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. 2007 Oct 31;26(21):4523-34.
doi: 10.1038/sj.emboj.7601872. Epub 2007 Oct 11.

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

Bekir Cinar  1 , Ping-Ke Fang, Mohini Lutchman, Dolores Di Vizio, Rosalyn M Adam, Natalya Pavlova, Mark A Rubin, Pamela C Yelick, Michael R Freeman
Affiliations

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

Bekir Cinar et al. EMBO J. .

Abstract

Akt kinases mediate cell growth and survival. Here, we report that a pro-apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft-enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full-length Mst1, and N- and C-terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane-targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone-resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3-kinase/Akt pathway and suggest an important role in cancer progression.

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Figures

Figure 1
Figure 1
Endogenous Mst1 and Akt1 interaction in Triton-X-100-insoluble subcellular fractions. (A) p-T308-reactive protein(s) co-precipitate with Akt1 from lipid raft-enriched (TI) fractions. Akt1 was immunoprecipitated and blots were generated with antibodies to total Akt, p-T308 and p-S473. TS=Triton X-100-soluble fraction. (B) Mst1 and Akt1 interact principally in detergent-insoluble fractions. Akt1 was immunoprecipitated from cytoplasmic (Cyt), nuclear (Nuc), and TI fractions prepared from LNCaP cells. Input shows protein levels before IP. Data are representative of two individual experiments. (C) Full-length Mst1 forms a complex with Myr-Akt1. Cell lysates were immunoprecipitated with anti-Mst1- or anti-Akt1-specific antibody. (D) Dose-dependent interaction of purified Mst1 with Myr-Akt1. Myr-Akt1 transiently expressed in HEK 293T cells was immunoprecipitated with anti-HA antibody. Upper panels: immune complexes were incubated with the indicated amounts of purified Mst1 and resolved by SDS–PAGE. Immunoblots are against Mst1 and Akt1. Lower panels: the reciprocal experiment, showing interaction of purified Akt1 with Mst1 in HEK 293T cells (500 ng Akt1). (E, F) Protein-binding assay with purified Akt1 and Mst1. Purified Akt1 was precipitated with anti-Akt1 antibody and subsequently incubated with or without purified Mst1. In the reciprocal experiment, purified Mst1 was precipitated with anti-Mst1 antibody and then incubated with or without purified Akt1.
Figure 2
Figure 2
Mst1-N and Mst1-C interaction with Akt1. (A) Interaction of Akt1 with Mst1-N. Myr-Akt1 was transiently expressed in the absence or presence of Mst1-N in HEK 293T cells. Akt1 or Mst1 was immunoprecipitated and immune complexes were blotted with the reciprocal antibody. (B) Interaction of Akt1 with Mst1-C. T7-tagged Akt1 was transiently expressed in the absence or presence of HA-Mst1-C in HEK 293T cells. (C) Competition experiments. Myc-Mst1-N and Myr-Akt1 were coexpressed with increasing amounts (0, 2, 3, and 4 μg) of Myc-Mst1-C. Akt1 was immunoprecipitated and the presence of Myc-Mst1-N in Akt1 immune complexes assessed with anti-Myc antibody. (D) Co-precipitation of HA-Mst1 or kinase dead Mst1-K59R or Mst1-N-K59R, and Myr-Akt1. Lysates from HEK 293T cells transiently expressing wild type or Mst1-K59R and Myr-Akt1 were immunoprecipitated anti-Akt1 antibody. (E) Dose-dependent inhibition of Mst1–Akt1 interaction and Mst1 complex in vitro kinase activity by dominant-negative Mst1 (K59R). Upper panels: competition experiments (upper panels). Myc-Mst1 and Myr-Akt1 were transiently coexpressed with increasing amounts (0, 0.5, 1, and 2 μg) of Mst1-K59R in HEK 293T cells. Lysates expressing wild type, Mst1-K59R, and Myr-Akt1 were immunoprecipitated with anti-Akt1 antibody. Lower panels: Mst1 complex kinase activity. Mst1 was immunoprecipitated from lysates with anti-Myc antibody subjected to kinase assay using radioactive 32P-γ-ATP, followed by autoradiography. (F) Co-precipitation of HA-Mst1-N or kinase dead Mst1-N-K59R, and Myr-Akt1. Lysates from HEK 293T cells expressing Mst1-N or Mst1-N-K59R and Myr-Akt1 were immunoprecipitated with anti-Mst1 antibody.
Figure 3
Figure 3
