Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity
<p>Microtubule-targeting drugs (paclitaxel and nocodazole) affect activator protein 1 (AP-1) activity in melanoma cells in vitro and in vivo. (<b>a</b>) Analyses of AP-1 luciferase reporter gene activity of human melanoma cells Mel Juso, Mel Ju, and Mel Im after treatment with the microtubule-stabilizing agent paclitaxel (PX; 5 µM) or nocodazole (NX; 30 µM), an agent promoting disruption of microtubule assembly. Control cells (ctrl) were treated with solvent DMSO (Dimethyl sulfoxide). (<b>b</b>) Electrophoretic mobility shift assays (EMSA) with nuclear extracts of PX or NX treated Mel Ju cells using the classical AP-1 consensus sequence (Oligo). Supershift experiments with an anti-c-Jun antibody demonstrate the direct involvement of c-Jun in the AP-1–DNA-binding complex. (<b>c</b>) Western blot analyses and densitometry of c-Jun in nuclear extracts of PX and NX treated Mel Juso cells and control cells (ctrl). Histone H2A was used as the loading control. (<b>d</b>,<b>e</b>) AP-1 luciferase reporter gene activity in (<b>d</b>) PX and (<b>e</b>) NX treated Hmb2-5 cells transfected with a c-Jun expression plasmid (c-Jun) or empty vector (pCDNA3; ctrl). (<b>f</b>) Immunohistochemical analyses of c-jun in melanoma tissues from <span class="html-italic">ret</span> transgenic mice (<span class="html-italic">n</span> = 3) after the treatment with PX (15 mg/kg) or PBS (ctrl). PX application was performed at day 0 and 5, and mice were sacrificed on day 7. (<b>g</b>) Immunohistochemical analyses of c- Jun in melanoma tissues from five patients before and after PX treatment. (<b>f</b>,<b>g</b>) The right panels depict the quantification (mean ± s.e.m.) of c-Jun positive nuclei per viewing field. (*: <span class="html-italic">p <</span> 0.05; ns: not significant).</p> "> Figure 2
<p>c-Jun protein interacts with TUB1A (Tubulin alpha chain) in melanoma cells and TUB1A affects AP-1 activity and stabilizes c-Jun protein. (<b>a</b>,<b>b</b>) Immunoprecipitation (IP) analyses of melanoma cell (Mel Juso, Mel Ju) lysates revealed co-precipitation of TUB1A with an (<b>a</b>) anti-c-Jun antibody and vice versa, (<b>b</b>) c-Jun with anti-TUB1A antibody. (<b>c</b>) Immunofluorescence analyses showed co-localization (white arrows) of c-Jun (red) and TUB1A (green) in the cytoplasm of melanoma cells. (<b>d</b>) Western blot analyses and densitometry of c-Jun and TUB1A in whole cell lysates of Mel Juso cells after TUB1A si-RNA (siTub1A) or control si-RNA (sictrl) transfection. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was used as a loading control. The bar graph depicts the quantification of protein amounts (mean ± s.d.) of three independent experiments. (<b>e</b>) Analyses of c-Jun protein expression in TUB1A-suppressed (siTub1A) and control (sictrl) Mel Juso cells after cycloheximide (CHX) treatment showed a faster decline of c-Jun levels in siTub1A compared to control cells. The bar graph (mean ± s.d. of three western blot analyses) depicts c-Jun levels normalized to GAPDH. (<b>f</b>) Western blot analyses and densitometry of nuclear extracts of Mel Juso cells showed lower c-Jun protein levels in TUB1A-suppressed (siTub1A) compared to control (sictrl) cells. The bar graph depicts c-Jun levels of three western blot analyses relative to LAMIN (Lamin A/C), which was used as a loading control. (<b>g</b>) AP-1 luciferase reporter gene analyses showed reduced AP-1 activity in TUB1A-suppressed (siTub1A) Mel Juso cells compared to control (sictrl) cells. Bars show the means ± s.d. of three independent experiments; measurements were performed in replicates. (*: <span class="html-italic">p <</span> 0.05).</p> "> Figure 3
