Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance

MITF (microphthalmia-associated transcription factor) is a basic-helix-loop-helix-leucine zipper (bHLHZip) factor which regulates expression of tyrosinase and other melanocytic genes via a CATGTG promoter sequence, and is involved in melanocyte differentiation. Mutations of MITF in mice or humans with Waardenburg syndrome type 2 (WS2) often severely disrupt the bHLHZip domain, suggesting the importance of this structure. Here, we show that Ser298, which locates downstream of the bHLHZip and was previously found to be mutated in individuals with WS2, plays an important role in MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, thereby enhancing the binding of MITF to the tyrosinase promoter. The same serine was found to be phosphorylated in vivo, and expression of dominant-negative GSK3 beta selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Moreover, mutation of Ser298, as found in a WS2 family, disabled phosphorylation of MITF by GSK3 beta and impaired MITF function. These findings suggest that the Ser298 is important for MITF function and is phosphorylated probably by GSK3 beta.