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F.B. Berry, F. Mirzayans, M.A. Walter; Regulation of FOXC1 Levels by Post–Translational Modifications: Implications for Glaucoma Pathologies Through Alterations in FOXC1 Stability . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3139.
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Purpose: To elucidate the role of FOXC1 phosphorylation on function. Methods: Mutations in the FOXC1 transcription factor gene result in Axenfeld–Rieger syndrome, where affected individuals present with a spectrum of anterior segment dysgeneses that result in a heightened propensity to develop glaucoma. OUrPrevious work has demonstrated an inverse correlation between FOXC1 activity and its phosphorylation state. To delineate a role for FOXC1 phosphorylation on its activity, four putative p42/44 MAP kinase phosphorylation sites (Thr68 Ser241 Ser259 and Ser272) were individually mutated to alanines and these substituted FOXC1 molecules were assayed for changes in activity. Results: Three out of four serine substitutions had a minimal effect on FOXC1 activity. However, the S272A substitution resulted in a severe reduction in FOXC1 steady–state protein levels and led to an impaired activation of a FOXC1 reporter gene. This S272A substitution led to a decrease in FOXC1 phosphorylation both in vitro and in vivo. The addition of the 26 proteasome inhibitor MG132 stabilized S272A levels, indicating that this FOXC1 S272A protein was degraded by the 26S proteasome pathway and furthermore suggests that phosphorylation of Ser272 was crucial for FOXC1 stability. Although the half–life of wild type FOXC1 was short lived (T1/2<60 min), the S272A substitution reduced the half–life to 30 minutes. Co–immunoprecipitation experiments indicated that FOXC1 was polyubiquitinated. To link the FOXC1 stability to specific phosphorylation events, we tested a battery of protein kinase inhibitors on the regulation of FOXC1 steady state protein levels. Treatment with the MEKK inhibitor PD98059 and the glycogen synthase 3 inhibitor LiCl greatly reduced full–length FOXC1 steady state levels. Further examination of the role of growth factors and MAP kinase signaling on FOXC1 protein levels revealed that FOXC1 stabilization may be regulated by cell cycle progression. Conclusions: These results indicate that FOXC1 protein levels are tightly regulated by post–translational modifications. Since genetic alterations in FOXC1 levels are pathogenic, these physiological mechanisms that modulate FOXC1 protein levels may be fundamental in glaucoma disease progression.
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