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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Cacybp protein, mouse
  • Calcium-Binding Proteins
  • Proteins
  • Siah1a protein, mouse
  • Ubiquitin-Protein Ligases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3