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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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Fascaplysin enhances PAX3-FOXO1 cytoplasmic localization in Rh30 cells.A) Scatter plot of GFP-PAX3-FOXO1 subcellular localization. The ratio of the average intensities of the GFP signals in the nucleus and cytoplasm was determined as described in Materials and Methods. Each dot in the plot represents one cell. Cells were transfected with wild-type (WT) GFP-PAX3-FOXO1 (GFP-PF), or GFP-PF S430A mutant (S430A) for 24 h, followed by treatment with either DMSO or 1 µM fascaplysin (FAS) for 30 min. Cells were then fixed and stained with DAPI. B) Representative images.
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pone-0058193-g005: Fascaplysin enhances PAX3-FOXO1 cytoplasmic localization in Rh30 cells.A) Scatter plot of GFP-PAX3-FOXO1 subcellular localization. The ratio of the average intensities of the GFP signals in the nucleus and cytoplasm was determined as described in Materials and Methods. Each dot in the plot represents one cell. Cells were transfected with wild-type (WT) GFP-PAX3-FOXO1 (GFP-PF), or GFP-PF S430A mutant (S430A) for 24 h, followed by treatment with either DMSO or 1 µM fascaplysin (FAS) for 30 min. Cells were then fixed and stained with DAPI. B) Representative images.

Mentions: The subcellular localization of a transcription factor is critical for its transcriptional activity and is well-studied for FOXO1 but not for PAX3-FOXO1 [14], [16]–[18]. Whereas activation of AKT signaling retains FOXO1 in the cytoplasm, PAX3-FOXO1 is insensitive to AKT activity and is predominantly nuclear-localized, suggesting that the regulation of subcellular localization by phosphorylation is different between FOXO1 and PAX3-FOXO1 [18]. Recently, Cdks was reported to phosphorylate Ser249 of FOXO1 and resulted in its cytoplasmic localization [16], [17]. Since Cdk4 phosphorylates Ser430 in PAX3-FOXO1 (corresponds to Ser249 in FOXO1), we tested whether inhibition of Cdk4 by fascaplysin alters the subcellular localization of PAX3-FOXO1. PAX3-FOXO1 is known to predominantly nuclear-localized [18]. When a cell analysis tool (Cellomics vHCS Scan NucTrans Bioapplication) [14] was used to analyze the images and determine the ratio of the average intensities of the GFP signals in the nucleus and cytoplasm, wild-type GFP-PAX3-FOXO1 is more nuclear localized than GFP-PAX3-FOXO1 S430A, and treatment with fascaplysin for 30 min significantly enhanced the cytoplasmic localization of wild-type GFP-PAX3-FOXO1 but not the phosphorylation-resistant mutant S430A (Fig. 5A). Representative images were shown in Fig. 5B to illustrate the fascaplysin-mediated cytoplasmic localization of PAX3-FOXO1 in a portion of the cells. The effect of fascaplysin in promoting cytoplasmic localization of PAX3-FOXO1 was also observed in NIH3T3 cells (Fig. S1). These findings suggest that Cdk4 enhances the transcriptional activity of PAX3-FOXO1 by phosphorylating and possibly either inhibiting the cytoplasmic translocation or promoting the nuclear translocation of PAX3-FOXO1 in a fascaplysin-sensitive manner.


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)

Fascaplysin enhances PAX3-FOXO1 cytoplasmic localization in Rh30 cells.A) Scatter plot of GFP-PAX3-FOXO1 subcellular localization. The ratio of the average intensities of the GFP signals in the nucleus and cytoplasm was determined as described in Materials and Methods. Each dot in the plot represents one cell. Cells were transfected with wild-type (WT) GFP-PAX3-FOXO1 (GFP-PF), or GFP-PF S430A mutant (S430A) for 24 h, followed by treatment with either DMSO or 1 µM fascaplysin (FAS) for 30 min. Cells were then fixed and stained with DAPI. B) Representative images.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585270&req=5

pone-0058193-g005: Fascaplysin enhances PAX3-FOXO1 cytoplasmic localization in Rh30 cells.A) Scatter plot of GFP-PAX3-FOXO1 subcellular localization. The ratio of the average intensities of the GFP signals in the nucleus and cytoplasm was determined as described in Materials and Methods. Each dot in the plot represents one cell. Cells were transfected with wild-type (WT) GFP-PAX3-FOXO1 (GFP-PF), or GFP-PF S430A mutant (S430A) for 24 h, followed by treatment with either DMSO or 1 µM fascaplysin (FAS) for 30 min. Cells were then fixed and stained with DAPI. B) Representative images.
Mentions: The subcellular localization of a transcription factor is critical for its transcriptional activity and is well-studied for FOXO1 but not for PAX3-FOXO1 [14], [16]–[18]. Whereas activation of AKT signaling retains FOXO1 in the cytoplasm, PAX3-FOXO1 is insensitive to AKT activity and is predominantly nuclear-localized, suggesting that the regulation of subcellular localization by phosphorylation is different between FOXO1 and PAX3-FOXO1 [18]. Recently, Cdks was reported to phosphorylate Ser249 of FOXO1 and resulted in its cytoplasmic localization [16], [17]. Since Cdk4 phosphorylates Ser430 in PAX3-FOXO1 (corresponds to Ser249 in FOXO1), we tested whether inhibition of Cdk4 by fascaplysin alters the subcellular localization of PAX3-FOXO1. PAX3-FOXO1 is known to predominantly nuclear-localized [18]. When a cell analysis tool (Cellomics vHCS Scan NucTrans Bioapplication) [14] was used to analyze the images and determine the ratio of the average intensities of the GFP signals in the nucleus and cytoplasm, wild-type GFP-PAX3-FOXO1 is more nuclear localized than GFP-PAX3-FOXO1 S430A, and treatment with fascaplysin for 30 min significantly enhanced the cytoplasmic localization of wild-type GFP-PAX3-FOXO1 but not the phosphorylation-resistant mutant S430A (Fig. 5A). Representative images were shown in Fig. 5B to illustrate the fascaplysin-mediated cytoplasmic localization of PAX3-FOXO1 in a portion of the cells. The effect of fascaplysin in promoting cytoplasmic localization of PAX3-FOXO1 was also observed in NIH3T3 cells (Fig. S1). These findings suggest that Cdk4 enhances the transcriptional activity of PAX3-FOXO1 by phosphorylating and possibly either inhibiting the cytoplasmic translocation or promoting the nuclear translocation of PAX3-FOXO1 in a fascaplysin-sensitive manner.

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