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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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Cdk4 phosphorylates PAX3-FOXO1 at Ser430in vitro and in vivo.A) In vitro kinase assay using GFP or GFP-PAX3-FOXO1 (GFP-PF; wild-type or S430A mutant) pulled down from transfected Rh30 cells. Top panel: 32P Phospho-image. Bottom panel: protein substrates revealed by Coomassie blue staining. B) Schematic diagram of three GST-PF fusion proteins (showing only the PAX3-FOXO1 portion). PF, full length PAX3-FOXO1; 1–423, the N-terminal 423 residues of PAX3-FOXO1; 1–459, N-terminal 459 residues of PAX3-FOXO1; S430, Serine 430; PAX3, the PAX3 portion of PAX3-FOXO1; FOXO1, the FOXO1 portion of PAX3-FOXO1. C) In vitro kinase assay using bacterially expressed PAX3-FOXO1. From top to bottom panel: 32P Phospho-image (32P); protein substrates revealed by Coomassie blue staining (Coomassie); Western blot using phosphor-ser CDKs substrate antibody (IB: Phospho-Ser); and Western blot using anti-GST monoclonal antibody (IB: GST). D) Detection of phosphorylation using GFP or GFP-PF (wild-type or S430A mutant) proteins pulled down from transfected Rh30 cells. Top panel: Western blot using anti-GFP monoclonal antibody; Bottom panel: Western blot using phosphor-ser CDKs substrate antibody. Rb, positive control for Cdk4 activity; M, protein marker indicating molecular weight (kD).
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pone-0058193-g002: Cdk4 phosphorylates PAX3-FOXO1 at Ser430in vitro and in vivo.A) In vitro kinase assay using GFP or GFP-PAX3-FOXO1 (GFP-PF; wild-type or S430A mutant) pulled down from transfected Rh30 cells. Top panel: 32P Phospho-image. Bottom panel: protein substrates revealed by Coomassie blue staining. B) Schematic diagram of three GST-PF fusion proteins (showing only the PAX3-FOXO1 portion). PF, full length PAX3-FOXO1; 1–423, the N-terminal 423 residues of PAX3-FOXO1; 1–459, N-terminal 459 residues of PAX3-FOXO1; S430, Serine 430; PAX3, the PAX3 portion of PAX3-FOXO1; FOXO1, the FOXO1 portion of PAX3-FOXO1. C) In vitro kinase assay using bacterially expressed PAX3-FOXO1. From top to bottom panel: 32P Phospho-image (32P); protein substrates revealed by Coomassie blue staining (Coomassie); Western blot using phosphor-ser CDKs substrate antibody (IB: Phospho-Ser); and Western blot using anti-GST monoclonal antibody (IB: GST). D) Detection of phosphorylation using GFP or GFP-PF (wild-type or S430A mutant) proteins pulled down from transfected Rh30 cells. Top panel: Western blot using anti-GFP monoclonal antibody; Bottom panel: Western blot using phosphor-ser CDKs substrate antibody. Rb, positive control for Cdk4 activity; M, protein marker indicating molecular weight (kD).

Mentions: Phosphorylation of PAX3-FOXO1 by protein kinases has been shown to regulate its transcriptional activity [1], [8]. We hypothesized that the activities of PAX3-FOXO1 would be regulated by its functional interactions with Cdk4 and sought to determine whether Cdk4 could phosphorylate the PAX3-FOXO1 protein. In an in vitro kinase assay, we found that purified Cdk4/cyclin D1 phosphorylates GFP-PAX3-FOXO1, but not GFP, both expressed and purified from Rh30 cells (Fig. 2A). Rb protein, a known substrate of Cdk4, was used as a positive control for Cdk4 activity. These findings indicate that Cdk4 directly phosphorylates PAX3-FOXO1 protein in vitro.


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)

Cdk4 phosphorylates PAX3-FOXO1 at Ser430in vitro and in vivo.A) In vitro kinase assay using GFP or GFP-PAX3-FOXO1 (GFP-PF; wild-type or S430A mutant) pulled down from transfected Rh30 cells. Top panel: 32P Phospho-image. Bottom panel: protein substrates revealed by Coomassie blue staining. B) Schematic diagram of three GST-PF fusion proteins (showing only the PAX3-FOXO1 portion). PF, full length PAX3-FOXO1; 1–423, the N-terminal 423 residues of PAX3-FOXO1; 1–459, N-terminal 459 residues of PAX3-FOXO1; S430, Serine 430; PAX3, the PAX3 portion of PAX3-FOXO1; FOXO1, the FOXO1 portion of PAX3-FOXO1. C) In vitro kinase assay using bacterially expressed PAX3-FOXO1. From top to bottom panel: 32P Phospho-image (32P); protein substrates revealed by Coomassie blue staining (Coomassie); Western blot using phosphor-ser CDKs substrate antibody (IB: Phospho-Ser); and Western blot using anti-GST monoclonal antibody (IB: GST). D) Detection of phosphorylation using GFP or GFP-PF (wild-type or S430A mutant) proteins pulled down from transfected Rh30 cells. Top panel: Western blot using anti-GFP monoclonal antibody; Bottom panel: Western blot using phosphor-ser CDKs substrate antibody. Rb, positive control for Cdk4 activity; M, protein marker indicating molecular weight (kD).
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pone-0058193-g002: Cdk4 phosphorylates PAX3-FOXO1 at Ser430in vitro and in vivo.A) In vitro kinase assay using GFP or GFP-PAX3-FOXO1 (GFP-PF; wild-type or S430A mutant) pulled down from transfected Rh30 cells. Top panel: 32P Phospho-image. Bottom panel: protein substrates revealed by Coomassie blue staining. B) Schematic diagram of three GST-PF fusion proteins (showing only the PAX3-FOXO1 portion). PF, full length PAX3-FOXO1; 1–423, the N-terminal 423 residues of PAX3-FOXO1; 1–459, N-terminal 459 residues of PAX3-FOXO1; S430, Serine 430; PAX3, the PAX3 portion of PAX3-FOXO1; FOXO1, the FOXO1 portion of PAX3-FOXO1. C) In vitro kinase assay using bacterially expressed PAX3-FOXO1. From top to bottom panel: 32P Phospho-image (32P); protein substrates revealed by Coomassie blue staining (Coomassie); Western blot using phosphor-ser CDKs substrate antibody (IB: Phospho-Ser); and Western blot using anti-GST monoclonal antibody (IB: GST). D) Detection of phosphorylation using GFP or GFP-PF (wild-type or S430A mutant) proteins pulled down from transfected Rh30 cells. Top panel: Western blot using anti-GFP monoclonal antibody; Bottom panel: Western blot using phosphor-ser CDKs substrate antibody. Rb, positive control for Cdk4 activity; M, protein marker indicating molecular weight (kD).
Mentions: Phosphorylation of PAX3-FOXO1 by protein kinases has been shown to regulate its transcriptional activity [1], [8]. We hypothesized that the activities of PAX3-FOXO1 would be regulated by its functional interactions with Cdk4 and sought to determine whether Cdk4 could phosphorylate the PAX3-FOXO1 protein. In an in vitro kinase assay, we found that purified Cdk4/cyclin D1 phosphorylates GFP-PAX3-FOXO1, but not GFP, both expressed and purified from Rh30 cells (Fig. 2A). Rb protein, a known substrate of Cdk4, was used as a positive control for Cdk4 activity. These findings indicate that Cdk4 directly phosphorylates PAX3-FOXO1 protein in vitro.

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