Volume 24, Issue 1, Pages 63-75 (October 2006) Spatiotemporal Regulation of c-Fos by ERK5 and the E3 Ubiquitin Ligase UBR1, and Its Biological Role  Takanori Sasaki, Hirotada Kojima, Rikiya Kishimoto, Ayu Ikeda, Hiroyuki Kunimoto, Koich Nakajima  Molecular Cell  Volume 24, Issue 1, Pages 63-75 (October 2006) DOI: 10.1016/j.molcel.2006.08.005 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Differential Regulation of the Stability and Subcellular Localization of c-Fos by IL-6- versus TPA-Induced Signaling (A) 293T-G133 cells were stimulated with soluble IL-6R/IL-6 (50 ng/ml each) or TPA (100 nM) for 90 min or with soluble IL-6R/IL-6, or TPA for 60 min, followed by the addition of CHX (10 μg/ml) or CHX plus PD98059 (PD, 30 μM) for 150 min. LMB (5 ng/ml) or MG132 (10 μM) was added 4 hr before stimulation. c-Fos was visualized by staining with an anti-c-Fos Ab. DAPI stains nuclei. (B) c-Fos degradation in the cytoplasm by gp130 stimulation and in the nucleus by TPA under the MEK inhibition. 293T-G133 cells were stimulated with G-CSF (25 ng/ml) or TPA for 90 min or with G-CSF or TPA for 60 min, followed by the addition of CHX, or CHX plus PD for the indicated times. LMB was added as in (A). The amounts of c-Fos and ERK2 were detected by immunoblotting. ERK2 was used as a loading control. (C) Ubiquitylation of c-Fos during gp130 signals, but not during TPA signals. c-Fos induced by gp130 stimulation, followed by PD treatment (G+PD), but not by TPA with PD-treatment (T+PD), showed its ubiquitylation. Gp130 signals alone (G) showed little c-Fos ubiquitylation. Cells were pretreated with MG132. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 ERK5 Is Responsible for the Nuclear Translocation and Stabilization of c-Fos, whereas a STAT3-Dependent Factor(s) Is Responsible for Its Cytoplasmic Localization (A) Gp130 stimulation in 293T-G133 cells causes sustained ERK5 activation and transient ERK1/2 activation. The immunoprecipitated ERK5 from 293T-G133 cells stimulated for the indicated times was subjected to an in vitro kinase assay; the incorporation of [32P]-ATP into MBP (top) and the amount of ERK5 (middle) are shown. The activated and phosphorylated ERK5, as seen in the shifted form, was assessed by immunoblotting with anti-ERK5 (bottom). The amount of phosphorylated ERK1/2 was detected by immunoblotting with an anti-phospho-ERK1/2 Ab (top). The amounts of ERK1/2 are shown (bottom). (B) Knockdown cells were made by the lentiviral siRNA expression system, and the efficiency of each knockdown (ERK5 and STAT3) is shown. CDK5 was used as a loading control. (C) c-Fos is localized to the cytoplasm of the ERK5-depleted cells in the presence of STAT3 activity. 293T-G133 (a), 293T-G133-ERK5KD (b, c, and d), 293T-G133-ERK5KD/STAT3KD (e and f), or 293T-G133-STAT3KD (g) cells were stimulated as indicated (G, G-CSF; and T, TPA, for 90 min or the last 10 min of a 90 min stimulation). c-Fos was visualized. DAPI stains nuclei. (D) c-Fos was destabilized in 293T-G133-ERK5KD cells, which were partially inhibited by LMB treatment. The amounts of c-Fos and ERK2 are shown. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 UBR1, a STAT3-Inducible E3 Ligase in 293T-G133 Cells, Interacts with Intact c-Fos in the Cytoplasm (A) The STAT3-dependent c-Fos expression cassette had five STAT3 binding sites (APRE) upstream of the junB gene promoter and cDNA for Flag- or CSF-tagged c-Fos. (B) The above construct for CSF-c-Fos was introduced into 293T-G133 or 293T-G133-ERK5KD cells by using the lentiviral system, and cells were stimulated with G-CSF for 90 min to express the protein. The CSF-c-Fos was detected in the nucleus of 293T-G133 or diffusely in 293T-G133-ERK5KD cells. (C) The c-Fos complexes from 293T-G133 or 293T-G133-ERK5KD cells were purified, digested with trypsin, and analyzed by using a nano-flow-LC/MS/MS system. (D) The lysates from G-CSF-stimulated 293T-G133 or 293T-G133-ERK5KD cells were