A screen for extracellular signal-regulated kinase-primed glycogen synthase kinase 3 substrates identifies the p53 inhibitor iASPP

Crystal Woodard, Gangling Liao, C. Rory Goodwin, Jianfei Hu, Zhi Xie, Thaila F. dos Reis, Rob Newman, Heesool Rho, Jiang Qian, Heng Zhu, S. Diane Hayward

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Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death.

Original languageEnglish (US)
Pages (from-to)9232-9241
Number of pages10
JournalJournal of virology
Volume89
Issue number18
DOIs
StatePublished - Jan 1 2015

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ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Woodard, C., Liao, G., Goodwin, C. R., Hu, J., Xie, Z., dos Reis, T. F., Newman, R., Rho, H., Qian, J., Zhu, H., & Hayward, S. D. (2015). A screen for extracellular signal-regulated kinase-primed glycogen synthase kinase 3 substrates identifies the p53 inhibitor iASPP. Journal of virology, 89(18), 9232-9241. https://doi.org/10.1128/JVI.01072-15