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MITF regulation – more hints from Wnt

Melanocytes and their precursor melanoblasts depend on the Microphthalmiaassociated transcription factor (MITF) for most decisions, including specification, differentiation, function, survival, and proliferation. MITF also plays an important role in melanoma where it is essential for maintaining tumor cell survival. As MITF plays multiple roles, it is not surprising that MITF activity and stability are regulated by signaling pathways, including the MAP kinase pathway which phosphorylates Ser73 and Ser409 of MITF (Wu et al., 2000), p38 which phosphorylates Ser307 (Mansky et al., 2002) and GSK3-beta which has been proposed to phosphorylate Ser298 (Takeda et al., 2000). As MITF is a key player in melanoma and a key mediator of resistance against BRAF inhibitors, finding additional signaling pathways which affect MITF is major news. Ploper et al. (2015) became interested in the MITF family of proteins when they discovered that three serine residues (Ser397, Ser401, and Ser405) located at the carboxyl end of these proteins are among the best conserved GSK3 phosphorylation motifs in the human proteome. They propose that the MITF Ser409 site, previously shown to be phosphorylated by p90Rsk, acts as a priming site for GSK3. Phosphorylation by GSK3 has been shown to target various proteins for degradation, including beta-catenin. During Wnt signaling, the endocytosis of a so-called beta-catenin destruction complex (containing GSK3 and other proteins) and subsequent sequestration in multivesicular bodies results in decreased levels of cytosolic GSK3 and increased stability of GSK3 target proteins (Dobrowolski and De Robertis, 2012). Ploper et al. investigated whether Wnt signaling has an effect on MITF stability. First, they showed that Wnt signaling increases stability of MITF in melanoma cells, leading to effects on MITF target gene expression. Effects on gene expression were also observed in Xenopus embryos injected with MITF and Wnt8 mRNA. Second, they raised and validated an antibody against doubly phosphorylated (Ser401, Ser405) MITF and used it to show that MITF phosphorylation at Ser401 and/or Ser405 does not take place in the presence of the GSK3 inhibitor BIO. Thus, they provide convincing evidence that GSK3 is involved in signaling to MITF and that Wnt signaling increases MITF stability. Furthermore, they show that the GSK3 phosphorylation sites are important, as injection into Xenopus embryos of mutant MITF mRNA where all three phosphorylation sites (Ser397, Ser401, and Ser405) are mutated to alanine resulted in increased protein levels compared with injection of wild-type MITF. However, whereas GSK3 inhibition by BIO treatment indicates increased total MITF levels in HEK293T cells, it apparently leads to decreased MITF levels in their C32 melanoma model. This suggests that the increased MITF stability by Wnt observed in the melanoma cells may not be directly via decreased GSK3 phosphorylation. The relationship between MITF and Wnt thus seems to be complex in melanoma cells. The data raise various additional questions such as: What is the role of the priming site Ser409 and MAP kinase signaling? Do phosphorylation events at Ser409 and Ser73 independently affect protein stability or is GSK3 always involved? Is the GSK3 pathway important in melanoma? The role of Ser298 also needs to be addressed as this site has been questioned as a bona fide GSK3-site (Grill et al., 2013). To determine the effects of MITF on Wnt signaling, the authors made use of a Xenopus animal cap system and detected increased expression from the SuperTopFlash Wnt reporter in the simultaneous presence of MITF and Wnt8. Interestingly, they find this to be dependent on the presence of components of the ESCRT (endosomal sorting complexes required for transport) machinery. Using a Wnt/beta-catenin activated reporter in inducible MITF melanoma cells, they show that MITF also enhances the effects of Wnt signaling in these cells, again in an ESCRT-dependent manner. Furthermore, they detect components of the beta-catenin destruction complex in the MVBs upon simultaneous Wnt3a treatment and MITF induction in melanoma cells. This suggests that MITF, in the presence of Wnt, leads to the translocation of the destruction complex proteins to the MVBs, thereby potentiating Wnt signaling. Whether this is due to increased production of MVBs upon MITF stimulation or if MITF also affects transfer mechanisms, remains to be established. TFEB, a close relative of MITF, has been shown to be an important regulator of lysosomal and autophagy genes. As MITF can bind the same DNA sequence as TFEB, Ploper et al. investigated whether MITF can also regulate the expression of lysosomal genes. Analysing the expression of 89 lysosomal genes in 51 melanoma cell lines they found that the cells with high MITF expression (mostly due to amplification of MITF) also had high levels of many, but not all, lysosomal genes. They then used Gene Set Enrichment Analysis to show significant correlation between MITF and lysosomal genes in an independent set of 83 melanoma cell lines; no such correlation was seen with TFEB. Next, they observed increased expression of lysosomal genes in melanoma cells upon MITF induction as determined by qPCR analysis. The authors also detected expansion of vesicular structures, increased lysosomal protein expression and Lysotracker staining, suggesting expansion of acidic organelles. Surprisingly, however, treatment with selfquenched BSA-DQ reagent which is endocytosed and digested into smaller fluorescent fragments in lysosomes, revealed decreased lysosomal activity upon MITF induction. The authors speculate that MITF regulates lysosomal genes in melanoma cells but Coverage on: Ploper et al. (2015) MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells. PNAS. 112, E420-E429.