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Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Liu L, Wu J, Ong SS, Chen T - PLoS ONE (2013)

Bottom Line: Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1.In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430).Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.

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Activation of Cdk4 enhances PAX3-FOXO1-mediated gene expression.A) NIH3T3 cells were co-transfected with wild-type GFP-PAX3-FOXO1 (left panel) or GFP-PAX3-FOXO1 S430A (right panel), 6 X PRS9 reporter, TK-renilla (PRL-TK), and one of the plasmids as indicated. Comparisons were made between other samples and a sample transfected with pcDNA3 (set as 100 relative luciferase units, or RLU). Cdk4, pCMV-Cdk4 (wild-type); Cdk4DN, pCMV-Cdk4DN (inactive Cdk4); cyclin D1, pCMV-cyclin D1. B) Protein expression in A) was confirmed by Western blot analysis using anti-GFP antibody (IB: GFP), anti-Cdk4 antibody (IB: Cdk4), anti-cycin D1 (IB: cyclin D1), and anti-actin (IB: actin). Actin was used as a loading control. GFP-PF, GFP-PAX3-FOXO1. C) NIH3T3 cells were transfected with 6 X PRS9 reporter, TK-renilla, and one of the plasmids indicated. PF WT, wild-type GFP-PAX3-FOXO1; PFS430A, GFP-PAX3-FOXO1S430A; PFS430D, GFP-PAX3-FOXO1S430D. Comparisons were made between other samples and a sample transfected with wild-type GFP-PAX3-FOXO1 (set as 100 RLUs). D) Protein expression in B) was confirmed by Western blot analysis. Actin was used as a loading control. Luciferase activity was measured 24 h after transfection. Firefly luciferase activity was normalized using Renilla luciferase activity. * p<0.05; *** p<0.001.
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pone-0058193-g003: Activation of Cdk4 enhances PAX3-FOXO1-mediated gene expression.A) NIH3T3 cells were co-transfected with wild-type GFP-PAX3-FOXO1 (left panel) or GFP-PAX3-FOXO1 S430A (right panel), 6 X PRS9 reporter, TK-renilla (PRL-TK), and one of the plasmids as indicated. Comparisons were made between other samples and a sample transfected with pcDNA3 (set as 100 relative luciferase units, or RLU). Cdk4, pCMV-Cdk4 (wild-type); Cdk4DN, pCMV-Cdk4DN (inactive Cdk4); cyclin D1, pCMV-cyclin D1. B) Protein expression in A) was confirmed by Western blot analysis using anti-GFP antibody (IB: GFP), anti-Cdk4 antibody (IB: Cdk4), anti-cycin D1 (IB: cyclin D1), and anti-actin (IB: actin). Actin was used as a loading control. GFP-PF, GFP-PAX3-FOXO1. C) NIH3T3 cells were transfected with 6 X PRS9 reporter, TK-renilla, and one of the plasmids indicated. PF WT, wild-type GFP-PAX3-FOXO1; PFS430A, GFP-PAX3-FOXO1S430A; PFS430D, GFP-PAX3-FOXO1S430D. Comparisons were made between other samples and a sample transfected with wild-type GFP-PAX3-FOXO1 (set as 100 RLUs). D) Protein expression in B) was confirmed by Western blot analysis. Actin was used as a loading control. Luciferase activity was measured 24 h after transfection. Firefly luciferase activity was normalized using Renilla luciferase activity. * p<0.05; *** p<0.001.

Mentions: Since PAX3-FOXO1 is phosphorylated by Cdk4/cyclin D1, and the selective inhibitor of Cdk4/cyclin D1 fascaplysin inhibited the transcriptional activity of PAX3-FOXO1, it is most likely that activation of Cdk4 will lead to the activation of PAX3-FOXO1. To test this hypothesis, we transfected PAX3-FOXO1 with or without co-transfection of Cdk4 or cyclin D1 into NIH3T3 cells, a cell line commonly used in studying PAX3-FOXO1 activity. Overexpression of a wild-type Cdk4 increased PAX3-FOXO1 transcriptional activity by more than 2-fold over that of an empty vector control (pcDNA3) (Fig. 3A). Overexpression of cyclin D1, which regulates the activity of Cdk4, also enhanced the activity of PAX3-FOXO1. However, an inactive kinase-dead Cdk4 (Cdk4DN) only marginally affected the activity of PAX3-FOXO1. The activation effect of Cdk4 on PAX3-FOXO1 was significantly reduced (to less than 50%) when a S430A mutation was introduced. Fig. 3B shows the expression levels of all constructs and indicates that the level of PAX3-FOXO1 was similar under all four transfection combinations. These data indicate that activation of Cdk4 leads to enhanced activity of PAX3-FOXO1. To provide further evidence that the activity of PAX3-FOXO1 is regulated by Cdk4 through phosphorylation, we tested the activity of a phosphorylation-deficient mutant (GFP-PAX3-FOXO1 S430A), in which the mutation of serine to alanine places a hydrophobic side chain at position 430 and renders it deficient of phosphorylation, and a phosphomimetic mutant (GFP-PAX3-FOXO1 S430D) in which serine was mutated to a negatively charged aspartate to mimic phosphorylation. As shown in Fig. 3C, the S430A mutant was 17% less active, whereas the S430D mutant was 32% more active than the wild-type PAX3-FOXO1. These findings, together with the results shown in Fig. 2, suggest that Cdk4 phosphorylates PAX3-FOXO1 to promote its transcriptional activity, and Ser430 is one of the phosphorylation sites.


Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Liu L, Wu J, Ong SS, Chen T - PLoS ONE (2013)

Activation of Cdk4 enhances PAX3-FOXO1-mediated gene expression.A) NIH3T3 cells were co-transfected with wild-type GFP-PAX3-FOXO1 (left panel) or GFP-PAX3-FOXO1 S430A (right panel), 6 X PRS9 reporter, TK-renilla (PRL-TK), and one of the plasmids as indicated. Comparisons were made between other samples and a sample transfected with pcDNA3 (set as 100 relative luciferase units, or RLU). Cdk4, pCMV-Cdk4 (wild-type); Cdk4DN, pCMV-Cdk4DN (inactive Cdk4); cyclin D1, pCMV-cyclin D1. B) Protein expression in A) was confirmed by Western blot analysis using anti-GFP antibody (IB: GFP), anti-Cdk4 antibody (IB: Cdk4), anti-cycin D1 (IB: cyclin D1), and anti-actin (IB: actin). Actin was used as a loading control. GFP-PF, GFP-PAX3-FOXO1. C) NIH3T3 cells were transfected with 6 X PRS9 reporter, TK-renilla, and one of the plasmids indicated. PF WT, wild-type GFP-PAX3-FOXO1; PFS430A, GFP-PAX3-FOXO1S430A; PFS430D, GFP-PAX3-FOXO1S430D. Comparisons were made between other samples and a sample transfected with wild-type GFP-PAX3-FOXO1 (set as 100 RLUs). D) Protein expression in B) was confirmed by Western blot analysis. Actin was used as a loading control. Luciferase activity was measured 24 h after transfection. Firefly luciferase activity was normalized using Renilla luciferase activity. * p<0.05; *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585270&req=5

pone-0058193-g003: Activation of Cdk4 enhances PAX3-FOXO1-mediated gene expression.A) NIH3T3 cells were co-transfected with wild-type GFP-PAX3-FOXO1 (left panel) or GFP-PAX3-FOXO1 S430A (right panel), 6 X PRS9 reporter, TK-renilla (PRL-TK), and one of the plasmids as indicated. Comparisons were made between other samples and a sample transfected with pcDNA3 (set as 100 relative luciferase units, or RLU). Cdk4, pCMV-Cdk4 (wild-type); Cdk4DN, pCMV-Cdk4DN (inactive Cdk4); cyclin D1, pCMV-cyclin D1. B) Protein expression in A) was confirmed by Western blot analysis using anti-GFP antibody (IB: GFP), anti-Cdk4 antibody (IB: Cdk4), anti-cycin D1 (IB: cyclin D1), and anti-actin (IB: actin). Actin was used as a loading control. GFP-PF, GFP-PAX3-FOXO1. C) NIH3T3 cells were transfected with 6 X PRS9 reporter, TK-renilla, and one of the plasmids indicated. PF WT, wild-type GFP-PAX3-FOXO1; PFS430A, GFP-PAX3-FOXO1S430A; PFS430D, GFP-PAX3-FOXO1S430D. Comparisons were made between other samples and a sample transfected with wild-type GFP-PAX3-FOXO1 (set as 100 RLUs). D) Protein expression in B) was confirmed by Western blot analysis. Actin was used as a loading control. Luciferase activity was measured 24 h after transfection. Firefly luciferase activity was normalized using Renilla luciferase activity. * p<0.05; *** p<0.001.
Mentions: Since PAX3-FOXO1 is phosphorylated by Cdk4/cyclin D1, and the selective inhibitor of Cdk4/cyclin D1 fascaplysin inhibited the transcriptional activity of PAX3-FOXO1, it is most likely that activation of Cdk4 will lead to the activation of PAX3-FOXO1. To test this hypothesis, we transfected PAX3-FOXO1 with or without co-transfection of Cdk4 or cyclin D1 into NIH3T3 cells, a cell line commonly used in studying PAX3-FOXO1 activity. Overexpression of a wild-type Cdk4 increased PAX3-FOXO1 transcriptional activity by more than 2-fold over that of an empty vector control (pcDNA3) (Fig. 3A). Overexpression of cyclin D1, which regulates the activity of Cdk4, also enhanced the activity of PAX3-FOXO1. However, an inactive kinase-dead Cdk4 (Cdk4DN) only marginally affected the activity of PAX3-FOXO1. The activation effect of Cdk4 on PAX3-FOXO1 was significantly reduced (to less than 50%) when a S430A mutation was introduced. Fig. 3B shows the expression levels of all constructs and indicates that the level of PAX3-FOXO1 was similar under all four transfection combinations. These data indicate that activation of Cdk4 leads to enhanced activity of PAX3-FOXO1. To provide further evidence that the activity of PAX3-FOXO1 is regulated by Cdk4 through phosphorylation, we tested the activity of a phosphorylation-deficient mutant (GFP-PAX3-FOXO1 S430A), in which the mutation of serine to alanine places a hydrophobic side chain at position 430 and renders it deficient of phosphorylation, and a phosphomimetic mutant (GFP-PAX3-FOXO1 S430D) in which serine was mutated to a negatively charged aspartate to mimic phosphorylation. As shown in Fig. 3C, the S430A mutant was 17% less active, whereas the S430D mutant was 32% more active than the wild-type PAX3-FOXO1. These findings, together with the results shown in Fig. 2, suggest that Cdk4 phosphorylates PAX3-FOXO1 to promote its transcriptional activity, and Ser430 is one of the phosphorylation sites.

Bottom Line: Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1.In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430).Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.

Show MeSH
Related in: MedlinePlus