E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma

Jean Pierre Obeid, Youssef Zeidan, Zafar Nawaz, Jimmy El Hokayem

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalMolecular Neurobiology
DOIs
StateAccepted/In press - Feb 18 2017

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Cyclic AMP-Dependent Protein Kinases
Neuroblastoma
Estrogens
Response Elements
Proteins
Chromatin Immunoprecipitation
Luciferases
Reporter Genes
Western Blotting
estrophilin
Angelman Syndrome
Estrogen Receptor alpha
Computational Biology
Oligonucleotides
Polymerase Chain Reaction
Messenger RNA

Keywords

  • Angelman syndrome
  • E6ap
  • Estrogen receptor
  • Neuroblastoma
  • Protein kinase A
  • UBE3A

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

Cite this

E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma. / Obeid, Jean Pierre; Zeidan, Youssef; Nawaz, Zafar; El Hokayem, Jimmy.

In: Molecular Neurobiology, 18.02.2017, p. 1-11.

Research output: Contribution to journalArticle

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