Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation

Abstract

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development and is key in the development of the childhood solid muscle tumor alveolar rhabdomyosarcoma (ARMS). ARMS is primarily characterized by a t(2;13)(q35;q14) chromosomal translocation, which fuses the 5′-coding sequences of Pax3 with the 3′-coding sequence of the forkhead transcription factor FOXO1 generating the oncogenic fusion protein Pax3-FOXO1. We previously demonstrated that Pax3 and Pax3-FOXO1 are phosphorylated by the protein kinase CK2 at serine 205 in proliferating primary myoblasts and that this phosphorylation event is rapidly lost from Pax3, but not Pax3-FOXO1 upon the induction of differentiation. However, reports suggested that additional sites of phosphorylation might be present on Pax3. In this report we use in vitro and in vivo analyses to identify serines 201 and 209 as additional sites of phosphorylation and along with serine 205 are the only sites of phosphorylation on Pax3. We provide solid evidence supporting the role of the protein kinase GSK3β as phosphorylating Pax3 at serine 201. Using phospho-specific antibodies we demonstrate a changing pattern of phosphorylation at serines 201, 205, and 209 throughout early myogenic differentiation and that this pattern of phosphorylation is different for Pax3-FOXO1 in primary myoblasts and in several ARMS cell lines. Taken together, our results allow us to propose a molecular model to describe the changing pattern of phosphorylation for Pax3 and the altered phosphorylation for Pax3-FOXO1 during early myogenic differentiation.

Caspase 3 cleavage of Pax7 inhibits self-renewal of satellite cells

“…This mechanism may also have implications in the regulation of the Pax7-FOXO1 fusion protein, the overexpression of which plays a causative role in the tumorigenicity of the skeletal muscle cancer, alveolar rhabdomyosarcoma (31). Indeed, CK2 phosphorylation of Pax3-FOXO1 enhances its protein stability in transformed cells (32). This study did not address the mechanism of Pax3 degradation; however, we predict a similar mechanism involving caspase 3, as we have demonstrated here for Pax7.…”

AKT and PAX3-FKHR cooperation enforces myogenic differentiation blockade in alveolar rhabdomyosarcoma cell

“…Several studies have reported phosphorylation in the PAX3 domain of PAX3-FKHR in association with its transcriptional activity. 1,[54][55][56][57][58] However, none of the above studies reported phosphorylation-induced we did perceive, during the assessment of PAX3-FKHR transactivation potency in ARMS cells, that PAX3-FKHR appears as a doublet of a faster and slower migrating form. Interestingly, the data showed the shift of PAX3-FKHR to a slower migrating form in ARMS cells grown in DM.…”

GSK-3 Promotes Cell Survival, Growth, and PAX3 Levels in Human Melanoma Cells

“…PAX3 is phosphorylated at multiple sites including Ser205 in mouse primary myoblasts and this phosphorylation is lost upon the progression of the differentiation program (27). A recent report finds Ser201, Ser205 and Ser209 are phosphorylated in PAX3, due to CK2 (formerly casein kinase II) at Ser205 and GSK-3β at Ser 201 with phosphorylation status affecting myogenic differentiation progression (29). The ubiquitously expressed CK2 often provides the priming phosphorylation for GSK-3, however, we found that GSK-3β alone was sufficient to phosphorylate PAX3 at both Ser205 and Ser197/Ser201 in-vitro (33, 48).…”

“…In melanoma, PAX3 activates downstream genes implicated in melanoma proliferation, survival and metastasis such as the receptor MET (20, 22–24). How PAX3 protein is regulated in melanoma is unknown, but post-translational modification of PAX3 in other cell types alters activity and stability thereby regulating differentiation and has been implicated in tumor development (25–29). Although the mechanism of action by PAX3 in melanoma is poorly understood, its regulatory functions towards cell proliferation and differentiation in the melanoblast may be paralleled in melanoma cells by promoting cell division and resistance to apoptosis.…”