Regulation of Neuronal Cell Death by c-Abl-Hippo/MST2 Signaling Pathway

BACKGROUND: Mammalian Ste20-like kinases (MSTs) are the mammalian homologue of Drosophila hippo and play critical roles in regulation of cell death, organ size control, proliferation and tumorigenesis. MSTs exert pro-apoptotic function through cleavage, autophosphorylation and in turn phosphorylation of downstream targets, such as Histone H2B and FOXO (Forkhead box O). Previously we reported that protein kinase c-Abl mediates oxidative stress-induced neuronal cell death through phosphorylating MST1 at Y433, which is not conserved among mammalian MST2, Drosophila Hippo and C.elegans cst-1/2. METHODOLOGY/PRINCIPAL FINDINGS: Using immunoblotting, in vitro kinase and cell death assay, we demonstrate that c-Abl kinase phosphorylates MST2 at an evolutionarily conserved site, Y81, within the kinase domain. We further show that the phosphorylation of MST2 by c-Abl leads to the disruption of the interaction with Raf-1 proteins and the enhancement of homodimerization of MST2 proteins. It thereby enhances the MST2 activation and induces neuronal cell death. CONCLUSIONS/SIGNIFICANCE: The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST2 suggests that the conserved c-Abl-MST signaling cascade plays an important role in oxidative stress-induced neuronal cell death

c-Abl-Mediated MST2 Phosphorylation at Y81 Promotes its Homodimerization and Disrupts the Interaction with Raf-1 Proteins.

<p>(A). Lysates of HEK 293T cells transfected with GFP-MST2 alone or together with FLAG-MST2 or Myc-c-Abl expression plasmid were immunoprecipitated with FLAG antibody and analyzed by immunoblotting against GFP antibody. (B). HEK 293T cells transfected with GFP-Raf-1 alone or together with FLAG-MST2 expression plasmid were treated with or without 10 µM STI571 for 1 hour. Lysates of cells were immunoprecipitated and analyzed as in (A). (C). Lysates of HEK293T cells transfected with GFP-Raf-1 alone or together with FLAG-MST2-WT or–Y81F expression plasmid were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with the indicated antibodies. (D). MST2 immunoprecipitates from Neuro2A cells treated with or without STI571 were immunoblotted with anti-Raf-1antibody or other indicated antibodies.</p

c-Abl-MST2 Signaling Mediates Rotenone-induced Neuronal Cell Death.

<p>(A). Neuro2A cells were left untreated or treated with 400 μM Rotenone for 1.5 hours with or without 10 μM STI571. Lysates of cells were immunoprecipitated with anti-MST2 antibody and analyzed by immunoblotting with anti-pan-tyrosine phosphorylation antibody. (B). Cerebellar granule neurons were transfected with the pEGFP alone or together with the FLAG-MST2 expression plasmid, and c-Abl RNAi or control vector as indicated. 72 hours after transfection, neurons were left untreated or treated with Rotenone for 20 hours. Transfected neurons were subjected to immunofluorescence analysis using GFP antibody together with the DNA dye Hoechst 33258 to reveal neuronal nuclei. Representative images of neurons are shown in the upper panel. White arrowheads indicating healthy transfected neurons and red arrowheads indicating transfected neurons that are undergoing apoptosis. The percentage of cell death of GFP-positive neurons is represented as the mean ± SEM. Exposure of MST2-transfected neurons to Rotenone induced neuronal cell death (ANOVA; p<0.01, n = 3), and the cell death was dramatically reduced by c-Abl knockdown (ANOVA; p<0.01, n = 3).(C). Cerebellar granule neurons were transfected with the pEGFP alone or together with the MST2 shRNA, c-Abl shRNA or control vector as indicated. 72 hours after transfection, neurons were treated and analyzed as in B. The percentage of cell death of GFP-positive neurons is represented as the mean ± SEM. Knockdown MST2 or c-Abl protects neurons from Rotenone induced cell death (ANOVA; p<0.01, n = 3). (D). The upper panel shows the expression of the MST2 rescue constructs in Neuro2A cells. Lower panel: Lysates of HEK 293T cells transfected with FLAG-MST2 or FLAG-MST2R expression plasmids together with MST2 RNAi or the control vector, were immunoblotted with the FLAG or ERK1/2 antibody. MST2 RNAi effectively knockdown endogenous MST2 in Neuro2A cells, but not the rescue form of MST2. (E). Cerebellar granule neurons transfected with pEGFP and MST2 RNAi or control vector, alone or together with MST2, MST2R, and MST2R-Y481F expression plasmids, were treated and analyzed as in B. MST2R but not Y81F mutants of MST2R enhanced Rotenone-induced cell death (ANOVA; p<0.01, n = 3). The percentage of cell death in GFP-positive neurons is represented as the mean ± SEM.</p

c-Abl Phosphorylates MST2 at Y81 <i>in vitro</i> and <i>in vivo.</i>

<p>(A). Sequence alignment of the mammalian MST2, <i>Drosophila</i> Hippo, <i>C. elegans</i> cst-1/2 and mammalian MST1. (B). Lysates of HEK 293T cells transfected with Myc-tagged c-Abl or the control vector were immunoprecipitated with anti-Myc antibody and subjected to an <i>in vitro</i> kinase assay using full-length GST-MST1 or–MST2 as substrate. Phosphorylation reactions were analyzed by immunoblotting with anti-pan-tyrosine phosphorylation antibody. MST2 and MST1 proteins were tyrosine phosphorylated by c-Abl kinase <i>in vitro.</i> (C). <i>In vitro</i> kinase assay using the recombinant full-length GST-MST2-WT or–Y81F as a substrate was performed and analyzed as in A. c-Abl phosphorylated MST2 at Y81 <i>in vitro</i>. (D). Lysates of HEK 293T cells transfected with FLAG-MST1-WT,–Y81F expression plasmid together with Myc-c-Abl were immunoprecipitated with anti-FLAG antibody and analyzed as in A. Y81 is the phosphorylation site of MST2 by c-Abl <i>in vivo</i>.</p

c-Abl Enhances MST2 Kinase Activity.

<p>(A). Lysates of Neuro2A cells stably transfected with c-Abl RNAi #1 or #2 or the control vector were immunoblotted with the indicated antibodies. (B). Lysates of HEK 293T cells transfected with FLAG-tagged MST2 alone or together with increasing amounts of Myc-tagged c-Abl expression plasmid were analyzed by immunoblotting with the indicated antibodies. (C). Anti-Myc immunoprecipitates from cells transfected with Myc-c-Abl or the control vector were subjected to the <i>in vitro</i> kinase reaction using the recombinant GST-MST2 or GST alone as substrate. GST-MST2 or GST from phosphorylation reactions was then subjected to the second <i>in vitro</i> kinase assay using GST-FOXO3-FD as substrate. Phosphorylation reactions were analyzed by immunoblotting with anti-pS207-FOXO3 antibody. The experiments were repeated for three times and quantative density is indicated. (D). Lysates of HEK 293T cells transfected with the FLAG-MST2 or–Y81F expression plasmid were immunoprecipitated with the anti-FLAG antibody and subjected to an <i>in vitro</i> kinase assay using Histone H2B as substrate in the presence of [³²P] ATP. Phosphorylation reactions were analyzed by electrophoresis and autoradiography. The experiments were repeated for three times and quantative density is indicated.</p