immunoprecipitated with anti-c-Fos, anti-UBR1, or control Abs, and the amount of UBR1 or c-Fos in the immunoprecipitates was assessed by immunoblotting with an anti-UBR1 or anti-c-Fos Ab (left). Flag-c-Fos was induced in 293T-G133 and 293T-G133-ERK5KD cells as in (B). The anti-Flag-immunoprecpitates from ERK5KD cells contained UBR1 more efficiently than that from parental cells did (right). (E) Northern blot analysis of ubr1 mRNA expression. The ubr1 mRNA levels increased after stimulation with gp130 signals in the parental 293T-G133 cells, but not in the STAT3-KD cells. A CHOB mRNA was used as a loading control. (F and G) The endogenous UBR1 was increased after 90 min stimulation with gp130 signals in a STAT3-dependent manner, detected by immunoblotting (F) or by immunocytochemistry (G). ERK2 was used as a loading control for (F). (H) HeLa cells show the constitutive UBR1 expression at a level much higher than the level in gp130-stimulated 293T-G133 cells. No increase in the level of UBR1 by EGF. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 UBR1 Inhibits the AP-1 Activity by Accelerating c-Fos Degradation through Its Ubiquitylation in the Cytoplasm (A and B) The AP-1 reporter gene assay in 293T-G133 cells with siRNA for ubr1 (A) or with increasing amounts of UBR1 expression plasmid (B). Cells were stimulated with G-CSF, TPA, or G-CSF plus TPA for 5 hr. Luciferase activities normalized with the transfection efficiency are shown (data represented as mean ± SD). (C and D) UBR1 depletion enhances c-Fos protein levels in both gp130-stimulated 293T-G133 and HeLa cells stimulated with various factors. Efficiency of UBR1 knockdown in 293T-G133 cells (C) or HeLa cells (D) is shown (left). STAT3 or ERK2 was used as a control. 293T-G133 or 293T-G133-UBR1KD cells (center), unstimulated or stimulated with G-CSF for 90 min, and HeLa and HeLa-UBR1KD cells, unstimulated or stimulated with EGF (50 ng/ml), TPA, or 20% serum, were tested for the levels of c-Fos. ERK2 was used as a loading control. The c-fos mRNA and control CHOB mRNA levels in 293T-G133 and 293T-G133-UBR1KD are shown (right). (E) The gp130-induced c-Fos in the 293T-G133-UBR1KD cells shows improved stability under the MEK inhibition with PD, comparing with that in the parental 293T-G133 cells. (F) Nuclear localization of c-Fos under the MEK inhibition in gp130-stimuated 293T-G133-UBR1KD cells. (G) c-Fos ubiquitylation in gp130 signals depends on the E3 ligase activity of UBR1. The UBR1-reconstituted cells (UBR1WT or UBR1CS) express UBR1 at a level similar to that in gp130-stimulated parental cells (left). 293T-G133, 293T-G133-UBR1KD, and UBR1KD cells reconstituted with UBR1WT or UBR1CS were tested for c-Fos ubiquitylation in gp130 signals as in Figure 1C. Flag-Ub was used. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Phosphorylation of c-Fos at Ser32 and Thr232 by ERK5 Causes the Nuclear Localization and Stabilization of c-Fos in the Presence of UBR1 (A and B) Full-length Flag-c-Fos and truncated mutants of Flag-c-Fos (1–314, 1–244, 1–217, and 45–380) were induced in 293T-G133 or 293T-G133-ERK5KD cells by using the STAT3-dependent expression system with gp130 stimulation for 90 min, or for 60 min, followed by CHX treatment for 150 min. Flag-c-Fos was visualized. Only representative photographs are shown from more than 100 cells observed. The two regions of c-Fos, 217–244 and 1–45, are required for the ERK5-dependent c-Fos nuclear localization and stabilization in the presence of induced UBR1, summarized in (B). (C) ERK5 phosphorylates c-Fos at Ser32 and Thr232 in in vitro kinase assays. GST-c-Fos, including GST-c-Fos(1–110) WT or S32A, or GST-c-Fos(212-314) WT or T232A were used as substrates. (D) Phosphorylation of Ser32 or Thr232 causes c-Fos nuclear localization and enhanced stability with different mechanisms in the presence of induced UBR1. Phosphorylation at Thr232 inhibits the c-Fos nuclear export. Full-length Flag-c-Fos (S32A, S32D, T232G, and T232D) was induced and treated as in (A). Flag-c-Fos was visualized. (E) The cytoplasmic localization of c-FosT232G largely depends on the induced UBR1. (F) The c-Fos NES is shown. c-FosL223A/L227A, named c-FosNESm, was localized to the nucleus with improved stability in gp130-stimulated 293T-G133-ERK5KD cells. