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. IMPORTANCEKSHV 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.T he Kaposi's sarcoma-associated herpesvirus (KSHV) latencyassociated nuclear antigen (LANA) is expressed in all KSHVinfected cells, including the tumor cells of the KSHV-associated malignancies Kaposi's sarcoma, primary effusion lymphoma (PEL), and muticentric Castleman disease. LANA functions in the replication and maintenance of latent, episomal KSHV genomes (1, 2) by binding to the KSHV origin of replication in the terminal repeats (3, 4), recruiting cellular replication proteins (5-11), and tethering the KSHV episomal genomes to host chromosomes (12-18). Sequences in the KSHV terminal repeats are bound by the C terminus of LANA, and the X-ray crystal structures of the human and murine gammaherpesvirus 68 (MHV68) LANA DNA-binding domains have been solved (19)(20)(21).KSHV infection leads to a global reprogramming of cel...

GSK-3β Can Regulate the Sensitivity of MIA-PaCa-2 Pancreatic and MCF-7 Breast Cancer Cells to Chemotherapeutic Drugs, Targeted Therapeutics and Nutraceuticals

“…Downstream of MEK is ERK. ERK can prime substrates for GSK-3β [83]. Some of substrates GSK-3β phosphorylates may alter the proliferation of the cells.…”

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

“…Protein Array Phosphorylation Assays-The HuProt array was fabricated by spotting down 16,368 unique full-length human recombinant proteins in duplicate along with several control proteins provided by CDI Laboratories, Inc. (31,32). The protocol for phosphorylation assays on HuProt arrays was modified from that of previous publications (23,24). Briefly, HuProt arrays were preincubated in Superblock (Pierce) containing 0.2% BSA and 1% Triton X-100 for 1 h at room temperature.…”

Cell Cycle Regulatory Functions of the KSHV Oncoprotein LANA

“…In addition, LANA has also been shown to interact with glycogen synthase kinase (GSK-3β), a kinase involved in phosphorylation and subsequent degradation of many cell-cycle regulators including c-Myc and Cyclin D ( Fujimuro et al, 2003 ; Bubman et al, 2007 ; Liu et al, 2007a , b ). Recent studies showed that the interaction of GSK-3 with LANA could lead to the accumulated expression of iASPP and in turn degradation of p53 for cell growth ( Woodard et al, 2015 ). Our previous studies further demonstrated that p53 can be degraded by LANA-mediated recruitment of the cellular EC 5 S (Elongin BC-Cullin 5-Rbx1) ubiquitin complex ( Cai Q.-L. et al, 2006 ), as well as involvement of the Serine/Threonine oncogenic kinase Aurora A ( Cai et al, 2012 ).…”

“…The stabilized p53 in turn up-regulates the Cdk inhibitor p21 at the G1 checkpoint, which eventually leads to cell arrest or apoptosis. Although no literatures reported that LANA could directly target Cdc25B/C or Cdk1-cyclin B complex, many molecules [including p73 ( Santag et al, 2013 ), ANG ( Sadagopan et al, 2009 ), Mdm2 ( Sarek et al, 2007 ), and GSK-3 ( Woodard et al, 2015 )] have been shown to interaction with LANA for blocking p53-mediated apoptosis during G2/M checkpoint. Recent studies showed that LANA upregulates the expression of survivin, and recruits Aurora kinase B to induce phosphorylation of Survivin at Tyrosine 34 ( Lu et al, 2009 , 2014 ), which potentially drive host cell proliferation during mitosis.…”