title: Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress.
abstract: CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.
authors.affiliation: *Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA; and International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Agnieszka M
authors.initials: AM
authors.lastname: Topolska-Woś
authors.affiliation: *Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA; and International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Shell
authors.affiliation: *Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA; and International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Kilańczyk
authors.affiliation: *Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA; and International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Roman H
authors.initials: RH
authors.lastname: Szczepanowski
authors.affiliation: *Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA; and International Institute of Molecular and Cell Biology, Warsaw, Poland a.filipek@nencki.gov.pl walter.j.chazin@vanderbilt.edu.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
authors.affiliation: *Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA; and International Institute of Molecular and Cell Biology, Warsaw, Poland a.filipek@nencki.gov.pl walter.j.chazin@vanderbilt.edu.
authors.forename: Anna
authors.initials: A
authors.lastname: Filipek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D010766:Phosphorylation; D011487:Protein Conformation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D017550:Spectroscopy, Fourier Transform Infrared; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
chemical_list: C111922:Cacybp protein, mouse; D002135:Calcium-Binding Proteins; D011506:Proteins; C484442:Siah1a protein, mouse; D044767:Ubiquitin-Protein Ligases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: CacyBP/SIP; ERK1/2; MAPK phosphatase; NB2a cells; protein structure