Liu, J., Prickett, T.D., Elliott, E., Meroni, G. & Brautigan, D.L. Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit 4. Proc. Natl Acad. Sci. USA 98, 6650-6655
Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the alpha4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged alpha4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of alpha4. Localization of Mid1 and alpha4 was influenced by one another in transiently transfected cells. Mid1 could recruit alpha4 onto microtubules, and high levels of alpha4 could displace Mid1 into the cytosol. Metabolic (32)P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length alpha4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein-Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by alpha4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.
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[Show abstract] [Hide abstract] ABSTRACT: The zinc-finger transcription factor GLI3 acts as a primary transducer of Sonic hedgehog (Shh) signaling in a context-dependent combinatorial fashion. GLI3 participates in the patterning and growth of many organs, including the central nervous system (CNS) and limbs. Previously, we reported a subset of human intronic cis-regulators controlling many known aspects of endogenous Gli3 expression in mouse and zebrafish. Here we demonstrate in a transgenic zebrafish assay the potential of two novel tetrapod-teleost conserved non-coding elements (CNEs) docking within GLI3 intronic intervals (intron 3 and 4) to induce reporter gene expression at known sites of endogenous Gli3 transcription in embryonic domains such as the central nervous system (CNS) and limbs. Interestingly, the cell culture based assays reveal harmony with the context dependent dual nature of intra-GLI3 conserved elements. Furthermore, a transgenic zebrafish assay of previously reported limb-specific GLI3 transcriptional enhancers (previously tested in mice and chicken limb buds) induced reporter gene expression in zebrafish blood precursor cells and notochord instead of fin. These results demonstrate that the appendage-specific activity of a subset of GLI3-associated enhancers might be a tetrapod innovation. Taken together with our recent data, these results suggest that during the course of vertebrate evolution Gli3 expression control acquired a complex cis-regulatory landscape for spatiotemporal patterning of CNS and limbs. Comparative data from fish and mice suggest that the functional aspects of a subset of these cis-regulators have diverged significantly between these two lineages.1Comment 0Citations
- "Congenital abnormalities associated with human GLI3 are listed under the term " GLI3 morphopathies " , including Greig cephalopolysyndactyly syndrome (GCPS), non-syndromic polydactyly, Pallister Hall syndrome (PHS), acrocallosal syndrome, preaxial polydactyly type IV (PPD-IV) and postaxial polydactyly type A (PAPA) (Vortkamp et al. 1991; Kang et al. 1997; Radhakrishna et al. 1997 Radhakrishna et al. , 1999 Elson et al. 2002 ). Moreover, GLI3 is also associated with oral-facial-digital syndrome (OFDS) and Opitz syndrome (OS) (Liu et al. 2001; Johnston et al. 2010). A dominant developmental syndrome, GCPS with polydactyly and craniofacial abnormalities, is linked with large deletions, translocations and truncating mutations resulting in functional haploinsufficiency of GLI3 (Shin et al. 1999; Johnston et al. 2010). "
[Show abstract] [Hide abstract] ABSTRACT: MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.0Comments 3Citations
- "Alpha4 was shown to bind and induce a conformational change that inactivated the PP2Ac subunit, and this complex apparently protected the PP2Ac subunit from degradation , . On the other hand, alpha4 is postulated to facilitate a MID1-alpha4-PP2Ac complex that promoted the ubiquitination of PP2Ac by MID1 , , . Our ubiquitination data suggest that MID1 could interact with and target PP2Ac in the absence of alpha4 (Figure 2A). "
[Show abstract] [Hide abstract] ABSTRACT: Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.0Comments 15Citations
- "MID14PP2A complex is localized to microtubules via the interaction of MID1 with microtubule structures and is thought to be involved in the maintenance of microtubule stability (6, 20, 21). Although the precise function of this complex in microtubule stabilization remains unclear, it likely involves PP2A-mediated dephosphorylation of various MAPs. "
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