Identification and Characterization of Target Proteins of Microtubule-associated Protein Phosphatase 2A (PP2A)
Final Report Abstract
Gain of function of the Sonic Hedgehog (SHH) signalling cascade has been found in a wide range of tumor disorders. Transcription of diverse genes, that are involved in cell-growth and cell proliferation is being regulated by the three SHH effectors GLI1, GLI2 and GLI3. We have demonstrated, that protein phosphatase 2A (PP2A), its regulatory subunit α4 and the mammalian target of rapamycin kinase (mTOR) inhibitor rapamycin regulate the nuclear localization and transcriptional activity of GLI3. We have shown, that both, an increase of PP2A activity and treatment with rapamycin lead to cytosolic retention and a reduction of transcriptional activity of the activator form of this transcription factor towards the cell cycle regulator Cyclin D1. While rapamycin treatment decreases expression of the GLI3 target gene Cyclin D1, inhibition of PP2A results in an increased expression of this central player of cell cycle progression. Furthermore we have found that this regulatory mechanism is mediated by hFu and have shown that this protein is polyubiquitinated in a MID1 dependent manner. We have also shown that the MID1 protein also associates with elongation factor 1α (EF−1α) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients loose the ability to interact with EF-1α. The composition of the MID1 protein complex was determined by several independent methods: (i) Yeast two Hybrid screening and (ii) immunofluorescence, (iii) a biochemical approach involving affinity purification of the complex, (iv) co-fractionation in a microtubule assembly assay and (v) immunoprecipitation. Furthermore, the cytoskeleton-bound MID1 / translation factor complex specifically associates with G- and U-rich RNAs and incorporates the MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. mRNAs that are associated to the MID1/PP2A mRNP complex are controlled in their stability and translation efficiency by MID1 and PP2A activity. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS. Increase of PP2A activity and dysfunctionality of the mTOR pathway that is involved in the regulation of cell growth and survival comprehend an attractive model for the pathogenesis of OS and sheds some light into the development of the ventral midline. Moreover, the described mechanism, that allows an exogeneously induced increase of activity of the tumor suppressor PP2A is an interesting target for the development of anti-tumor drugs. Another, also highly interesting application for molecules interfering with the degradation of microtubule-associated PP2A is a putative therapy for Alzheimer’s disease. One of the two important steps in the pathogenesis of this disorder is the development of intracellular tau-fibers that consist of hyperphosphorylated tau protein which leads to cell death of thereby affected neurons. Since tau protein is a target of microtubule-associated PP2A, the possibility to induce this particular pool of the enzyme via the MID1/PP2A mRNP complex promises much for the development of a prophylactic anti-Alzheimer drug.
Publications
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Aranda-Orgillés B., Winter J., Aigner J., Köhler A., Jastrzebska E., Stahl J., Müller E.C., Otto A., Wanker E.E., Schneider R., Schweiger S. The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex. (2008) Hum. Genet 123(2):163-76
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Krauß S., Foerster J., Schneider R., Schweiger S. (2008) PP2A and rapamycin regulate the nuclear localization and activity of the transcription factor GLI3. Cancer Research 68(12): 4658-65
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So J., Suckow V., Kijas Z., Kalscheuer V., Moser B., Baars M., Firth H., Lunt P., Hamel B., Moraine C., Odent S., Schinzel A., van der Smagt J.J., Devriendt K., Passarge E., van der Burgt I., Cox T., Opitz J., Ropers H.H. & Schweiger S. (2005) Mild phenotypic spectrum in a series of Opitz BBB/G syndrome patients with MID1 mutations. Am. J. Med. Genet. 132A:1-7
