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. 2012 Apr 12;3(4):e297.
doi: 10.1038/cddis.2012.37.

Mss4 Protein Is a Regulator of Stress Response and Apoptosis

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Free PMC article

Mss4 Protein Is a Regulator of Stress Response and Apoptosis

B M Walter et al. Cell Death Dis. .
Free PMC article

Abstract

Mss4 (mammalian suppressor of Sec4) is an evolutionarily highly conserved protein and shows high sequence and structural similarity to nucleotide exchange factors. Although Mss4 tightly binds a series of exocytic Rab GTPases, it exercises only a low catalytic activity. Therefore Mss4 was proposed to work rather as a chaperone, protecting nucleotide free Rabs from degradation than as a nucleotide exchange factor. Here we provide further evidence for chaperone-like properties of Mss4. We show that expression levels of cellular Mss4 mRNA and protein are rapidly changed in response to a broad range of extracellular stress stimuli. The alterations are regulated mostly via the (c-jun NH(2)-terminal kinase) JNK stress MAPK signaling pathway and the mode of regulation resembles that of heat shock proteins. Similar to heat shock proteins, upregulation of Mss4 after stress stimulation functions protectively against the programmed cell death. Molecular analysis of the Mss4-mediated inhibition of apoptosis showed that interaction of Mss4 with eIF3f (eukaryotic translation initiation factor 3 subunit f), a member of the translation initiation complex and a protein with distinct pro-apoptotic properties, is the critical event in the anti-apoptotic action of Mss4.

