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. 2013 Dec 3;6(304):ra105.
doi: 10.1126/scisignal.2004125.

PLC-γ and PI3K link cytokines to ERK activation in hematopoietic cells with normal and oncogenic Kras

Ernesto Diaz-Flores  1 , Hana Goldschmidt, Philippe Depeille, Victor Ng, Jon Akutagawa, Kimberly Krisman, Michael Crone, Michael R Burgess, Olusegun Williams, Benjamin Houseman, Kevan Shokat, Deepak Sampath, Gideon Bollag, Jeroen P Roose, Benjamin S Braun, Kevin Shannon
Affiliations

PLC-γ and PI3K link cytokines to ERK activation in hematopoietic cells with normal and oncogenic Kras

Ernesto Diaz-Flores et al. Sci Signal. .

Abstract

Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

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Figures

Fig. 1
Fig. 1. PI-90 and U73122 impair Ras and ERK activation in wild-type and Mx1-Cre, KrasG12D hematopoietic cells and reduce PLC-γ phosphorylation
(A) M+G+ cells from wild-type (WT) and Mx1-Cre, KrasG12D (KrasG12D) mice were incubated for 30 min with the indicated inhibitors (each at final concentration of 5 μM), stimulated with GM-CSF for 10 min, and then analyzed by phospho-flow cytometry to determine the abundance of pERK. Basal indicates cells that were treated neither with inhibitor nor cytokine. The red vertical lines indicate basal median fluorescence intensity (MFI) values. (B) K+M−G− cells from WT and KrasG12D mice were incubated for 30 min with the same inhibitors used in (A) before they were stimulated with SCF for 15 min and then analyzed by phospho-flow cytometry to determine pERK abundance. (C) Ras-GTP and pERK abundances were measured in bone marrow cells from WT and KrasG12D mice (as described in the Methods) that were treated with the indicated inhibitors before being stimulated with GM-CSF. Actin was used as a loading control. (D) The amounts of phosphorylated PLC-γ1 (pPLC-γ1) and PLC-γ2 (pPLC-γ2) were determined by flow cytometric analysis of WT and KrasG12D mouse bone marrow M+G+ cells that were pretreated with the indicated inhibitors before being stimulated with GM-CSF. Data from additional independent experiments are shown in figs. S2 B and C, and S3.
Fig. 2
Fig. 2. Effects of inhibiting PLC-γ and PI3K on signaling in BMMPCs
(A) BMMPCs that were not transduced (Unt) or were transduced with a retroviral vector expressing mCherry (mCh) or a vector encoding mCherry and one of two independent shRNAs specific for PLC-γ2 (shγ2-1 or shγ2-2) were lysed, and the abundances of PLC-γ2, pAkt, and pERK were measured by Western blotting analysis, with actin used as a loading control. (B) Effects of PMA and GM-CSF on Ras-GTP, pERK, and pAkt abundances in BMMPCs from WT and KrasG12D mice. BMMPCs from the indicated mice were treated with PMA or GM-CSF for the indicated times and lysed. Ras-GTP abundance was assessed by a Ras-RBD pull down assay and abundance of pAkt, and pERK was analyzed by Western blotting on the same lysates. Actin was used as a loading control. (C) BMMPCs were pretreated with PI-90 or U73122 (U73) and then were left unstimulated or were stimulated with GM-CSF or PMA for 15 min. Cells were then analyzed by Western blotting for the abundances of pAkt and pERK, with actin used as a loading control. (D) Analysis of CFU-GM growth from bone marrow cells from WT and KrasG12D mice that were cultured in a saturating concentration of GM-CSF (10 ng/ml) in the absence or presence of the inhibitors JakI, PI-90, U73122, or AKT VIII. Data from independent experiments corresponding the panels (B) and (C) are shown in figs S8 – S10. Data in (D) are mean values ± SD from three independent experiments.
Fig. 3
Fig. 3. Effects of the knockdown of RasGRP isoforms on ERK phosphorylation in BMMPCs from WT and Mx1-Cre, KrasG12D mice
