and BAG5 amplified samples. Mean ± S.E.M values. *, p = 0.02, **, p = 0.0048. Unpaired t test with Welch’s correction. (E) Highest correlated DUBs with BAG5 in

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Expressing, Amplification

Journal: International journal of molecular sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Figure 5. Phosphorylation of BAG5 by Akt correlates with a reduction in the interaction between them. (A) Hypothetical model showing the phosphorylation of BAG5 by Akt. The structure of BAG5 is shown as predicted by AlphaFold, and the indicated phosphorylation site is the most prominent in the phosphoproteomic analysis available at https://www.phosphosite.org/ (accessed on 14 November 2023). (B) Akt activation attenuates its interaction with BAG5. HeLa cells transfected with HA-Akt, Myc-BAG5, or both were incubated overnight in serum-free media and then stimulated, or not, with HGF for 15 min, as indicated. Akt was immunoprecipitated from total cell lysates using an anti-HA antibody (IP:HA), and interacting-BAG5 was detected by anti-Myc Western blot. The expression of Akt and BAG5 was confirmed in total cell lysates (TCL) using anti-HA and anti-Myc antibodies, respectively. The effect of HGF on Akt and Erk activation was detected with phospho-specific antibod- ies. Expression of Erk in total cell lysates served as a loading control. (C) Akt degradation induced by BAG5 preferentially occurs under serum starvation conditions. HeLa cells were cotransfected with HA-Akt and increasing amounts of Myc-BAG5. Thirty-six hours post-transfection, cells were incubated with serum-free or serum-supplemented media for an additional 12 h. Expression of Akt, GSK-3β, and BAG5 was analyzed by immunoblot in total cell lysates. GSK-3β served as a loading control. (D) Akt phosphorylates BAG5. Cells were transfected with Myc-BAG5 and one of the following Akt constructs: HA-Akt (wild type), myr-Akt (N-myristoylated-Akt, constitutively active), or kinase-negative mutant Akt (Akt-K179M). Twenty-four hours post-transfection, cells that

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Phospho-proteomics, Activation Assay, Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Control, Construct, Mutagenesis

Journal: International journal of molecular sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Figure 6. BAG5 modulates Akt activation and HGF-dependent cell migration. (A) BAG5 attenuates the activation of Akt in response to HGF without affecting the activation of Erk. HeLa cells transfected with the control vector, or HA-BAG5, were serum starved and stimulated with increasing amounts of HGF for 15 min. Then, activation of Akt and Erk was detected by Western blot using phospho-specific antibodies, whereas their expression was confirmed using antibodies that detect endogenous proteins. The expression of transfected BAG5 was confirmed using anti-HA antibodies. Graph at the top represents the analysis of Akt phosphorylation determined in three independent experiments. Mean ± S.E.M values. *, p < 0.05. (B,C) BAG5 expression reciprocally correlates with Akt activation in conditions in which Akt stability is not affected. (B) HeLa cells transiently transfected with increasing amounts of shRNA-BAG5 (B) or Myc-BAG5 (C) were used to detect Akt activation by Western blot in total cell lysates with a phospho-Akt (Ser473) specific antibody. The expression of total Akt, as well as transfected Myc-BAG5, was confirmed in the same samples, as indicated. The numbers below the top panels (A–C) indicate the normalized densitometric value with respect to the condition indicated at 1. (D) BAG5 diminishes HGF-dependent chemotactic cell migration. HeLa cells transfected with BAG5 or control plasmid were subjected to chemotaxis assays in Boyden chambers in which they were stimulated with HGF or 10% FBS, as indicated. Graph represents the analysis of relative cell migration determined in three independent experiments. Mean ± S.E.M values. *, p < 0.05. A representative result is shown at the bottom of the graph. (E) Model depicting the proposed mechanism of Akt regulation by BAG5. Under starved conditions and BAG5 overexpression, Akt interacts with BAG5, which promotes Akt inhibition and, with the participation of Hsp70, leads to its degradation. When the Akt signaling pathway is stimulated, the interaction between Akt and BAG5 decreases, coincident with Akt-dependent BAG5 phosphorylation.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Activation Assay, Migration, Transfection, Control, Plasmid Preparation, Western Blot, Expressing, Phospho-proteomics, shRNA, Chemotaxis Assay, Over Expression, Inhibition