To explore the mutations’ functional impact directly, we evaluated MAF phosphorylation status. The disease-causing p.Ser54Leu, p.Thr58Ala, p.Thr58Ile, p.Pro59Leu, p.Pro59His, and p.Pro69Arg (FLS-like disorder), and p.Arg288Pro (c.863G>C) changes, the latter considered as representative of lesions associated with isolated cataract,13 were introduced into the MAF cDNA cloned in pCS2+ vector using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). Consistent with previous reports,26 Western blot analysis of transiently transfected COS1 cell lysates documented two MAF states: a slower-migrating, fully phosphorylated form, and a faster-migrating, unphosphorylated form. In cells expressing wild-type MAF, the phosphorylated protein (upper band) predominated, while unphosphorylated MAF (lower band) was barely detectable (Figure 3A, upper panel). Similarly, the mutant carrying the p.Arg288Pro substitution in the DNA binding domain, previously associated with isolated lens and eye defects, was efficiently phosphorylated. This was in sharp contrast to all MAF mutants identified in the present study, which accumulated in cells as unphosphorylated proteins. GSK3-mediated phosphorylation represents a regulatory mechanism promoting MAFA ubiquitination and degradation.24,25 Based on the high conservation of the GSK3 recognition motif and MAF being a GSK3 substrate, we hypothesized that the amino acid changes in our affected subjects might mediate inefficient protein clearance. On Western blot analyses, we noted increased protein levels (Figure 3A, upper panel) and decreased ubiquitination (Figure 3A, middle panel) for the disease-causing MAF mutants when compared to the wild-type protein (Figures 3A and 3B). Treatment with cycloheximide (CHX), a protein synthesis inhibitor, showed that the half-life of wild-type MAF was much shorter than that of the mutants (Figure 3B). Indeed, a complete disappearance of the protein was observed upon 4 hr CHX treatment, while the steady-state level of the mutants was largely unchanged. Consistently, treatment with MG132, which specifically inhibit proteasomal function, stabilized the protein level of wild-type MAF, while it did not have any significant effects on mutants (Figure 3B). Taken together, these results showed that mutations prevented MAF degradation and enhanced their stability. Of note, a partial phosphorylation was apparent for the p.Pro69Arg MAF mutant, which was associated with increased degradation via proteasome, even though less efficiently compared to wild-type MAF. This finding suggests a milder perturbing role of the proline-to-arginine substitution on GSK3-mediated phosphorylation at Ser66 compared to the other disease-causing amino acid changes, possibly due to the peculiar effect of the introduced arginine residue, which was documented to primarily affect MAF interaction with the GSK priming site. Confocal microscopy of transfected COS1 cells confirmed the nuclear localization of all tested mutants and their higher abundance within cells (Figure 3C). Moreover, treatment with CSK buffer prior fixation indicated that the syndrome-causing mutants retained efficient interaction with chromatin suggesting that they bind to DNA, in contrast to the DNA binding-impaired cataract-associated p.Arg288Pro mutant (Figure 3C and Table S7). Transactivation assays using luciferase as reporter under control of the IL4 promoter documented that COS1 cells transiently expressing the cataract-causing mutant allele had barely detectable reporter induction (Figure 3D). In contrast, cells expressing the p.Ser54Leu, p.Thr58Ala, p.Thr58Ile, p.Pro59Leu, p.Pro59His, or p.Pro69Arg MAF coding alleles showed efficient induction of luciferase levels, though not reaching the levels of the wild-type protein, suggesting that, despite their stabilization and much higher levels, these mutants are less active, at least under these specific conditions.