(A) Rasgrp3 and Rasgrp4 mRNA and RasGRP3 and RasGRP4 in BMMPCs from WT and KrasG12D mice were determined by quantitative RT-PCR. (B) Protein abundances were assessed by Western blotting (see also fig. S14) (C) BMMPCs that were incubated with or without siRNAs (10 nM) specific for Rasgrp3 (RG3) or RasGrp4 (RG4) or with a control scrambled siRNA (Sc) were stimulated with GM-CSF for 15 min. The cells were lysed and Western blotting analysis was performed to determine the amounts of RasGRP3, RasGRP4, and pERK, with actin used as a loading control. (D) BMMPCs were starved of serum and cytokines overnight, incubated with PI-90, U73122, KN-62, or PD0325901 for 30 min, and then stimulated for 15 min with GM-CSF alone or in the presence of 10 μM PLX4720. Cells were then lysed and analyzed by Western blotting to determine the abundances of pAkt and pERK, with actin used as a loading control. Data from additional independent experiments are presented in fig. S15.
Fig. 4
Fig. 4. Inhibition of PLC-γ and PI3K reduces SCF-induced ERK phosphorylation in WT and Mx1-Cre, KrasG12D cells highly enriched for stem cells
(A to C) Bone marrow cells from WT and Mx1-Cre, KrasG12D mice were left unpretreated or were pretreated with (A) PD0325901, (B) PI-90, or (C) U73122 before being stimulated with SCF in the absence or presence of PMA. Cells were analyzed by phospho-flow cytometry to determine the abundance of pERK in KLS 48- cells. (D) Quantitation of two independent experiments.
Fig. 5
Fig. 5. Treatment of mice with inhibitors of MEK or PI3K impairs cytokine-induced ERK activation in bone marrow cells
(A and B) WT mice were left untreated or received either PD0325901 (901, 5 mg/kg) or GDC-0941 (GDC, 100 mg/kg) by oral gavage, and were euthanized 2 or 6 hours later. Bone marrow cells were harvested and left untreated or were stimulated with GM-CSF for 10 min, and the abundances of pERK (A, left panel) and pSTAT5 (A, right panel) were determined by flow cytometric analysis. Data are representative of three mice for each time point. The red vertical lines display basal MFI values. (B) MFI plots of the data shown in (A) depict the effects of GM-CSF on pERK and pSTAT5 abundances, as well as the extent of inhibition observed in mice that were treated with the MEK inhibitor PD032501 or the PI3K inhibitor GDC-0941. Data are medians ± SD from three mice for each time point.
Fig. 6
Fig. 6. Proposed mechanism of cytokine-induced Ras and ERK activation in hematopoietic cells expressing WT and oncogenic K-Ras
In response the binding of GM-CSF to its receptor, JAK2 trans-phosphorylates multiple tyrosine residues on the β common chain of the GM-CSF receptor, creating docking sites for adaptor proteins and signaling molecules. PLC-γ1 and PLC-γ2 are recruited to the receptor complex and becomes activated, generating DAG. The p85 regulatory subunit of PI3K binds to the receptor through adaptor proteins, which results in PI3K activation and PIP3 production. Second messengers generated by PI3K and PLC-γ stimulate nucleotide exchange on Ras, likely by both localizing Ras to the plasma membrane and activating RasGRPs. The DAG mimetic PMA activates Ras and ERK independently of PLC-γ and PI3K. This scheme does not directly address the interaction between PLC-γ and PI3K; however, multiple lines of evidence suggested that PLC-γ is upstream of PI3K. The levels at which the different inhibitors used in this study exerted their effects are indicated by dashed red bars. Raf activation by PLX4720 is indicated as a dashed green arrow. Studies using many of these inhibitors support a similar role for PLC-γ and PI3K in linking the activated receptor tyrosine kinase c-Kit to Ras and ERK signaling.
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