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. 2014 Nov;5(11-12):393-406.
doi: 10.18632/genesandcancer.41.

mTORC2 modulates feedback regulation of p38 MAPK activity via DUSP10/MKP5 to confer differential responses to PP242 in glioblastoma

Angelica Benavides-Serrato  1 , Lauren Anderson  2 , Brent Holmes  2 , Cheri Cloninger  2 , Nicholas Artinian  2 , Tariq Bashir  1 , Joseph Gera  3
Affiliations

mTORC2 modulates feedback regulation of p38 MAPK activity via DUSP10/MKP5 to confer differential responses to PP242 in glioblastoma

Angelica Benavides-Serrato et al. Genes Cancer. 2014 Nov.

Abstract

Dual-specificity phosphatases (DUSPs) dephosphorylate MAP kinases (MAPKs) resulting in their inactivation. Activation of MAPK signaling leads to enhanced DUSP expression, thus establishing feedback regulation of the MAPK pathway. The DUSPs are subject to regulation at the post-translational level via phosphorylation resulting in alterations of protein stability. Here we report that mTORC2 function leads to stabilization of the p38 MAPK phosphatase, DUSP10, thereby inhibiting p38 activity. We demonstrate that mTORC2 binds DUSP10 and phosphorylates DUSP10 on serine residues 224 and 230. These phosphorylation events block DUSP10 turnover resulting in inactivation of p38 signaling. We further show that insulin-stimulated PI3K/mTORC2 signaling regulates DUSP10 stability and p38 activity. Importantly, knockdown of DUSP10 or ectopic overexpression of nonphosphorylatable or phosphomimetic DUSP10 mutants was sufficient to confer differential mTOR kinase inhibitor responses to GBM cells in vitro and in murine xenografts. Finally, DUSP10 was shown to be overexpressed in a significant number of GBM patients. These data demonstrate the ability of the mTORC2 pathway to exert regulatory effects on the DUSP10/p38 feedback loop to control the cellular effects of mTOR kinase inhibitors in GBM and support the use of DUSP10 expression as a surrogate biomarker to predict responsiveness.

Keywords: DUSP; mTOR; p38 MAPK.

