Prohibitin 2 represents a novel nuclear AKT substrate during all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells

FASEB J. 2014 May;28(5):2009-19. doi: 10.1096/fj.13-244368. Epub 2014 Feb 12.

Abstract

The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

Keywords: SRp20/SRSF3; mass spectrometry; phosphorylation; proteomics; signal transduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Cell Cycle
  • Cell Differentiation*
  • Cell Nucleus / metabolism
  • Cell Proliferation
  • Cell Survival
  • HEK293 Cells
  • Humans
  • Leukemia, Promyelocytic, Acute / metabolism*
  • Mutagenesis, Site-Directed
  • Oxidative Stress
  • Phosphorylation
  • Prohibitins
  • Proteomics
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Repressor Proteins / metabolism*
  • Signal Transduction
  • Tretinoin / metabolism*

Substances

  • PHB protein, human
  • PHB2 protein, human
  • Prohibitins
  • Repressor Proteins
  • Tretinoin
  • Proto-Oncogene Proteins c-akt