Limits...
Functional Interactions between BM88/Cend1, Ran-binding protein M and Dyrk1B kinase affect cyclin D1 levels and cell cycle progression/exit in mouse neuroblastoma cells.

Tsioras K, Papastefanaki F, Politis PK, Matsas R, Gaitanou M - PLoS ONE (2013)

Bottom Line: We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM.However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization.Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, Athens, Greece.

ABSTRACT
BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors.

Show MeSH

Related in: MedlinePlus

Dyrk1B protein expression and turn-over in Neuro 2a cells.(a) Dyrk1B expression in transiently transfected Neuro 2a cells was examined at 16 h and 48 h post-transfection in the presence or in the absence of the specific proteasome inhibitor MG132. Dyrk1B is not detectable in non-transfected Neuro 2a cells (CTL) with available antibody, while transgene Dyrk1B, obvious at 16h, is decreased overtime in a proteasome-specific manner (upper panel). In the absence of MG132, cyclin D1 levels are inversely related to Dyrk1B (middle panel) while in the presence of MG132 cyclin D1, also degraded via the proteasome, is maintained despite persisting Dyrk1B expression (middle panel); β-tubulin indicates protein loading (lower panel). (b) Dyrk1B is localized in the nucleus of Dyrk1B+ Neuro 2a cells 16 h post-transfection, while at 48 h it is mainly cytoplasmic as shown by immunocytochemistry and confocal microscopy. The cells depicted by arrows at 16h and 48h, respectively, are shown at higher magnification. Different image acquisition settings were used at 16h and 24h to compensate for signal reduction at 48h. Scale bar: 10 μm. (c) Dyrk1B protein turn-over in Neuro 2A cells. Cells were transfected with Dyrk1B and allowed for expression 16 h after transfection. Cells were then treated with cycloheximide for different times as indicated and subjected to Western blot analysis. (d) Dyrk1B reduces BrdU incorporation in transiently transfected Neuro 2a cells after 16 h of expression. **Student’s t-test, p=0.00146, n=5. (e) In the presence of 10 μM harmine, a specific kinase inhibitor of the Dyrk1 protein family, the Dyrk1B-dependent down-regulation of cyclin D1 is inhibited in Neuro 2a cells (middle panel) without affecting Dyrk1B protein levels (upper panel). Lanes show Neuro 2a cells transiently transfected with Dyrk1B and allowed 16 h for expression, or non-transfected cells (CTL), harvested and immunoblotted with the indicated antibodies. (f) Quantification of Dyrk1B protein levels normalized relative to β-actin, in the presence or absence of 10 μΜ harmine. (g) Cyclin D1 is wiped out from the nuclei of Dyrk1B+ transiently transfected Neuro 2a cells (arrows, i-iv) but is clearly maintained in the nucleus of Dyrk1B+ cells in the presence of harmine (arrows, v-viii). Cells were double labeled for Dyrk1B (green) and cyclin D1 (red) while nuclei were visualized using TO-PRO-3 (blue). The merged pictures are shown (iv, viii). Scale bar: 8 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842983&req=5

pone-0082172-g005: Dyrk1B protein expression and turn-over in Neuro 2a cells.(a) Dyrk1B expression in transiently transfected Neuro 2a cells was examined at 16 h and 48 h post-transfection in the presence or in the absence of the specific proteasome inhibitor MG132. Dyrk1B is not detectable in non-transfected Neuro 2a cells (CTL) with available antibody, while transgene Dyrk1B, obvious at 16h, is decreased overtime in a proteasome-specific manner (upper panel). In the absence of MG132, cyclin D1 levels are inversely related to Dyrk1B (middle panel) while in the presence of MG132 cyclin D1, also degraded via the proteasome, is maintained despite persisting Dyrk1B expression (middle panel); β-tubulin indicates protein loading (lower panel). (b) Dyrk1B is localized in the nucleus of Dyrk1B+ Neuro 2a cells 16 h post-transfection, while at 48 h it is mainly cytoplasmic as shown by immunocytochemistry and confocal microscopy. The cells depicted by arrows at 16h and 48h, respectively, are shown at higher magnification. Different image acquisition settings were used at 16h and 24h to compensate for signal reduction at 48h. Scale bar: 10 μm. (c) Dyrk1B protein turn-over in Neuro 2A cells. Cells were transfected with Dyrk1B and allowed for expression 16 h after transfection. Cells were then treated with cycloheximide for different times as indicated and subjected to Western blot analysis. (d) Dyrk1B reduces BrdU incorporation in transiently transfected Neuro 2a cells after 16 h of expression. **Student’s t-test, p=0.00146, n=5. (e) In the presence of 10 μM harmine, a specific kinase inhibitor of the Dyrk1 protein family, the Dyrk1B-dependent down-regulation of cyclin D1 is inhibited in Neuro 2a cells (middle panel) without affecting Dyrk1B protein levels (upper panel). Lanes show Neuro 2a cells transiently transfected with Dyrk1B and allowed 16 h for expression, or non-transfected cells (CTL), harvested and immunoblotted with the indicated antibodies. (f) Quantification of Dyrk1B protein levels normalized relative to β-actin, in the presence or absence of 10 μΜ harmine. (g) Cyclin D1 is wiped out from the nuclei of Dyrk1B+ transiently transfected Neuro 2a cells (arrows, i-iv) but is clearly maintained in the nucleus of Dyrk1B+ cells in the presence of harmine (arrows, v-viii). Cells were double labeled for Dyrk1B (green) and cyclin D1 (red) while nuclei were visualized using TO-PRO-3 (blue). The merged pictures are shown (iv, viii). Scale bar: 8 μm.