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 c-Fos Interacts with UBR1, but Not with UBR2, through Its Unphosphorylated N-Terminal Region (1–50) (A) HA-UBR1(1–1175) was localized mostly to the cytoplasm. DAPI was used for nuclear staining. Only a representative result is shown. (B) FLAG-c-Fos, coexpressed with HA-UBR1(1–1175), was localized to the cytoplasm and translocated to the nuclei in response to 10 min gp130 stimulation (G). This translocation is dependent on ERK5. (C) 35S-labeled in vitro-translated UBR1(1–1175) interacts with various GST-c-Fos through the N-terminal region of c-Fos. The interaction was largely disrupted by the S32D mutation, suggesting the inhibitory role of the Ser32 phosphorylation in the c-Fos-UBR1 interaction. (D) 35S-labeled in vitro-translated UBR2(1–1167) does not interact with full-length GST-c-Fos. (E) A model for the specific c-Fos recognition by UBR1. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 The ERK5 and UBR1 Regulatory System Controls the Serum-Dependent Cell Proliferation of HeLa Cells in a c-Fos-Dependent Manner (A) ERK5 depletion reduced the serum-induced c-Fos expression at the posttranslational level as well as at the mRNA expression level. HeLa and HeLa-ERK5KD cells, unstimulated and stimulated with serum for 90 min (top) or 45 min (bottom), were tested for the c-Fos protein (top) and the c-fos mRNA (bottom) expression. ERK2 was used as a loading control. 28S RNA was used as a loading control for the total RNA. (B) UBR1 depletion enhanced the HeLa cell proliferation in the presence of serum, whereas ERK5 depletion reduced it. HeLa, HeLa-UBR1KD, and HeLa-ERK5KD cells were assayed for their cell proliferation with MTT assay on days 0, 1, 2, and 3. (C) Stable introduction of either Flag-c-FosWT or Flag-c-FosNESm into HeLa-ERK5KD cells with the lentiviral system. The c-fos mRNA levels, uninduced or induced with serum for 45 min, in parental HeLa and HeLa-ERK5KD cells with the Flag-c-FosWT or Flag-c-FosNESm are shown. (D) Flag-c-FosWT was expressed diffusely, but Flag-c-FosNESm was localized to the nucleus in growing HeLa-ERK5KD cells. (E) Exogenous c-FosNESm, but not c-FosWT, recovered the reduced cell proliferation of HeLa-ERK5KD cells to the level of that of parental HeLa cells. Cell proliferation was measured with the MTT assay from day 0 to 4 (data represented as mean ± SD). (F) A model for the spatiotemporal regulation of c-Fos by ERK5 and UBR1. See text for the detailed explanation. Briefly, c-Fos shuttles between the nucleus and the cytoplasm with the use of its nuclear import mechanisms and its nuclear export mechanism, which is dependent on the c-FosNES, located at the 221–233 region. The E3 ligase UBR1 is also involved in the cytoplasmic localization of c-Fos in addition to its ubiquitylation activity to c-Fos. ERK5 inhibits the nuclear export of c-Fos by phosphorylating Thr232 and disrupts the interaction of c-Fos with UBR1 by phosphorylating Ser32. The expression of UBR1 at a high level makes c-Fos a good sensor for the intensity and duration of the ERK5 activity. The expression level of UBR1 is also rapidly induced by STAT3 in some cells so that the balance between UBR1 and the ERK5 activity can tightly regulate the c-Fos/AP-1 activity. Importantly, this system regulates c-Fos independently of another regulation system (data not shown), using an unidentified ubiquitin-independent degradation mechanism and multiple kinases phosphorylating the c-Fos C-terminal region. Molecular Cell 2006 24, 63-75DOI: (10.1016/j.molcel.2006.08.005) Copyright © 2006 Elsevier Inc. Terms and Conditions