Figures

Figure 1
Figure 1
Regulation of the Mss4 promoter activity by HSF1 and AP-1. (a) Luciferase reporter gene construct with Mss4 promoter. The 47 and 34 potential binding sites for AP-1 and HSF1 transcription factors, respectively, present in the 3.6-kB long promoter sequence are shown schematically. The 1.0 kb fragment contains only 10 binding sites for AP-1 and 8 for HSF1. (b) A7 melanoma cells were transiently transfected with reporter construct driven by the 3.6-kb Mss4 promoter fragment along with indicated transcription factors and 48 h later the reporter gene activity was determined. (c) HeLa cells were transfected with reporter construct shown in A for 24 h, starved overnight and stimulated for 5h with indicated growth factors, chemical compounds or cytokines. (d) HeLa cells were treated with 10 μM celastrol and additionally incubated at 42 °C for up to 4h. Western blot analysis shows expression and phosphorylation of HSF1. β-actin immunoblot served as loading control. (e) HeLa cells transfected for 40 h with reporter construct shown in A were stimulated with 6 or 10 μM celastrol or DMSO as control for additional 5 h before the reporter gene activity was determined. Relative luciferase activity measured in unstimulated or vector transfected cells was taken as unity. Mean values±S.D. from at least three repeated experiments are shown. *P<0.05 and ***P<0.001 relative to vector-transfected or unstimulated control cells, t-test
Figure 2
Figure 2
Endogenous Mss4 mRNA and protein response to stress stimuli. (a–c) HeLa cells were stimulated with 42 °C for up to 24h and changes in cell numbers (by counting, (a)), in Mss4 mRNA levels (by qRT-PCR, (b)) or protein levels (by western blotting, (c)) were determined. The upper image of (c) represents changes in Mss4 protein expression estimated densitometrically as the relative intensity of the Mss4 bands of the lower image to the loading β-actin controls. Values at time point 0 were taken as unity. (d) Mss4 mRNA decay in control or heat shocked (for 2 h) A7 melanoma cells after 5 μg/ml actinomycin D treatment. (e–h) Changes in cell number, Mss4 mRNA and protein levels as well as Mss4 mRNA decay were determined as in (a–d) but after stimulation of HeLa cells with 2mM H2O2. H, Mss4 mRNA decay in control or H2O2 stimulated A7 melanoma cells (2 mM). All experiments were carried out 2–4 times. Mean±S.D. values from a representative experiment are shown. *P<0.05; **P<0.01 and ***P<0.001 relative to unstimulated cells, t-test
Figure 3
Figure 3
JNK and p38 stress MAPK pathways dowregulate Mss4 expression. A7 melanoma cells were co-transfected with reporter construct driven by the 3.6-kb Mss4 promoter fragment and different c.a. or d.n. members of the p38 (a) or JNK (b) signaling pathways. The SB203580 (c) and SP600125 inhibitors (d) were added 24 h after transfection with c.a. MKK6 (c) and MKK7 (d) constructs. Reporter gene activity was determined 48 h later. Values of control cells transfected with reporter gene construct plus empty vector were taken as unity. Mean values±S.D. from three repeated experiments are shown. *P<0.05, **P<0.01 and ***P<0.001 relative to vector-transfected control cells, t-test. The asterisks over the lines show the significance between inhibitor- and DMSO-treated cells
Figure 4
Figure 4
Mss4 downregulation induces cell apoptosis. (a) A7 melanoma cells stably overexpressing human myc-tagged Mss4 were transfected with control or Mss4 specific siRNA for 48 h and the knockdown efficiency of endogenous and recombinant Mss4 protein was evaluated by Western blotting. Immunoblot for β-actin served as loading control. (b and c) A7 melanoma cells stably overexpressing human myc-tagged Mss4 were transfected with control or Mss4 specific siRNA. Forty-eight hours later the cells were either stimulated with 42 °C (b) or 5 mM H2O2 (c) for times indicated, stained with PI and the number of apoptotic cells was determined by flow cytometry. Mean values±S.D. from 2–3 repeated experiments are shown. (d) A7 melanoma cells were transiently transfected with myc-tagged Mss4 or empty vector for 40h. Then the cells were stressed with 2.5 or 5 mM H2O2 for 8h, lysed with RIPA buffer and the expression of Mss4 protein, as well as cleaved PARP was evaluated by western blotting. The β-actin immunoblot served as loading control. The n-fold PARP cleavage was estimated densitometrically as the relative intensity of cleaved PARP bands to the loading controls. Values of vector transfected and non-stressed cells were taken as unity. All experiments were carried out at least three times. *P<0.05, **P<0.01 and ***P<0.001 relative to ctrRNA transfected unstressed cells, t-test
Figure 5
Figure 5
Interaction of Mss4 with eIF3F prevents the eIF3F-mediated apoptosis. (a) To measure the interaction between different Mss4 mutants and eIF3f in a direct yeast-binding assay, yeast Y190 cells were transformed with GAL4 DNA-binding domain (BD) and GAL-4 activation domain (AD) chimeric constructs as indicated and a β-Gal filter assay was performed as described in Materials and Methods. (b) HEK 293 cells were transiently transfected with DNA constructs as indicated, left untreated (lanes 1–3) or stimulated with 5 mM H2O2 for 8h (lanes 4–6) and lysed with TLB. The presence of myc-tagged eIF3f protein in GST–Mss4 wt or GST-Mss4 F75A pull downs (PD) were detected by immunoblotting (IB) with myc antibody (top blot). To ascertain equal amounts of precipitated Mss4 or GST proteins, the blot was redeveloped with antibodies against GST. The blot on the bottom shows the expression in transfected cells of eIF3f protein. 10 μg of total cell lysates were loaded. (c) A7 melanoma cell stably overexpressing Mss4 wt or Mss4 F75A were stressed with 2.5 or 5 mM H2O2 for 16h and phase-contrast pictures were taken to document morphological changes. (d) A7 melanoma cell stably overexpressing Mss4 wt or Mss4 F75A were transfected with ctr or Mss4 specific siRNA and stimulated 48h later with 5 mM H2O2 for additional 8h. Numbers over the columns represent the amount of apoptotic cells that were determined by flow cytometry after staining of cells with PI. Mean values±S.D. from three repeated experiments are shown. **P<0.01 and ***P<0.001 relative to vector-transfected control cells, t-test. (e) A7 melanoma cells were transiently transfected with GST-tagged Mss4 and myc-tagged eIF3f, either separately or together and stressed with 5 mM H2O2 for 8h. Phase-contrast images were taken to document morphological changes in stressed (lower panels) compared with control (upper panels) cells. (f) Western blot analysis of cells examined in (g) demonstrating different expression levels of endogenous and recombinant Mss4 proteins. β-actin immunoblot served as loading control. (g) To stably overexpress the Mss4 protein, A7 melanoma cell were retrovirally transduced with myc-tagged wt or F75A mutated Mss4 variants. Vector-transduced cells served as control. To stably downregulate the endogenous Mss4, A7 cells were infected with lentiviruses containing either scrambled or Mss4 specific shRNA. The five A7 cell lines with different amounts of endogenous or recombinant Mss4 proteins were transfected with myc-tagged eIF3f or empty vector and 40 h later stimulated with 5 mM H2O2 for further 8h. The amount of apoptotic cells was documented by flow cytometry after PI staining. Mean values±S.D. from two repeated experiments are shown. *P<0.05, **P<0.01 and ***P<0.001 relative to control vector-transfected cells, t-test
Figure 6
Figure 6
Expression of Mss4 abrogates the eIF3f-mediated inhibition of translation. A7 melanoma cells were transfected with pRL-CMV vector along with either empty pCS2+MT vector or pCS2+MT-Mss4, pCS2+MT-eIF3f or both plasmids, and 24 h later cell lysates were tested for luciferase activity (a), amounts of mRNA for luciferase (b), Mss4 (c) and eIF3f (d). Mean values±S.D. from two repeated experiments are shown. ***P<0.001 relative to vector-transfected cells, t-test
Figure 7
Figure 7
Proposed scheme of eIF3f and Mss4 interaction in context of apoptosis. In case of regular conditions or low stress amounts Mss4 is tightly bound to eIF3f, inhibiting its phosphorylation and subsequent association with eIF3 protein complex and pro-apoptotic functions. (left picture). If a certain threshold of stress is crossed, Mss4 is downregulated, leading to a release of eIF3f, which can now be phosphorylated by the caspase-activated CDK11p46 kinase, incorporated into the eIF3 protein compound, which in turn results in inhibition of translation and induction of apoptosis (right picture)

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