<p>Stabilization of c-Jun by TUB1A influences nuclear import of c-Jun by IPO13. (<b>a</b>) AP-1 luciferase reporter gene activity in Mel Juso cells with si-RNA mediated suppression of several importins (siIPO7, siIPO8, siIPO9, siIPO13, siIPOβ) and cells transfected with control si-RNA (sictrl). The bar graph shows the means ± s.d. of three independent experiments. (*: <span class="html-italic">p <</span> 0.05 compared to sictrl; ns: not significant). (<b>b</b>,<b>c</b>) Analyses of c-Jun protein levels in (<b>b</b>) nuclear extracts and (<b>c</b>) whole cell extracts of melanoma cells with IPO13 suppression (siIPO13) and control cells (sictrl) by western blot and densitometry. GAPDH or LAMIN served as loading controls. (<b>d</b>–<b>f</b>) Immunoprecipitation (IP) analyses of whole cell lysates from Mel Juso and Mel Ju cells performing co-precipitation of (<b>d</b>) IPO13 using an anti-c-Jun antibody, (<b>e</b>) TUB1A using an anti-IPO13 antibody, and (<b>f</b>) IPO13 using an anti-TUB1A antibody. (<b>g</b>) Co-IP of Mel Juso cell lysates with si-RNA mediated suppression of TUB1A (siTub1A) or IPO13 (siIPO13) and sictrl using an anti-c-Jun-antibody. (<b>h</b>) Co-IP of extracts from Hmb2-5 cells transfected with wildtype JUN nuclear localization sequence (Jun NLS WT) or a Jun plasmid with mutated NLS (Jun NLS Mut) applying anti-TUB1A- or anti-IPO13-antibodies. Depicted are western blots using an anti-HA-tagged antibody for c-Jun detection. (<b>i</b>) Western blot analyses and densitometry showing the expression of HA-tagged c-Jun in Hmb2-5 protein lysates after transfection with Jun WT NLS or Jun MUT NLS. β-Actin served as a loading control. (<b>j</b>) Western blot analyses and densitometry showing the expression of HA-tagged c-Jun in Hmb2-5 nuclear extracts after transfection with Jun WT NLS or Jun MUT NLS. LAMIN served as a loading control. (<b>k</b>) AP-1 luciferase reporter gene activity of Hmb2-5 cells after transfection with Jun NLS WT or Jun NLS Mut. Bar graph shows the means ± s.d. of three independent experiments. (*: <span class="html-italic">p <</span> 0.05).</p> "> Figure 4
<p>Schematic overview of the c-Jun/TUB1A/IPO13 complex in melanoma cells. Monomeric α-tubulin (TUB1A) stabilizes the transcription factor c-Jun for nuclear transport via importin 13 (IPO13). The complex assembly of c- Jun, TUB1A, and IPO13 occurs in a nuclear localization sequence (NLS)- dependent manner, however, TUB1A remains in the cytoplasm whereas c-Jun translocates into the nucleus via IPO13, and hence, affects AP-1 activity in melanoma cells.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
3. Materials and Methods
3.1. Cells and Cell culture
3.2. Analysis of Gene Expression by Quantitative PCR
3.3. Western Blot Analysis
3.4. Co-Immunoprecipitation
3.5. Reporter Gene Analysis
3.6. Gene Suppression Using siRNA
3.7. Spin-Down Assay
3.8. Immunofluorescence Staining
3.9. Gel Shift Experiments
3.10. Mutation of the c-Jun Nuclear Localization Sequence
3.11. Paclitaxel Treatment of Melanoma Bearing Mice
3.12. Melamoma Tissues from Patients before and after Paclitaxel Treatment
3.13. Immunohistochemical Analysis
3.14. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Nucleotide Sequence (fwd/rev) |
---|---|
β-actin | 5′-CTACGTCGCCCTGGCTTCGAGC-3′ 5′-GATGGAGCCGCCGATCCACACGG-3′ |
importin β/ karyopherin | 5’-CAGCAGAACAAGGACGGCCCC-3′ 5′-TGCTGCTTTGCAGGGGTTCCA-3′ |
importin 7 | 5′-AGTGAGTGGCGCTATTCCTG-3′ 5′-CCCTGGTGCTGTTTCTCGAT-3′ |
importin 8 | 5′-GAACCTCCACCAGGAGAAGC-3′ 5′-AGCTTGCACTGCTCTGTGAT-3′ |
importin 9 | 5′-AATTCAGACCAGGCTCACCG-3′ 5′-AGGCGGGGCAAAATAATCCA-3′ |
importin 13 | 5′-TTCCCTGAGGCACCTACTGT-3′ 5′-GCCTCCTTGATCCACATGCT-3′ |
α-tubulin | 5′-GAAGCAGCAACCATGCGTGA-3′ 5′-GTGCCAGTGCGAACTTCATC-3′ |
MUTPrimer NLS c-Jun R273A/R275A | 5′-CCTCCAAGTGCGCGAAAGCGAAGCTGGAGAGAATCGCCC-3′ 5′-GGGCGATTCTCTCCAGCGCCGCCGCCGCGCACTTGGAGG-3′ |
MUTPrimer NLS c-Jun K274A/K276A | 5′-GCCTCCAAGTGCGCGGCGGCGGCGCTGGAGAG-3′ 5′-CTCTCCAGCGCCGCCGCCGCGCACTTGGAGGC-3′ |
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Kappelmann-Fenzl, M.; Kuphal, S.; Krupar, R.; Schadendorf, D.; Umansky, V.; Vardimon, L.; Hellerbrand, C.; Bosserhoff, A.-K. Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity. Cancers 2019, 11, 1806. https://doi.org/10.3390/cancers11111806
Kappelmann-Fenzl M, Kuphal S, Krupar R, Schadendorf D, Umansky V, Vardimon L, Hellerbrand C, Bosserhoff A-K. Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity. Cancers. 2019; 11(11):1806. https://doi.org/10.3390/cancers11111806
Chicago/Turabian StyleKappelmann-Fenzl, Melanie, Silke Kuphal, Rosemarie Krupar, Dirk Schadendorf, Viktor Umansky, Lily Vardimon, Claus Hellerbrand, and Anja-Katrin Bosserhoff. 2019. "Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity" Cancers 11, no. 11: 1806. https://doi.org/10.3390/cancers11111806