title: A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.
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.
  
  
  OBJECTIVE
  KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Crystal
authors.initials: C
authors.lastname: Woodard
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Gangling
authors.initials: G
authors.lastname: Liao
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: C Rory
authors.initials: CR
authors.lastname: Goodwin
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jianfei
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xie
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Thaila F
authors.initials: TF
authors.lastname: Dos Reis
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Rob
authors.initials: R
authors.lastname: Newman
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Heesool
authors.initials: H
authors.lastname: Rho
authors.affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.affiliation: High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA hzhu4@jhmi.edu dhayward@jhmi.edu.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA hzhu4@jhmi.edu dhayward@jhmi.edu.
authors.forename: S Diane
authors.initials: SD
authors.lastname: Hayward
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000956:Antigens, Viral; D004791:Enzyme Inhibitors; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C119612:PPP1R13L protein, human; D010879:Piperazines; D012097:Repressor Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C109540:latency-associated nuclear antigen; C482205:nutlin 3; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3