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Figures

Figure 1
Figure 1. Interaction of Rictor and DUSP10 in yeast and mammalian cells
A). The indicated deletion mutants of Gal4DBD-Rictor or Gal4AD-DUSP10 were cotransfected into AH109 cells to determine whether an interaction between the proteins was detectable via activation of the HIS3 reporter (+++, strong growth; ++, moderate growth; -, no growth). Colonies which grew were assayed for β-gal activity. B) Rictor, Raptor or DUSP10 was immunoprecipitated from U87 cells and precipitates subjected to Western analysis for the indicated proteins. Lane 1, beads, no antibody; Lane 2, immunoprecipitation with an irrelevant antibody (control IgG); Lane 3, input cell lysate; Lane 4, indicated immunoprecipitate probed with antibodies for the indicated proteins. As a control, in Raptor immunoprecipitates DUSP10 was not detected.
Figure 2
Figure 2. DUSP10 is phosphorylated by mTORC2
A). U87Rictor cells harboring active mTORC2, display a slower migrating DUSP10 species (lane 1) which is eliminated by protein phosphatase lambda (ppλ) in vitro (lane 2) or by treating cells with PP242 (50 nM, 24 h) (lane 3). B). Immunoprecipitated mTORC2 phosphorylates recombinant DUSP10 in vitro. High-resolution SDS-PAGE of mTORC2 kinase reactions utilizing recombinant DUSP10 as a substrate and [γ32P]ATP for the indicated time (min) and treated with ppλ as shown. Phosphorylated and unphosphorylated species are as indicated, as well as partially dephosphorylated DUSP10 (asterisk). C). DUSP10 was mutated to produce the single mutants S224A and S230A and the double mutant S224A-S230A and treated with mTORC2 as described in (B). Wild-type (WT) DUSP10 and the mutants were subjected to an in vitro kinase assay with mTORC2 and [γ32P]ATP. Reactions were immunoprecipitated and detected by immunoblotting and autoradiography. D). U87Rictor cells were transfected with expression plasmids encoding DUSP10 or the double mutant S224A-S230A (SA/SA) and 24 h following transfection cells were labeled with 32P (500 μCi/ml) in phosphate-free media for 4 h. DUSP10 was immunoprecipitated, resolved by SDS-PAGE and revealed by autoradiography (top) or immunoblotted (bottom). Results in A, B were performed three times with similar results.
Figure 3
Figure 3. Half-life of DUSP10 is altered in response to modulation of mTORC2
A). Basal half-life of DUSP10 in U373MG (left panel), U87 (middle panel) and LN229 (left panel) glioblastoma cells. Cells were pulsed with 35S-methionine and DUSP10 levels monitored as described in the Methods section. Solid circles are in the absence, while open circles are in presence of the proteosome inhibitor MG-132 (25 nM) B). Differential stability of DUSP10 in U87Rictor (open squares) cells versus U87shRNARictor (solid squares) cells [18]. C). Destabilization of DUSP10 following PP242 exposure (10 nM, 6 h) in U373MG cells. Solid circles are in the absence of PP242, while open circles are in the presence of PP242. D). DUSP10 knockdown in U87 glioblastoma cells leads to enhanced p38 activity. Lysates from cells expressing shRNA targeting DUSP10 or empty vector (EV) were immunoprecipitated for p38 and immunoprecipitates subjected to an in vitro kinase assay using ATF2 as a substrate. Phosphorylated ATF2 was detected using phosphospecific antibodies. Input lysates were immunoblotted for the indicated proteins.
Figure 4
Figure 4. Insulin-PI3K signaling regulates DUSP10 stability and phosphorylated p38 levels
A) Half-life of DUSP10 in U87 cells under basal (solid squares) and following insulin stimulation (open squares). B). Signaling effects of insulin stimulation in U87 cells. Insulin-stimulated cells (10 nM), treated for the indicated time points, were lysed and extracts immunoblotted for the indicated proteins. C). DUSP10 half-life (left panel) and signaling (right panel) in U87 cells treated with LY294002 (50 μM) (untreated, solid squares; LY294002 treated, open squares). D). siRNA-mediated knockdown of PTEN in LN229 cells leads to stabilization of DUSP10 (left panel; untreated solid squares; PTEN siRNA treated, open squares). Extracts from cells transfected with the indicated siRNA and immunoblotted for the indicated proteins (right panel).
Figure 5
Figure 5. Effects of DUSP10 knockdown and nonphosphorylatable or phosphomimetic mutant overexpression on GBM responses to mTOR kinase inhibition
A) Relative proliferation rates as determined from XTT proliferation assays of U87 cells expressing shRNA targeting DUSP10 as compared to empty vector (EV) transfected cells in the presence or absence of PP242 (25 nM, 24 h). Values in parentheses above bars correspond to percent apoptotic cells as determined via Annexin V staining. * P < 0.05 as determined by Student's t-test. B) Relative p38 and mTORC2 signaling in cells from (A). Extracts were prepared from the indicated lines and treatment groups and subjected to immunoblot analysis for the proteins shown. C&D) Effects of PP242 on proliferation, apoptosis and signaling in U87 cells overexpressing nonphosphorylatable DUSP S224A/S230A. * P < 0.05. E&F) As in (C&D), except performed using cells overexpressing the phosphomimetic DUSP10 S224E/S230E mutant. * P < 0.05.
Figure 6
Figure 6. In vivo effects of PP242 on DUSP10 nonphosphorylatable or phosphomimetic expressing GBM lines
A-C). Effects of PP242 treatment on U87, U87 DUSP10 S224A/S230A or U87 DUSP10 S224E/230E expressing lines in xenografts as indicated. Mice with established tumors (200 mm3) received either vehicle (closed circles) or PP242 (20 mg/kg/day, open circles) for 10 consecutive days and tumor growth was assessed every 2 days following initiation of treatment (start, day 0). D). Quantification of in situ TUNEL assay results from xenografts harvested at day 10 as indicated. The data are expressed as the number of positive apoptotic bodies divided by high power field (hpf; 10-12 hpf/tumor). Values are the means + SD. * P < 0.05. E). Effects of PP242 therapy on signaling pathways from day 10 harvested xenografts from the indicated lines. Immunoblot analysis was performed for the indicated proteins.
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