GSK-3-Mediated Phosphorylation Enhances Maf-Transforming Activity Nathalie Rocques, Nancy Abou Zeid, Karine Sii-Felice, Laure Lecoin, Marie-Paule Felder-Schmittbuhl, Alain Eychène, Celio Pouponnot  Molecular Cell  Volume 28, Issue 4, Pages 584-597 (November 2007) DOI: 10.1016/j.molcel.2007.11.009 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 GSK-3 Triggers Maf Phosphorylation (A) Amino acid sequence comparison of large Maf proteins (MafA, MafB, and c-Maf) from different species. GSK-3 phosphorylation consensus sequence is shown. GSK-3 putative phosphorylation sites are indicated with solid arrowheads. The priming phosphorylation site is indicated with an open arrowhead. (B) INS-1 cells were metabolically labeled with 32P orthophosphate and treated as indicated. Extracts were immunoprecipitated with MafA antibody, except for the first lane, in which a nonimmune serum was used. The immunoprecipitates were analyzed for 32P incorporation by SDS-PAGE followed by autoradiography. Total-cell extracts were also analyzed by western blotting using MafA and β-actin antibodies. (C) COS-7 cells were transfected with plasmids encoding quail, human, or mouse MafA and treated for 2 hr with LiCl (10 mM) or kenpaullone (20 μM), as indicated. Total-cell extracts were analyzed by western blotting using MafA, β-catenin (β-cat), phospho-β-catenin (p-β-cat), and β-actin antibodies. (D) 293T cells were cotransfected with the HA-MafA vector and with plasmids encoding a control shRNA (Ctrl) or shRNAs complementary to human GSK-3α or GSK-3β, as indicated. Different combinations of two independent shRNAs targeting each GSK-3 isotype were used. Lysates were subjected to anti-GSK-3, anti-HA, and anti-β-actin western blot analysis. (E) COS-7 cells were cotransfected with plamids encoding HA-MafA and either Frat1-Myc or Frat2-Myc. Total-cell extracts were analyzed by western blotting using HA, Myc, β-catenin (β-cat), phospho-β-catenin (p-β-cat), and β-actin antibodies. (F) COS-7 cells were transfected by HA-MafA-encoding plasmid. Cells were treated by insulin for the indicated time. Total-cell extracts were analyzed using HA, GSK-3, phospho-GSK-3 (p-GSK3), Akt, phospho-Akt (p-Akt), and β-actin antibodies. Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 GSK-3 Triggers MafA Phosphorylation on S49, T53, T57, and S61 (A) COS-7 cells were transfected with HA-WT- or HA-4A-encoding constructs, metabolically labeled with 32P orthophosphate, and treated or not with LiCl. MafA proteins were immunoprecipitated and 32P incorporation was analyzed by SDS-PAGE followed by autoradiography. Western blots were carried out on total-cell lysates using HA and β-actin antibodies. (B) Anti-HA and anti-β-actin western blot was carried out on protein extracts from COS-7 cells transfected with empty vector or with HA-tagged WT or mutant (S49A, T53A, T57A, S61A, S65A, 4A) MafA-expressing constructs. (C) COS-7 cells were transfected as in (B) and metabolically labeled with 32P orthophosphate. MafA proteins were immunoprecipitated and 32P incorporation was analyzed by SDS-PAGE followed by autoradiography. The amount of MafA in the immunoprecipitates was assessed by anti-HA western blot. (D) The specificity of the T53-T57 MafA phospho-specific antibody (p-T53/T57) was tested by western blotting on protein extracts from COS cells transfected with the indicated MafA phosphorylation mutants. The antibody detected MafA only when phosphorylated on both T53 and T57. Note that the preimmune did not react with any of these proteins. Total MafA was analyzed using anti-HA antibody. β-actin western blot was performed as a loading control. (E) COS-7 cells were transfected with the indicated HA-tagged expression vectors and treated or not with LiCl. Protein extracts were analyzed by western blotting using p-T53/T57, HA, and β-actin antibodies. (F) INS-1 cells were treated with different pharmacological inhibitors: LiCl (10 mM), kenpaullone (20 μM), U0126 (20 μM), or roscovitine (20 μM) for 2 hr. Extracts were analyzed by western blotting using p-T53/T57, MafA, and β-actin antibodies. The amount of MafA relative to that of β-actin is indicated, MafA amount in untreated cells being set at 1. Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 GSK-3 Directly Phosphorylates MafA In Vitro (A) Purified GST, GST-MafA-WT, GST-MafA-Nter, or GST-MafA-Cter fusion proteins were incubated with recombinant GSK-3β, and in vitro kinase assays were performed. (B) The same experiment was carried out using purified GST, GST-MafA-WT, or GST-MafA-4A. Kinase reactions were incubated for 5 or 10 min. (C) Purified GST, GST-MafA-WT, or GST-MafA-S65A fusion proteins were first incubated with recombinant active ERK kinase in the presence of unlabeled ATP. Fusion proteins were then washed in order to eliminate ERK and subsequently incubated with recombinant GSK-3β and [γ-32P]ATP. (A–C) Reaction products were run on SDS-PAGE and submitted to both autoradiography (upper panels) and Coomassie blue staining (lower panels). GSK-3 autophosphorylation is indicated (p-GSK-3). Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 GSK-3-Mediated Phosphorylation Triggers Ubiquitination and Proteasome-Dependent Degradation of MafA (A) COS-7 cells were transfected with the HA-tagged WT or 4A MafA-expressing vectors and treated with 20 μg/ml cycloheximide (Chx) for the indicated times. Cell extracts were analyzed by western blot using HA or β-actin antibodies. Band intensity was measured and plotted as a percentage of the initial band intensity. (B and C) COS-7 cells were transfected with the HA-tagged indicated constructs. Cells were treated with MG132 (B) or LiCl for 3 hr (C). To assess ubiquitination, immunoprecipitated MafA was subjected to anti-ubiquitin western blot. Whole-cell extracts were subjected to HA or β-actin western blot. (D) INS-1 cells were treated or not with LiCl for 1 hr followed by Chx treatment, as indicated. Anti-MafA and anti-β-actin western blots were carried out on the resulting extracts. Band intensity was measured and plotted as a percentage of the initial band intensity. (E) INS-1 cells were treated or not with LiCl or kenpaullone for 1 hr and then with MG132 for 3 hr. Anti-MafA and anti-β-actin western blots were carried out on cell extracts. The amount of MafA relative to that of β-actin is indicated, the MafA amount in untreated cells being set at 1. Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 GSK-3-Mediated MafA Phosphorylation Increases Its Transcriptional Activity (A) COS-7 cells were cotransfected with the [4xAbox-Tk10-luciferase] reporter vector; the [actin-β-galactosidase] normalization vector; and 1, 2, or 4 ng of HA-tagged expression vectors. Luciferase activities were measured. (B) The same experiments as in (A) were performed using 4 ng of expression vectors. Cells were treated with 0, 10, or 20 μM kenpaullone for 16 hr. (A and B) Results represent mean values of three independent experiments after normalization with the β-galactosidase activity; error bars represent the standard deviation. Expression levels for WT and mutant MafA were analyzed by western blotting using anti-HA antibody. (C–F) NR cells were transfected with empty RCAS, RCAS-MafA-WT, RCAS-MafA-S65A, or RCAS-MafA-4A constructs and subsequently cultured for 2 weeks. (C) Transcription of αA-, βB1-, and δ1-crystallin genes was analyzed by qPCR. (D) The effect of kenpaullone (20 μM for 18 hr) on crystallin transcripts levels was assessed by qPCR. (C and D) Results represent mean values of three experiments after normalization by quantification of the HPRT transcript; error bars represented the standard deviation. (E) Whole-cell extracts were analyzed for the expression of crystallin proteins by western blot using the indicated antibodies. (F) Formation of lentoid bodies in NR cultures is illustrated by phase-contrast photographs. Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 MafA Phosphorylation Increases P/CAF Recruitment, which Protects MafA from Ubiquitination (A) Flag-tagged P/CAF and HA-tagged WT or 4A MafA expression vectors were transfected into COS-7 cells. MafA immunoprecipitates were analyzed for the presence of P/CAF by anti-Flag western blot. Whole-cell extracts were analyzed using anti-Flag, anti-β-actin, and anti-HA antibodies. (B) Cells were transfected as in (A) and treated or not with kenpaullone. P/CAF was immunoprecipitated using anti-Flag antibody followed by anti-HA western analysis. Whole-cell extracts were analyzed using anti-Flag, anti-β-actin, and anti-HA antibodies. (C) INS-1 cells were treated as indicated, and nuclear extracts (NEs) were used to immunoprecipitate endogenous MafA using anti-MafA or control nonimmune antibodies followed by western blotting using anti P/CAF antibody. Expression levels were controlled using anti-MafA and anti-P/CAF antibodies on NEs. Anti-SAM68 western blot was performed as a loading control. (D) Luciferase assays were performed using 10 ng of control, HA-MafA-WT, or HA-MafA-4A expression vectors together with 15 ng of Flag-P/CAF expression vector. Results represent mean values from three experiments after normalization with the β-galactosidase activity; error bars represent the standard deviation. (E) HA-tagged WT or 4A MafA expression vectors together with increasing amount of Flag-tagged P/CAF (0.5, 1, 2 μg) plasmids were transfected into COS-7 cells. Immunoprecipitated MafA was subjected to anti-ubiquitin western blot. Whole-cell extracts were subjected to anti-HA, Flag, or β-actin western blot. (F) HA-tagged WT or 4A MafA expression vectors together with Flag-tagged P/CAF plasmids were transfected into COS-7 cells treated with cycloheximide for the indicated times. MafA amount was measured by western blot (Figure S6B) quantification and plotted as a percentage of the initial band intensity. Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 GSK-3-Mediated MafA Phosphorylation Is Required for Its Transforming Activity (A) CEFs were transfected with replication-competent retroviral RCAS constructs encoding WT, 4A MafA, or v-Jun proteins and subsequently cultured for 3 weeks (see Figure S7A for expression levels). Cells (2 × 105) were then cultured for 3 weeks in soft agar with different doses of LiCl as indicated. Plates were scanned to visualize the macroscopic colonies and photographed to depict the characteristic morphology of the colonies. (B) Schematic representation of the results obtained by microarray analysis performed in cultures obtained as in (A). (C) Expression of selected MafA-regulated genes from each group depicted in (B). RT-PCR analyses were performed, and band intensity was quantified using the GeneTools software. Data were normalized on ribosomal S17 transcript. Results represent mean values from three RT-PCR experiments; error bars represented the standard deviation. (D) COS-7 cells were transfected with plasmids encoding HA-tagged MafA, MafB, or c-Maf and treated with LiCl or kenpaullone. Total-cell extracts were analyzed by western blotting using HA, β-catenin, phospho-β-catenin, and β-actin antibodies. (E) c-Maf western blot was performed on two multiple myeloma cell lines (MM), RPMI-8226 and LP-1 and treated or not with LiCl or kenpaullone. As a control, extracts were analyzed by western blotting using β-catenin, phospho-β-catenin, and β-actin antibodies. The amount of c-Maf relative to that of β-actin is indicated, the c-Maf amount in untreated cells being set at 1. Molecular Cell 2007 28, 584-597DOI: (10.1016/j.molcel.2007.11.009) Copyright © 2007 Elsevier Inc. Terms and Conditions