14-3-3γ regulates Copine1-mediated neuronal differentiation in HiB5 hippocampal progenitor cells

Exp Cell Res. 2017 Jul 1;356(1):85-92. doi: 10.1016/j.yexcr.2017.04.015. Epub 2017 Apr 12.

Abstract

Copine1 (CPNE1), known as a calcium-dependent membrane-binding protein, has tandem C2 domains and an A domain. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Protein kinase B (AKT) phosphorylation in the hippocampal progenitor cell line, HiB5. To better understand its cellular function, we carried out a yeast two-hybrid screening to find CPNE1 binding partners. Among the identified proteins, 14-3-3γ appears to directly interact with CPNE1. Between CPNE1 and 14-3-3γ, the physical interaction as well as the specific binding regions of CPNE1 was confirmed in vitro and in vivo. Furthermore, among the seven 14-3-3 isotypes, only 14-3-3γ directly interacts with CPNE1. Our results also demonstrate that AKT phosphorylation, neurite outgrowth and expression of the neuronal marker protein are increased when 14-3-3γ is overexpressed in CPNE1 high expressed HiB5 cells. Furthermore, the neighboring Ser54 amino acids residue of C2A domain in CPNE1 has an important role in binding with 14-3-3γ, and in differentiation-related function of CPNE1. Moreover, mutation of Ser54 amino acids residue in CPNE1 effectively decreased association with 14-3-3γ and neuronal differentiation of HiB5 cells. Collectively, our findings indicate that 14-3-3γ regulates the differentiation ability of CPNE1 through the binding with C2A domain of CPNE1 in HiB5 cells.

Keywords: 14-3-3γ; C2A domain; Copine1; HiB5 cell; Protein-protein interaction.

MeSH terms

  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism*
  • Animals
  • COS Cells
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Cell Line
  • Chlorocebus aethiops
  • HEK293 Cells
  • Hippocampus / cytology*
  • Humans
  • Neurogenesis / genetics*
  • Phosphorylation
  • Protein Binding / genetics
  • Protein Domains
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA Interference
  • RNA, Small Interfering / genetics
  • Stem Cells / cytology*

Substances

  • 14-3-3 Proteins
  • CPNE1 protein, human
  • Calcium-Binding Proteins
  • RNA, Small Interfering
  • Proto-Oncogene Proteins c-akt