Mentions: Next we proceeded to analyze the effect of RanBPM interaction with Dyrk1B on cell cycle progression and cyclin D1 stability, first in the absence and then in the presence of Cend1, in Neuro 2a cells. Endogenous Dyrk1B expression was not detectable by immunoblotting in non-transfected or mock transfected Neuro 2a cells cultured in standard conditions in the presence of 10% fetal bovine serum, with the antibody used in this study (Figure 5a). In Neuro 2a cells transiently transfected with Dyrk1B cDNA, its expression was detected by immunoblotting 16 h post transfection, but declined by 48 h (Figure 5a). This was unlike transgene Cend1 and RanBPM which remained strongly expressed at 48 h post-transfection (see Figure 3a). Quantification by real-time RT-qPCR showed a significant decline of Dyrk1B mRNA between 16 h and 48 h, suggesting that the transgene cDNA expression became extinct over time (Figure S3a).


Functional Interactions between BM88/Cend1, Ran-binding protein M and Dyrk1B kinase affect cyclin D1 levels and cell cycle progression/exit in mouse neuroblastoma cells.

Tsioras K, Papastefanaki F, Politis PK, Matsas R, Gaitanou M - PLoS ONE (2013)

Dyrk1B protein expression and turn-over in Neuro 2a cells.(a) Dyrk1B expression in transiently transfected Neuro 2a cells was examined at 16 h and 48 h post-transfection in the presence or in the absence of the specific proteasome inhibitor MG132. Dyrk1B is not detectable in non-transfected Neuro 2a cells (CTL) with available antibody, while transgene Dyrk1B, obvious at 16h, is decreased overtime in a proteasome-specific manner (upper panel). In the absence of MG132, cyclin D1 levels are inversely related to Dyrk1B (middle panel) while in the presence of MG132 cyclin D1, also degraded via the proteasome, is maintained despite persisting Dyrk1B expression (middle panel); β-tubulin indicates protein loading (lower panel). (b) Dyrk1B is localized in the nucleus of Dyrk1B+ Neuro 2a cells 16 h post-transfection, while at 48 h it is mainly cytoplasmic as shown by immunocytochemistry and confocal microscopy. The cells depicted by arrows at 16h and 48h, respectively, are shown at higher magnification. Different image acquisition settings were used at 16h and 24h to compensate for signal reduction at 48h. Scale bar: 10 μm. (c) Dyrk1B protein turn-over in Neuro 2A cells. Cells were transfected with Dyrk1B and allowed for expression 16 h after transfection. Cells were then treated with cycloheximide for different times as indicated and subjected to Western blot analysis. (d) Dyrk1B reduces BrdU incorporation in transiently transfected Neuro 2a cells after 16 h of expression. **Student’s t-test, p=0.00146, n=5. (e) In the presence of 10 μM harmine, a specific kinase inhibitor of the Dyrk1 protein family, the Dyrk1B-dependent down-regulation of cyclin D1 is inhibited in Neuro 2a cells (middle panel) without affecting Dyrk1B protein levels (upper panel). Lanes show Neuro 2a cells transiently transfected with Dyrk1B and allowed 16 h for expression, or non-transfected cells (CTL), harvested and immunoblotted with the indicated antibodies. (f) Quantification of Dyrk1B protein levels normalized relative to β-actin, in the presence or absence of 10 μΜ harmine. (g) Cyclin D1 is wiped out from the nuclei of Dyrk1B+ transiently transfected Neuro 2a cells (arrows, i-iv) but is clearly maintained in the nucleus of Dyrk1B+ cells in the presence of harmine (arrows, v-viii). Cells were double labeled for Dyrk1B (green) and cyclin D1 (red) while nuclei were visualized using TO-PRO-3 (blue). The merged pictures are shown (iv, viii). Scale bar: 8 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842983&req=5

