Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress.

Topolska-Woś AM, Shell SM, Kilańczyk E, Szczepanowski RH, Chazin WJ, Filipek A
FASEB J. 2015 29 (5): 1711-24

PMID: 25609429 · PMCID: PMC4415008 · DOI:10.1096/fj.14-264770

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.

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MeSH Terms (22)

Amino Acid Sequence Animals Blotting, Western Calcium-Binding Proteins Flow Cytometry Fluorescent Antibody Technique Immunoprecipitation Mice Mitogen-Activated Protein Kinase 1 Mitogen-Activated Protein Kinase 3 Molecular Sequence Data Neuroblastoma Oxidative Stress Phosphorylation Protein Conformation Protein Multimerization Protein Processing, Post-Translational Proteins Scattering, Small Angle Sequence Homology, Amino Acid Spectroscopy, Fourier Transform Infrared Tumor Cells, Cultured

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