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

Center for Cell Signaling, University of Virginia School of Medicine, P.O. Box 800577, Charlottesville, VA 22908-0577, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2001; 98(12):6650-5. DOI: 10.1073/pnas.111154698
Source: PubMed


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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    • "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 [4], [21]. On the other hand, alpha4 is postulated to facilitate a MID1-alpha4-PP2Ac complex that promoted the ubiquitination of PP2Ac by MID1 [17], [24], [37]. Our ubiquitination data suggest that MID1 could interact with and target PP2Ac in the absence of alpha4 (Figure 2A). "
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    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.
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    • "The amount of Tap42 associated PP2A/C was predicted to be about 2% of the whole PP2A/C pool. However, there were only 28% shared sequence identity between α4 and Tap42, thus the amount of α4 associated PP2A/C was still unknown to our knowledge 12. It is notable that the complex of α4 and PP2A/C is independent of the A and B subunits, as the binding sites between α4 and PP2A/C partially overlap with that of the PP2A A and C subunits 13. "
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    ABSTRACT: Protein phosphatase 2A (PP2A) is a major protein phosphatase with important cell functions. Known and utilized as a potent inhibitor of PP2A, microcystin-LR (MCLR) targets PP2A as a core element that affects numerous cellular mechanisms. But apart from direct inhibition, the exact effect of MCLR on PP2A in cell is largely unknown, specifically with regard to cellular response and autoregulation. Here, we show that a low concentration of MCLR stimulates, rather than inhibits, PP2A activity in HEK293 cells. Immunoprecipitation and immunofluorescence assays reveal that the catalytic subunit and a regulatory subunit of PP2A, termed α4, dissociate from inactive complex upon MCLR exposure, suggesting that the released catalytic subunit regains activity and thereby compensates the activity loss. At high concentrations of MCLR, PP2A activity decreases along with dissociation of the core enzyme and altered post-translational modification of its catalytic subunit. In addition, the dissociation of α4 and PP2A may contribute to destabilization of HEK293 cells cytoskeleton architecture, detachment to extracellular matrix and further anoikis. Our data provide a novel PP2A upregulation mechanism and challenge the recognition of MCLR only as a PP2A inhibitor in cells.
    Preview · Article · Jun 2011 · International journal of biological sciences
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    • "These observations provide additional support for the critical role of primary cilia in Gli3 regulation and suggest that GLI3 is involved in the pathogenesis of OFDS. GLI3 is also linked to Opitz syndrome (OS), a genetically heterogeneous disorder characterized by defects of the ventral midline (Liu et al. 2001). "
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    ABSTRACT: Gli zinc-finger proteins are transcription factors involved in the intracellular signal transduction controlled by the Hedgehog family of secreted molecules. They are frequently mutated in human congenital malformations, and their abnormal regulation leads to tumorigenesis. Genetic studies in several model systems indicate that their activity is tightly regulated by Hedgehog signaling through various posttranslational modifications, including phosphorylation, ubiquitin-mediated degradation, and proteolytic processing, as well as through nucleocytoplasmic shuttling. In vertebrate cells, primary cilia are required for the sensing of Hedgehog pathway activity and involved in the processing and activation of Gli proteins. Two evolutionarily conserved Hedgehog pathway components, Suppressor of fused and Kif7, are core intracellular regulators of mammalian Gli proteins. Recent studies revealed that Gli proteins are also regulated transcriptionally and posttranslationally through noncanonical mechanisms independent of Hedgehog signaling. In this review, we describe the regulation of Gli proteins during development and discuss possible mechanisms for their abnormal activation during tumorigenesis.
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