Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

Abstract

BackgroundMutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function.Principal FindingsWe have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity.ConclusionsThe present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations.

The E3 Ubiquitin Ligase MID1 Catalyzes Ubiquitination and Cleavage of Fu

“…Transcriptional Activity of GLI3-As we have reported previously, the MID1-␣4-PP2A complex regulates the subcellular localization and transcriptional activity of GLI3 in cancer cell lines (16,17). However, our data also suggested that this regulation indirectly involves other GLI3-interacting proteins.…”

“…As we have shown previously, a protein complex consisting of the microtubule-associated ubiquitin ligase MID1, the ␣4 protein, and protein phosphatase 2A (PP2A) 2 regulates the nuclear localization and transcriptional activity of one of the three transcriptional effector molecules of mammalian SHH signaling, GLI3, in cancer cell lines with autonomously activated SHH signaling (16,17). Via its ubiquitin ligase activity, the RING finger protein MID1 targets PP2A toward ubiquitindependent degradation by the proteasome and thereby decreases PP2A activity at the microtubules (18).…”

The Antagonistic Action of B56-containing Protein Phosphatase 2As and Casein Kinase 2 Controls the Phosphorylation and Gli Turnover Function of Daz Interacting Protein 1

“…In vertebrates, PP2A regulates Hh signaling at the level of Gli as well. It has been observed that PP2As regulate the nuclear localization of Gli3 (24,25). We previously reported that B56⑀ positively regulates transcriptional activation function of Gli during Xenopus embryonic development (26).…”

New insights into genotype–phenotype correlation for GLI3 mutations

“…By contrast, haploinsufficiency resulting from chromosomal rearrangements, but also missense, splicing or truncating mutations elsewhere in the gene cause GCPS by loss of the DNA-binding capacity 11 or activation of nonsense-mediated mRNA decay, 12 or by the formation of an unstable or mislocalized protein. 13,14 In this study, we report on the clinical and molecular data of a French cohort of 76 individuals from 55 families carrying a GLI3 molecular defect. Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation.…”