pone-0082172-g005: Dyrk1B protein expression and turn-over in Neuro 2a cells.(a) Dyrk1B expression in transiently transfected Neuro 2a cells was examined at 16 h and 48 h post-transfection in the presence or in the absence of the specific proteasome inhibitor MG132. Dyrk1B is not detectable in non-transfected Neuro 2a cells (CTL) with available antibody, while transgene Dyrk1B, obvious at 16h, is decreased overtime in a proteasome-specific manner (upper panel). In the absence of MG132, cyclin D1 levels are inversely related to Dyrk1B (middle panel) while in the presence of MG132 cyclin D1, also degraded via the proteasome, is maintained despite persisting Dyrk1B expression (middle panel); β-tubulin indicates protein loading (lower panel). (b) Dyrk1B is localized in the nucleus of Dyrk1B+ Neuro 2a cells 16 h post-transfection, while at 48 h it is mainly cytoplasmic as shown by immunocytochemistry and confocal microscopy. The cells depicted by arrows at 16h and 48h, respectively, are shown at higher magnification. Different image acquisition settings were used at 16h and 24h to compensate for signal reduction at 48h. Scale bar: 10 μm. (c) Dyrk1B protein turn-over in Neuro 2A cells. Cells were transfected with Dyrk1B and allowed for expression 16 h after transfection. Cells were then treated with cycloheximide for different times as indicated and subjected to Western blot analysis. (d) Dyrk1B reduces BrdU incorporation in transiently transfected Neuro 2a cells after 16 h of expression. **Student’s t-test, p=0.00146, n=5. (e) In the presence of 10 μM harmine, a specific kinase inhibitor of the Dyrk1 protein family, the Dyrk1B-dependent down-regulation of cyclin D1 is inhibited in Neuro 2a cells (middle panel) without affecting Dyrk1B protein levels (upper panel). Lanes show Neuro 2a cells transiently transfected with Dyrk1B and allowed 16 h for expression, or non-transfected cells (CTL), harvested and immunoblotted with the indicated antibodies. (f) Quantification of Dyrk1B protein levels normalized relative to β-actin, in the presence or absence of 10 μΜ harmine. (g) Cyclin D1 is wiped out from the nuclei of Dyrk1B+ transiently transfected Neuro 2a cells (arrows, i-iv) but is clearly maintained in the nucleus of Dyrk1B+ cells in the presence of harmine (arrows, v-viii). Cells were double labeled for Dyrk1B (green) and cyclin D1 (red) while nuclei were visualized using TO-PRO-3 (blue). The merged pictures are shown (iv, viii). Scale bar: 8 μm.
Mentions: Next we proceeded to analyze the effect of RanBPM interaction with Dyrk1B on cell cycle progression and cyclin D1 stability, first in the absence and then in the presence of Cend1, in Neuro 2a cells. Endogenous Dyrk1B expression was not detectable by immunoblotting in non-transfected or mock transfected Neuro 2a cells cultured in standard conditions in the presence of 10% fetal bovine serum, with the antibody used in this study (Figure 5a). In Neuro 2a cells transiently transfected with Dyrk1B cDNA, its expression was detected by immunoblotting 16 h post transfection, but declined by 48 h (Figure 5a). This was unlike transgene Cend1 and RanBPM which remained strongly expressed at 48 h post-transfection (see Figure 3a). Quantification by real-time RT-qPCR showed a significant decline of Dyrk1B mRNA between 16 h and 48 h, suggesting that the transgene cDNA expression became extinct over time (Figure S3a).

Bottom Line: We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM.However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization.Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, Athens, Greece.

ABSTRACT
BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors.

Show MeSH
Related in: MedlinePlus