Progestin-inducible EDD E3 ubiquitin ligase binds to α4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac

Mol Cell Endocrinol. 2014 Jan 25;382(1):254-261. doi: 10.1016/j.mce.2013.09.033. Epub 2013 Oct 18.

Abstract

Mammalian α4 phosphoprotein binds to the protein phosphatase 2A catalytic subunit (PP2Ac) to regulate PP2A activity, and to poly(A)-binding protein (PABP) and progestin-inducible EDD E3 ubiquitin ligase. This study showed induction of the EDD protein by progesterone, 17β-estradiol and prolactin in breast cancer cells. Co-immunoprecipitation analyses, using lysates of COS-1 cells transfected with α4-deletion constructs, showed the α4 N-terminus binding to endogenous PP2Ac and PABP, and the C-terminus to EDD. Monoubiquitinated α4 in MCF-7 cells was unaffected by EDD-targeting siRNA (siEDD) nor by non-targetting siNT, thus, EDD does not ubiquitinate α4. PP2Ac is polyubiquitinated, and 36-kDa PP2Ac only was detected in siEDD- or siNT-transfected cells. However, treatment with proteasomal inhibitor MG132 showed polyubiquitinated-PP2Ac molecules (∼65-250kDa) abundantly in siNT controls but low in siEDD-transfectants, implicating PP2Ac as an EDD substrate. Finally, progesterone induction of EDD in MCF-7 cells correlated with decreased PP2Ac levels, further implicating hormone-inducible EDD in PP2Ac turnover.

Keywords: 17β-Estradiol; EDD E3 Ubiquitin ligase; PP2Ac; Progesterone; Prolactin; α4 Phosphoprotein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • COS Cells
  • Chlorocebus aethiops
  • Enzyme Induction / drug effects
  • Female
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • MCF-7 Cells
  • Molecular Chaperones
  • Mutant Proteins / metabolism
  • Poly(A)-Binding Proteins / metabolism
  • Polyubiquitin / metabolism
  • Progestins / pharmacology*
  • Protein Binding / drug effects
  • Protein Phosphatase 2 / metabolism*
  • Proteolysis / drug effects*
  • Substrate Specificity / drug effects
  • Ubiquitin-Protein Ligases / biosynthesis*
  • Ubiquitination / drug effects*

Substances

  • Adaptor Proteins, Signal Transducing
  • IGBP1 protein, human
  • Intracellular Signaling Peptides and Proteins
  • Molecular Chaperones
  • Mutant Proteins
  • Poly(A)-Binding Proteins
  • Progestins
  • Polyubiquitin
  • UBR5 protein, human
  • Ubiquitin-Protein Ligases
  • Protein Phosphatase 2