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FASEB J. 2015 May;29(5):1711-24. doi: 10.1096/fj.14-264770. Epub 2015 Jan 21.

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

Author information

1
*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.
2
*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.

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.

KEYWORDS:

CacyBP/SIP; ERK1/2; MAPK phosphatase; NB2a cells; protein structure

PMID:
25609429
PMCID:
PMC4415008
DOI:
10.1096/fj.14-264770
[Indexed for MEDLINE]
Free PMC Article

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