Regulation of FOXC1 Stability and Transcriptional Activity by an Epidermal Growth Factor-activated Mitogen-activated Protein Kinase Signaling Cascade

Abstract

Mutations in the FOXC1 transcription factor gene result in Axenfeld Rieger malformations, a disorder that affects the anterior segment of the eye, the teeth, and craniofacial structures. Individuals with this disorder possess an elevated risk for developing glaucoma. Previous work in our laboratory has indicated that FOXC1 transcriptional activity may be regulated by phosphorylation. We report here that FOXC1 is a short-lived protein (t1 ⁄ 2 <30 min), and serine 272 is a critical residue in maintaining proper stability of FOXC1. Furthermore, we have demonstrated that activation of the ERK1/2 mitogen-activated protein kinase through epidermal growth factor stimulation is required for maximal FOXC1 transcriptional activation and stability. Finally, we have demonstrated that FOXC1 is targeted to the ubiquitin 26 S proteasomal degradation pathway and that amino acid residues 367-553, which include the C-terminal transactivation domain of FOXC1, are essential for ubiquitin incorporation and proteolysis. These results indicate that FOXC1 protein levels and activity are tightly regulated by post-translational modifications.Protein phosphorylation provides a rapid means of altering the function of a protein in response to changes in the cellular environment. In the case of transcription factors, phosphorylation can alter the activity of these proteins through regulation of their nuclear localization (1, 2), modulation of their protein-protein (2, 3) and/or protein-DNA (4) interactions, and by controlling their stability (5). Therefore, the phosphorylation state of a transcription factor can dictate activity and act as a molecular switch from an inactive to an active form or vice versa.The Forkhead Box transcription factor FOXC1 is an integral component for the proper formation and function of structures derived from mesoderm and neural crest lineages (6 -10). In humans, mutations in the FOXC1 gene cause Axenfeld Rieger malformations, a disorder that is characterized by a spectrum of dysgeneses of the anterior segment of the eye (7,8). The most serious consequence of this disorder is a heightened propensity to develop glaucoma, with 50% of affected individuals developing this progressively blinding disease. In addition to the ocular findings, patients can present with a variable array of nonocular findings, including dental, craniofacial, umbilical, and cardiac anomalies. In mice, targeted deletion of both Foxc1 alleles results in neonatal lethality, hydrocephalus, ocular and skeletal abnormalities (6, 10 -12), establishing Foxc1 as an essential developmental transcription factor.Previous work in our laboratory has demonstrated that the FOXC1 transcription factor is a phosphoprotein and that amino acid residues 215-366 contribute to a phosphorylation-dependent mobility shift in the FOXC1 protein as detected by SDS-PAGE (13). Removal of this region prevents this mobility shift and results in a transcriptionally hyperactive FOXC1 molecule. We sought to identify the residues that are phosphorylated in FOXC1 and the kinases ...

Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

“…Furthermore, these residues are also conserved in the related transcription factor FOXC1, suggesting that both TFs may be similarly regulated by phosphorylation. In accordance with this possibility, half (4 of 8) of the conserved phosphorylation sites have already been experimentally confirmed in FOXC1 (29,31,32). These findings once again underscore the importance of phosphorylation in the regulation of FOXC2-and FOXC1-mediated transcription and further imply that the regulation involves complex molecular interactions, rather than simple addition of cGOF , and FOXC2 ecGOF embryos.…”

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“…FoxC1 expression is undetectable in adult tissues, but FoxC1 expression is activated by Wnt and EGF/ERK signaling pathway during embryogenesis 10,11 . Recent studies reported that FoxC1 is a master regulator of cancer invasion and metastasis.…”

Cortical dysplasia and skull defects in mice with a Foxc1 allele reveal the role of meningeal differentiation in regulating cortical development

“…It is likely that the dramatic overexpression of Foxc1 in our transfection assays is the reason for the apparently normal transactivation by mutant protein in our transcriptional assays. Stability of Foxc1 has been linked to the C-terminal phosphorylation of the protein (27), and evaluation of the phosphorylation state of F107L-Foxc1 protein revealed reduced phosphorylation compared with wildtype controls (Fig. 3e).…”