Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblasts

Abstract

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development. Previously, others and we have shown that the stability of Pax3 is regulated on a post-translational level. Evidence in the literature and from our laboratory suggests that phosphorylation, a common form of regulation, may play a role. However, at present, the sites of Pax3 phosphorylation are not known. We demonstrate here the first evidence that Pax3 exists as a phosphoprotein in proliferating mouse primary myoblasts. Using an in vitro kinase assay, deletion, and point mutant analysis, we conclusively identify Ser205 as a site of phosphorylation. The phosphorylation of Ser205 on endogenously expressed Pax3 was confirmed in vivo using antibodies specific for phosphorylation at Ser205. Finally, we demonstrate for the first time that the phosphorylation status of endogenous Pax3 changes rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating protein-protein interactions, and the fact that phosphorylation is lost upon induction of differentiation, allow for speculation on the biological relevance of phosphorylation.

BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

“…The phosphorylation sites at serine residues 201, 205, 209 and 222 attracted our particular attention, since all four sites are localized within the octapeptide domain of PAX3 known to mediate several protein-protein interactions (Miller et al, 2008). To analyze whether any of these phosphorylation sites are instrumental for the ability of PAX3 to stimulate muscle cell migration, we replaced the respective serine residues by alanine.…”

“…The PAX3 protein is posttranslationally modified by acetylation (Ichi et al, 2011), ubiquitination (Boutet et al, 2007), and phosphorylation (Amstutz et al, 2008; Iyengar et al, 2012; Miller et al, 2008). The functional relevance of PAX3 phosphorylation is largely unknown although the presence of phosphorylation sites at Ser201, Ser205 and Ser209 near the octapeptide domain, a region crucial for mediating protein-protein interactions and DNA binding suggests important regulatory functions.…”

“…Additional PAX3 and anti-LBX1 antibodies were purchased from Abcam (Cambridge, Massachusetts). Anti-phospho-PAX3 (Serine 205) was a kind gift of Dr. A. D. Hollenbach (Miller et al, 2008). The antibody against CD31 and recombinant Scatter Factor/Hepatocyte Growth factor (HGF) was obtained from R and D Systems (Minneapolis, Minnesota).…”

“…For pull down assays cell lysates from wild type Braf or mutant Braf transfected cultures were incubated overnight with PAX3-GST fusion proteins coupled to glutathione-Sepharose beads and processed by standard methods for Western Blot analysis. Whole-mount in situ hybridization with digoxigenin-labeled antisense cRNA probes was performed using Lbx1, Myf5, Myod, Myogenin cRNA probes as described previously (Miller et al, 2008; Schäfer and Braun, 1999). The in vitro BRAF kinase assay was performed with purified GST-proteins according to manufacturer´s instructions (Millipore, Billerica, Massachusetts).…”

PAX3 across the spectrum: from melanoblast to melanoma

“…A Ser205 phosphorylation site has recently been identified; but to date phosphorylated Pax3 has only been seen in proliferating mouse primary myoblasts (Miller and Hollenbach, 2007;Miller et al, 2008). Pax3 protein stability is also regulated by ubiquitination and proteasomal degradation during adult muscle stem cell activation (Boutet et al, 2007).…”