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Division of Molecular Biology

Department of Medical Biochemistry

Division of Molecular Biology

Research Groups

 

Regulation of Enzyme Biogenesis

Group Leader:         Egon Ogris

Postdoc:                  Stefan Schüchner

Diploma students:   Christian Pongracz
                                David Weismann

Technician:              Ingrid Mudrak (part time)





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Egon Ogris Lab


   |    Lectures   |

 

Protein phosphatase 2A (PP2A)

All life is regulated by reversible protein phosphorylation, controlled by a balance between kinase and phosphatase activity. Protein phosphatase 2A (PP2A) is a prime example for the multi-subunit architecture of protein-serine/threonine phosphatases (PSTPs). PP2A contains an abundant catalytic C subunit, whose catalytic activity is tightly controlled by a novel mechanism, which we have discovered recently (Fellner et al., 2003). Malfunctions of PP2A biogenesis are implicated in diseases as diverse as Alzheimer and cancer. We study the molecular mechanisms of PP2A biogenesis by biochemical, immunological and genetic approaches in yeast and mammalian cells. Moreover, we focus on the identification of PP2A substrates to dissect the multiple PP2A-regulated pathways.

Biogenesis of PP2A

The PP2A catalytic C subunit is amazingly abundant, making up, dependent on the cell type, between 0.3-1% of total cellular protein. Substrate specificity and targeting of the catalytic C subunit is achieved through the interaction of C subunit with different regulatory subunits. Stable complex formation with these regulatory subunits requires methylation of the carboxy-terminal leucine of the C subunit. Until methylation occurs and complexes assemble, newly synthesized C subunit would be a highly active but unspecific phosphatase that constitutes a risk to the cell. Therefore mechanisms must exist that keep in check the catalytic activity of free C subunit until methylation-dependent holoenzyme assembly can occur.
More recently we discovered a novel and conserved mechanism that indicated how the cell avoids the danger of highly active free C subunit, namely by synthesis of C subunit in a low active conformation that requires the functional interaction with PTPA (phosphotyrosyl phosphatase activator) for the switch into an active enzyme (Fellner et al., 2003). However, the molecular mechanism of the activation is still unknown. Other important questions are how C subunit activation by PTPA is connected to the methylation-regulated holoenzyme assembly and how extracellular signals affect PP2A biogenesis.

             LOW ACTIVE CONFORMATION                         HIGH ACTIVE CONFORMATION




PP2A and Alzheimer disease

More recently a correlation between Alzheimer disease pathology and decreased PP2A levels and activity has been found but the cause for the decreased PP2A levels/activity remain unknown (Sontag et al., 2004). We are investigating in collaboration with Estelle Sontag (University of Texas, Dallas) whether dysfunction of C subunit biogenesis might be involved in the pathogenesis of Alzheimer disease.

PP2A and chromosome condensation

We isolated a novel condensin-related complex as a potential PP2A substrate (Yeong et al., 2003). In addition, we obtained evidence for the phosphorylation-dependent association of the condensin subunit hHCP-6 to chromosomes. Currently we are investigating whether hHCP-6 is indeed a substrate of PP2A in vivo and whether and how PP2A regulates chromosome condensation.

                           CONDENSIN I                                             CONDENSIN II



My research team possesses expertise in biochemical (phosphatase enzyme assays, protein expression), immunological (immunoprecipitation, immunoblotting and immunofluorescence) and cell biological techniques (yeast and mammalian cell culture, FACS) and in methods like the yeast two-hybrid system, two-dimensional gel electrophoresis, the generation of hybridomas and the production of poly- and monoclonal antibodies.
We generated the novel anti-Myc Tag Monoclonal 4A6, which shows improved detection properties in IB, IP, IF applications (for details please go to: http://www.upstate.com/browse/Search.asp?query=4a6#Myc).

