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

Fred B Berry,Farideh Mirzayans,Michael A Walter

doi:10.1074/jbc.m513629200

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 (t 1/2< 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.

Highlights

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)
We focused on ERK1/2 as a potential FOXC1 kinase, because it is well established that ERK1/2 activity can regulate growth and differentiation events and because many of the developmental anomalies observed from FOXC1 mutations are suggested to result from impaired differentiation processes [6, 10, 17]
When the mutated FOXC1 proteins were assayed for activation of a FOXC1-responsive luciferase reporter, we found that T68A, S241A, and S259A had little effect on FOXC1 transactivation (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—Site-directed mutagenesis of FOXC1 was performed using the QuikChange mutagenesis kit (Stratagene) as described previously [14]. For the detection of endogenous FOXC1 proteins, 40 ␮g of HeLa nuclear extracts were fractionated by SDS-PAGE and proteins transferred to a nitrocellulose membrane. FOXC1 Half-life Determination—HeLa cells were transfected with Xpress-tagged FOXC1 expression vectors. Protein concentrations were determined by Bradford assays, and protein expression was detected by SDS-PAGE and immunoblotting as described above. HeLa cells were harvested in lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 50 mM NaF, 5 mM ␤-glycerophosphate, 2 mM EDTA, 1 mM Na3VO4, and 1% Triton X-100). One hundred micrograms of HeLa cell extract were incubated with 2.5 ␮g of recombinant FOXC1 protein, 10 ␮Ci of [␥-32P]ATP, and 0.3 mM ATP in 1ϫ kinase buffer (25 mM Tris, pH 7.5, 5 mM ␤-glycerophosphate, 2 mM dithiothreitol, 1 mM Na3VO4, 50 mM NaF, and 10 mM MgCl2) for 30 min at 30 °C. Bound proteins were removed by boiling in 2ϫ SDS sample buffer and analyzed by SDS-PAGE and immunoblotting

RESULTS

Activation of MAPK signaling by EGF stimulation led to a robust

DISCUSSION