Potential Kinases Regulating the Transcription Factor Twist1

Twist proteins are basic helix-loop-helix (bHLH) transcription factors that play essential roles during development. Mutations in TWIST1 cause Saethre-Chotzen syndrome characterized by craniosynostosis, a narrow or cleft palate, and limb defects. Furthermore, Twist proteins transiently inhibit osteoblast differentiation during skeletogenesis through their interaction with Runx2. Motif scans for possible kinases that may phosphorylate Twist1 identified Akt and PKA as potential candidates. Mutations in the Twist1 Akt and PKA motifs have been identified in patients with Saethre-Chotzen syndrome suggesting that phosphorylation may affect Twist1 function.

Objective: The aim of this study was to determine if Twist1 is phosphorylated on specific serine and threonine residues which are part of the motif RERQRTQS and are conserved in most Twist family members.

Methods: To study Twist1 phosphorylation by Akt in cells, we co-transfected epitope tagged Twist1 with constitutively active (myristoylated) Akt (caAkt) or PKA (caPKA), and examined Twist1 phosphorylation using an antibody directed against the phosphorylated form of the Akt or PKA consensus motif respectively.

Results: Twist1 is strongly phosphorylated by caAkt and mutating both threonine and serine residues in the motif described above to alanines abolished this phosphorylation while mutating only the serine to alanine decreased it by a significant amount suggesting that Akt does phosphorylate Twist1 at these residues. Contrary to these findings, mutating both residues to alanines reduced but did not abolish phosphorylation by caPKA, suggesting that additional Twist1 sites are potentially phosphorylated by PKA.

Conclusion: Twist1 is phosphorylated by both Akt and PKA. This observation will be extended in future studies to include experiments aimed at the identification of all Twist1 sites essential for PKA phosphorylation and ultimately, the functional significance of Twist1 phosphorylation by Akt and PKA. Funding: K22DE016614 (TD)