Volume 15, Issue 5, Pages 713-725 (September 2004) Distinct Roles for JNK1 and JNK2 in Regulating JNK Activity and c-Jun-Dependent Cell Proliferation  Kanaga Sabapathy, Konrad Hochedlinger, Shin Yuen Nam, Anton Bauer, Michael Karin, Erwin F. Wagner  Molecular Cell  Volume 15, Issue 5, Pages 713-725 (September 2004) DOI: 10.1016/j.molcel.2004.08.028

Figure 1 JNK1 and JNK2 Differentially Regulate Cellular Proliferation (A) Primary MEFs or early passage 3T3 fibroblasts were labeled with BrdU and the BrdU+ cells were quantified by flow cytometry. Vertical bars represent the standard deviations. (B) Basal apoptotic rates were evaluated by determining the percentage of Annexin-V positive cells by flow cytometry. (C) Jnk2−/− fibroblasts exit the G1 phase of the cell cycle earlier. Various mutant 3T3 cells were labeled for an hour with BrdU and these cells were followed for the indicated periods of time to determine the percentage of BrdU-labeled cells in the S phase of the cell cycle. Arrows indicate time point at which cells exit the G1 phase of the cell cycle and re-enter the S phase. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)

Figure 2 JNK2 Negatively Regulates Proliferation of Erythroid Progenitors (A and B) Splenocytes from normal or anemic mice (PHZ–induced) were equally seeded on semi-solid media supporting either CFU-E (A) or BFU-E (B) formation. Representative photographs of colonies and colony numbers are shown. (C) Splenocytes were stained with antibodies to CD34 and c-Kit and analyzed by flow cytometry. At least four independent mice were used in each group. *, p < 0.01 by one-tail unpaired t test. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)

Figure 3 Increased Levels of Total and Phosphorylated c-Jun in Jnk2−/− Fibroblasts (A) Western blot analysis was performed on serum-starved or stimulated early passage 3T3 fibroblast extracts using the indicated antibodies. The relative levels of the indicated proteins (with respect to loading) were quantified by imaging and represented graphically. The levels of proteins from serum-starved wild-type cells were set as 1.0. (B) Northern blot analysis of c-jun, c-fos, and mkp-1 using total mRNA from serum-starved and released primary MEFs. (C) Levels of the indicated proteins were determined after UV irradiation. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)

Figure 4 Altered AP-1 Activity in JNK Mutant Cells (A) AP-1 transactivation assays were performed after transfections with the plasmid carrying the multimerized AP-1 consensus binding site (5 TRE) expressing the luciferase gene, and a plasmid expressing the β-galactosidase gene, and luciferase activity was measured 30 hr post transfection (left panel). Similarly, Jnk1−/−Jnk2−/− cells were transfected with the indicated plasmids, together with the 5× TRE plasmid and AP-1 activity was determined (right panel). Results are representative of three independent experiments. (B and C) MEFs were elutriated into fractions and their cell cycle status was determined by flow cytometry (A represents asynchronous cell culture). Extracts prepared from these cells (B) and from serum-starved and restimulated wild-type and Jnk2−/− 3T3 fibroblasts (C) were used for Western blot analysis. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)

Figure 5 c-Jun Kinase Activity Is Elevated and c-Jun Is More Stable in Jnk2−/− Fibroblasts (A) Extracts from serum-starved and released 3T3 cells were used to determine total c-Jun kinase activity by the solid-state kinase assay with GST-c-Jun as a substrate. The relative levels are represented graphically. (B) Total c-Jun kinase activity was determined after UV irradiation. Fold induction is indicated below. (C) Half-life of c-Jun. Cells were pulse-labeled with 35S-methionine for 1 hr and chased for the indicated periods of time. c-Jun-immunoprecipitated extracts were analyzed by SDS-PAGE and radioautography. Relative c-Jun levels are represented as percentage of total 35S-labeled c-Jun remaining, normalized to loading. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)

Figure 6 Re-Expression of JNK1 or JNK2 in Jnk2−/− Fibroblasts (A) Pools of Jnk2−/− early passage 3T3 fibroblasts stably transfected with either empty vector, HA-tagged Jnk1, or Jnk2 vectors were analyzed by Western blotting. (B) These cells were grown for the indicated time periods and cumulative cell numbers were determined. (C) Total c-Jun kinase activity and c-Jun levels were determined after UV irradiation. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)

Figure 7 Specific Binding of c-Jun to the JNKs (A) Untreated or UV-irradiated wild-type 3T3 fibroblasts were immunoprecipitated with anti-JNK1 or anti-JNK2 antibodies and c-Jun kinase activity in the immunocomplexes was determined. Western blot analysis was performed with the indicated antibodies. Blots were overexposed to detect the c-Jun-specific upshift. *, a nonspecific band. (B) Untreated or UV-irradiated wild-type, Jnk1−/− and Jnk2−/− 3T3 fibroblasts were immunoprecipitated with anti-c-Jun antibody and blots were immunodetected with the indicated antibodies. The relative levels of ubiquitin were quantified by imaging and represented graphically. The level of ubiquitin from untreated wild-type cells were set as 1.0. Molecular Cell 2004 15, 713-725DOI: (10.1016/j.molcel.2004.08.028)