Mst1 Is an Interacting Protein that Mediates PHLPPs' Induced Apoptosis Meng Qiao, Yaqi Wang, Xiaoen Xu, Jing Lu, Yongli Dong, Wufan Tao, Janet Stein, Gary S. Stein, James D. Iglehart, Qian Shi, Arthur B. Pardee  Molecular Cell  Volume 38, Issue 4, Pages 512-523 (May 2010) DOI: 10.1016/j.molcel.2010.03.017 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 PHLPP1 and PHLPP2 Inhibit Tumor Cell Growth and Migration and Induce Apoptosis (A) The expression of PHLPP1 and PHLPP2 in 21T series of breast cells. Top: Western blot showing protein levels of PHLPP1 and PHLPP2, with their correlation to S473- Akt phosphorylation. Bottom: mRNA levels of PHLPP1 and PHLPP2 were obtained with quantitative PCR. (B) Pictures of 21T series of breast cells 7 days after transfection with PHLPP expression vectors. (C) Growth of Panc1 cells after transfection with PHLPP vectors. (D) Colony-formation assay in Panc1 cells transfected with PHLPP expression vectors. Three independent experiments were performed and representative images and quantification were shown. (E) PHLPPs inhibit tumor cell migration. Transwell assays were performed in Panc1 cells transfected with PHLPP plasmids (left) and in NT cells transfected with PHLPP siRNA (right). (F) Annexin V-based apoptosis analysis of breast cancer cell MT2 (left) and pancreatic cancer cell Panc1 (right) transfected with PHLPP expression vectors. Annexin V staining followed by fluorescence-activated cell sorting (FACS) analysis were performed. All data are from at least two biological replicates. Error bars represent standard error of the mean (SEM). Student's t tests were used to compare PHLPP-transfected cells with control vector-transfected cells. Asterisks denote statistical significance (p < 0.05 compared with vector-only controls). See also Figure S2. Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 PHLPP's Functions Are Independent of Akt, PKC, and ERK (A and B) PHLPP can modify Akt phosphorylation in 21T series of breast cells. 21T series of breast cells were transfected with control vector or a vector expressing PHLPP1 or PHLPP2. (C–E) PHLPP signaling was independent of Akt in many other cells. Breast cell lines HMEC and BT474 (C), kidney cell 293T, glioma cell U87, pancreatic cancer cells Colo357 (D), and Panc1 (E). (F) Phosphatase assay using immunoprecipitated PHLPP proteins. HA-tagged PHLPP1 or 2 was transfected into Panc1 cells, pulled down using anti-HA antibody, and then incubated with Akt substrate, the phosphorylated Akt was detected using specific antibodies. Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 PHLPP Interacts with Mst1 and Dephosphorylates T387 of Mst1 (A) Endogenous PHLPP1 interacts with endogenous Mst1. Immunoprecipitation was performed on protein extracts from 16N cells. (B) PHLPP1 and PHLPP2 interact with Mst1 in vivo. 293T cells were transfected with vectors as indicated and immunoprecipitation experiments were performed. Interaction partners were detected with antibodies against HA and Mst1. (C) The interaction between PHLPP1 and Mst1 was further confirmed with GST-fusion proteins of Mst1 and Mst1/K59R. (D) PP2C domain of PHLPP is required for the interaction of PHLPP with Mst1. GST tagged full-length and deletion mutants of PHLPP (−1, −2, −3) were cotransfected with c-Myc tagged Mst1 in 293T cells, and IP experiments were performed with GST antibody to determine the domains essential for the binding of PHLPP to Mst1. (E) PHLPP1 directly interacts with Mst1 in a cell-free system. Bacteria purified PP2C domain of PHLPP1 and full length of Mst1 proteins were incubated as indicated and the complex was pulled down with glutathione beads and immunoblotting (IB) was performed with anti-His antibody. (F) PHLPP1 dephosphorylates Mst1 on T387. Upper panel: GST-Mst1 and HA-PHLPP expression vectors were co-transfected into Panc1 cells. Phosphorylation of T387 of Mst1 was detected using specific antibody. Lower panel: Purified PP2C domain of PHLPP1 was incubated with purified Mst1 in a phosphatase assay buffer. Western blot was performed with phospho-Akt substrate antibody that detects phosphorylation of T387 on Mst1. Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 PHLPPs Activate Mst1 Signaling (A) Panc1 cells were transfected with PHLPP vectors and Mst1 vectors alone or in combination as indicated. Total proteins and phosphorylation of proteins were detected using specific antibodies. (B) NT cells were transfected with PHLPP vectors and western blot were performed to detect changes in phosphorylation of Mst1 and its target proteins. (C) Inhibition of PHLPPs reduced the Mst1 signaling. siRNAs against PHLPP1 and PHLPP2 were transfected into NT cells and Mst1 signaling was examined by western blot. See also Figure S3. Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 PHLPPs Synergize with Mst1 in Inducing Apoptosis and Inhibiting Cell Growth (A) Vectors expressing PHLPP1, PHLPP2, Mst1, and Mst1/K59R mutant were transfected alone or in combination into Panc1 cells and apoptosis assays were performed. ∗p < 0.05 compared with single vector transfection controls, i.e., PHLPP1, PHLPP2, or Mst1 transfection alone. ∗∗p < 0.05 compared with PHLPP1 or PHLPP2 transfection alone. (B) Similar transfections were carried out and the growth of Panc1 cells was measured by MTT assay. (C) PHLPP-induced apoptosis is abolished in Mst1 knockout MEFs. MEFs from Mst1−/− or wild-type embryos were transfected with vectors as indicated. (a) Expression of Mst1 and PHLPP1 were confirmed by WB using specific antibodies. The Mst1 antibody detects both mouse (lower band) and human (myc-tagged) Mst1 (upper band). (b) Apoptosis was analyzed by Annexin V-FITC staining followed by FACS. The percentage of apoptotic cells was quantitated. All data are from at least two biological replicates. Error bars represent SEM. ∗p < 0.05 compared with control; ∗∗p < 0.05 compared with PHLPP transfection. Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Interaction of PHLPP, Akt, and Mst1 (A) Effect of Akt inhibition on PHLPP-induced apoptosis. Panc1 cells were transfected with control or PHLPP and placed in low serum medium containing 1% FBS in the presence or absence of PI3K/Akt inhibitor LY294002 (20μM) for 48 hr. (a) The phosphorylation of Mst1 at Thr183 was increased in PHLPP transfected Panc1 cells by Western blot analysis. The phosphorylation of Akt at Ser473 was reduced by LY294002 treatment. (b) Quantified results of apoptosis in Panc1 cells. The bar graph summarizes two independent experiments showing the mean and standard error of the mean. ∗p < 0.05 compared with control; ∗∗p < 0.05 compared with PHLPP transfection or LY treatment alone. (B) Diagram for an autoinhibitory triangle of PHLPP-Mst1-Akt. PHLPP activates Mst1 by direct dephosphorylation of T387 in the regulatory domain of Mst1 (Mst1-C, C terminal of Mst1). PHLPP may also activate Mst1 indirectly via dephosphorylation of S473 of Akt, which leads to decreased activity of Akt and diminished phosphorylation of T387 on Mst1. Arrows and bars indicate activation and inhibition effect, respectively. Blocked arrows indicate high or low activities of enzymes or stress levels in normal or cancer conditions. Open blocked arrows: normal. Filled blocked arrows: cancer. Molecular Cell 2010 38, 512-523DOI: (10.1016/j.molcel.2010.03.017) Copyright © 2010 Elsevier Inc. Terms and Conditions