Publications (1999 - 2005)
  1. Gsandtner, I., C. Charalambous, E. Stefan, E. Ogris, M. Freissmuth, and J. Zezula. (2005). Heterotrimeric G protein independent signaling of a G protein-coupled receptor: Dircet binding of arno/cytohesin-2 to the carboxyl terminus of the A2A-adenosine receptor is necessary for sustained activation of the ERK/MAP-kinase pathway. J Biol Chem, in press

  2. Kurtev, V., R. Margueron, K. Kroboth, E. Ogris, V. Cavailles, and C. Seiser. 2004. Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases. J Biol Chem. 279:24834-43

  3. Sontag, E., C. Hladik, L. Montgomery, A. Luangpirom, I. Mudrak, E. Ogris, and C.L. White, 3rd. 2004. Downregulation of protein phosphatase 2A carboxyl methylation and methyltransferase may contribute to Alzheimer disease pathogenesis. J Neuropathol Exp Neurol. 63:1080-91.

  4. Sontag, E., Luangpirom, A., Hladik, C., Mudrak, I., Ogris, E., Speciale, S., and White, C. L., 3rd (2004). Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology. J Neuropathol Exp Neurol 63, 287-301.

  5. Fellner, T., Lackner, D. H., Hombauer, H., Piribauer, P., Mudrak, I., Zaragoza, K., Juno, C., and Ogris, E. (2003). A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo. Genes Dev 17, 2138-2150.

  6. Fellner, T., Piribauer, P., and Ogris, E. (2003). Altering the holoenzyme composition and substrate specificity of protein phosphatase 2A. Methods Enzymol 366, 187-203.

  7. Yeong, F. M., Hombauer, H., Wendt, K. S., Hirota, T., Mudrak, I., Mechtler, K., Loregger, T., Marchler-Bauer, A., Tanaka, K., Peters, J. M., and Ogris, E. (2003). Identification of a Subunit of a Novel Kleisin-beta/SMC Complex as a Potential Substrate of Protein Phosphatase 2A. Curr Biol 13, 2058-2064.

  8. Nunbhakdi-Craig, V., Machleidt, T., Ogris, E., Bellotto, D., White, C. L., 3rd, and Sontag, E. (2002). Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol 158, 967-78.

  9. Wei, H., Ashby, D. G., Moreno, C. S., Ogris, E., Yeong, F. M., Corbett, A. H., and Pallas, D. C. (2001). Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p. J Biol Chem 276, 1570-7.

  10. Yu, X. X., Du, X., Moreno, C. S., Green, R. E., Ogris, E., Feng, Q., Chou, L., McQuoid, M. J., and Pallas, D. C. (2001). Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Balpha Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen. Mol Biol Cell 12, 185-99.

  11. Ogris, E., Du, X. X., Nelson, K., Mak, E.; and Pallas, D. C. (1999) A Protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A. J Biol Chem 274, 14382-91.

  12. Ogris, E., Mudrak, I., Mak, E., Gibson, D., and Pallas, D. C. (1999). Catalytically inactive protein phosphatase 2A can bind to polyomavirus middle tumor antigen and support complex formation with pp60(c-src). J Virol 73, 7390-8.

Collaborators:
Dr. Estelle Sontag, Department of Pathology, University of Texas Southwestern Medical Center, Dallas;
Dr. David Barford, Section of Structural Biology, Institute of Cancer Research, London

Diploma theses:
Silvia Prieler (1999), Thomas Loregger (1999), Helene Klug (2001), Eduard Stefan (2001, supervised together with Univ. Prof. Dr. M. Freissmuth, Institute of Pharmacology), Patrick Piribauer (2002), Katrin Zaragoza Dörr (2002), Christian Gausterer (2002), Daniel Lackner (2002), Mathias Gebhart (2004), Katharina Maderböck (2004)

PhD theses:
Foong May Yeong (1998), Thomas Fellner (2002), Denis Mehic (2004), Hans Hombauer (2004)

Research awards: Aventis Prize 2003 awarded to Dr. Thomas Fellner for the best publication (Fellner et al., Genes Dev 17, 2138-2150.) at the Medical University of Vienna in 2003 (for more information please take a look at Aventis Prize 2003

Contact
Egon Ogris, M.D., phone + 43 1 4277 61730, fax + 43 1 4277 9617
e-mail: egon.ogris@univie.ac.at
  or      egon.ogris@meduniwien.ac.at

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