Dimerization and phosphatase activity of calcyclin‐binding protein/Siah‐1 interacting protein: the influence of oxidative stress | Zendy

TopolskaWoś Agnieszka M. | Zendy; Shell Steven M. | Zendy; Kilańczyk Ewa | Zendy; Szczepanowski Roman H. | Zendy; Chazin Walter J. | Zendy; Filipek Anna | Zendy

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

Author(s) -

TopolskaWoś Agnieszka M.,

Shell Steven M.,

Kilańczyk Ewa,

Szczepanowski Roman H.,

Chazin Walter J.,

Filipek Anna

Publication year - 2015

Publication title -

the faseb journal

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 1.709

H-Index - 277

eISSN - 1530-6860

pISSN - 0892-6638

DOI - 10.1096/fj.14-264770

Subject(s) - oxidative stress , phosphatase , chemistry , microbiology and biotechnology , protein phosphatase 1 , oxidative phosphorylation , stress (linguistics) , biochemistry , biophysics , biology , enzyme , linguistics , philosophy

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.—Topolska‐Woś, A. M., Shell, S. M., Kilańczyk, E., Szczepanowski, R. H., Chazin, W. J., Filipek, A. Dimerization and phosphatase activity of calcyclin‐binding protein/Siah‐1 interacting protein: the influence of oxidative stress. FASEB J. 29, 1711‐1724 (2015). www.fasebj.org

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