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A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

ABSTRACT:

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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.

Importance

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.

SUBMITTER: Woodard C

PROVIDER: S-EPMC4542386 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

Woodard Crystal C Liao Gangling G Goodwin C Rory CR Hu Jianfei J Xie Zhi Z Dos Reis Thaila F TF Newman Rob R Rho Heesool H Qian Jiang J Zhu Heng H Hayward S Diane SD

Journal of virology 20150624 18

<h4>Unlabelled</h4>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 microar ...[more]

PMID: 26109723

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Project description:BackgroundTumor necrosis factor-α-induced protein 8-like 3 (TNFAIP8L3, also called TIPE3) has been shown to activate PI3K-AKT and MEK-ERK pathways. However, the roles of TIPE3 in progression of lung cancer are largely unknown.MethodsImmunohistochemistry and western blotting were carried out to analyze the expression of TIPE3 in lung cancer clinical tissues and cells. TIPE3-overexpressing and knock-down NSCLC cell lines were established by transfer of TIPE3 coding sequence and shRNA, respectively. In vitro functional assays were performed to assess the effects of TIPE3 on proliferation and metastasis of NSCLC cells. Tumor xenograft mouse model was used to examine the roles of TIPE3 in growth of NSCLC cells in vivo. Western blotting, immunofluorescence, and immunohistochemistry were conducted to evaluate the association of TIPE3 and molecules related to AKT/ERK1/2-GSK3β-β-catenin/Snail pathway. PI3K, MEK, or GSK3β kinase and proteasome inhibition assays as well as β-Trcp and STUB1 siRNA assays were employed to determine the contribution of AKT/ERK1/2-GSK3β signaling and ubiquitin-proteasome pathway to the regulatory effects of TIPE3 on expression of β-catenin, Snail1, and Slug.ResultsWe demonstrated that TIPE3 was elevated in lung cancer tissues and cells. The expression level of TIPE3 was positively correlated with malignant clinicopathological characteristics of lung cancer patients, such as tumor size, pathologic stage, and lymph node metastasis. Knockdown of TIPE3 suppressed the proliferation and growth of NSCLC cells as well as their migration and invasion ability, whereas TIPE3 overexpression facilitated these biological processes. Mechanistic data showed that TIPE3 promoted AKT and ERK1/2 signaling, inactivated GSK3β activity, and enhanced the expression and transcriptional activity of β-catenin, Snail1, and Slug in NSCLC cells. Kinase or proteasome inhibition and β-Trcp or STUB1 knockdown assays further revealed that TIPE3 upregulated β-catenin, Snail1, and Slug via the AKT/ERK1/2-GSK3β pathway, in an ubiquitin-proteasome-dependent manner. More importantly, clinical data demonstrated that the expression level of TIPE3 was positively associated with the activation of AKT/ERK1/2-GSK3β-β-catenin/Snail pathway in lung cancer.ConclusionsOur findings indicate that upregulation of TIPE3 promotes the progression of human NSCLC considerably by activating β-catenin, Snail1, and Slug transcriptional signaling via the AKT/ERK1/2-GSK3β axis. Therefore, TIPE3 may represent a potential therapeutic target for NSCLC.

Dataset Information

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

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Publications

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

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