Mst1 and caspase-cleaved Mst1 fragments interact with the Akt1 C-terminal region. (A) HA-Akt1 truncation and point mutants used for experiments in (B) and (E). (B) Co-precipitation of HA-Akt1 mutants with Myc-Mst1. Akt1 mutants were co-transfected with Myc-Mst1 in HEK 293T cells. Co-IP was performed with anti-HA antibody. Arrows on the right identify the relative location of Akt1 reactive bands. (C) Lysates from HEK 293T cells expressing wild-type Myc-Mst1 and wild-type Akt1, the phosphorylation site mutant Akt1-T308A, or the ATP-binding site mutant Akt1-K179M were immunoprecipitated with anti-Myc antibody and blotted for Akt1. Data in all panels are representative of at least two individual experiments. IgG heavy (H) chain is indicated. (D) Co-precipitation of Akt1 with Mst1. HEK 293T cells were co-transfected with Mst1 and Myr-Akt1, followed by treatment with PI3K inhibitor (LY294002; 20 μM) or vehicle (DMSO) for 60 min. Co-precipitation assay was performed in lysate. (E) Co-precipitation of Myc-tagged Mst1, Mst1-N, and Mst1-C with Akt1 truncation mutants. Mst1, Mst1-N, and Mst1-C were coexpressed with Akt1 mutants in HEK 293T cells. * Indicates nonspecific band. IgG heavy (H) and light (L) chains are indicated.
Figure 4
Figure 4
Immunofluorescent localization of Mst1 and Akt1. (A) Colocalization of Mst1 and Myr-Akt1 in COS cells. Confocal images of cells co-transfected with full-length HA-Mst1 and Myr-Akt1 or vector only (VO). Mst1 (Cy3, red) or Akt1 (Cy5, purple; shown here as pseudocolored ‘white'). Nuclei (DAPI, blue); lipid raft membranes (GM1, cholera toxin B (CTxB; green). (B, C) Localization of Mst1-N and Mst1-C, and GFP-Myr-Akt1 coexpressed in DU145 cells (B). Colocalization of Mst1 truncation mutants with endogenous phospho-Akt in LNCaP cells (C). (D) Relocalization of HA-Mst1 in response to Akt1 activation by IGF-1. DU145 cells were co-transfected with Mst1 (Cy3, Red) and GFP vector only or GFP-Akt1. After starvation, cells were treated with IGF (50 ng/ml) or BSA control. Data in (A–D) are representative of three individual experiments.
Figure 5
Figure 5
Mst1 antagonizes Akt1 signaling. (A) Knockdown of Mst1, Mst2, or Mst1/2 increases Akt phosphorylation (p-S473). LNCaP cells were transfected with a pool of siRNA oligos (100 nM) targeting Mst1 or Mst2. Scrambled siRNA oligos were a negative control. Lysates were prepared 48 h after transfection. The graph shows normalized signal intensity. (B–D) Mst1 inhibition of Akt activity and Mst1-mediated apoptosis in LNCaP cells. Mst1, Mst1-N, and Mst1-C constructs were expressed for 36 h (B, C). Mst1, Mst2, or Mst1 and Mst2 (Mst1/2) were knocked down for 36 h, followed by treatment with LY294002 (20 μM) or DMSO for 60 min (D, E). Cell death was measured using cells grown overnight under serum-free conditions. Akt phosphorylation (S473) and kinase activity in (B) (blots) and (D) were determined in lysates. (F) The effects of Mst1/2 knockdown on Akt substrate phosphorylation. Mst1 and Mst2 were knocked down for 36 h. Detergent-soluble (TS) and lipid raft-enriched (TI) fractions were prepared from Mst1/2 or control (scrambled pool) siRNA-transfected LNCaP cells. Phosphorylated Akt substrates were recognized by phospho-Akt substrate antibody.
Figure 6
Figure 6
Mst1–Akt complex formation and inhibition of EGF-induced Akt activity. (A) MC3T3 cells were treated with 100 nM staurosporine (STR) or DMSO vehicle for 3 h. Akt1 was immunoprecipitated from lysates and subjected to kinase assay. Relative Akt1 kinase activity was determined by immunoblotting of IP eluates with antibodies to phosphorylated GSK3 (p-GSK3α/β) and Akt. Inputs were blotted with antibodies to total and phosphorylated Mst1 to confirm STR-induced phosphorylation of Mst1. Right panel: co-precipitation of endogenous Akt1 with exogenous Mst1. MC3T3 cells were transfected with Mst1. At 24 h, cells were treated with STR or DMSO. Co-precipitation assay was performed in lysate. (B, C) Endogenous Akt1 is inhibited by wild-type Mst1, Mst1-N, and Mst1-C. COS cells were transfected with respective plasmids. Cells were stimulated with EGF (20 ng/ml) or vehicle for 10 min under serum-free conditions. Akt phosphorylation and kinase activity were determined in lysates. Dominant negative (Mst1-KD) inhibits the effect of Mst1 on Akt1 activity (C).
Figure 7
Figure 7
Mst1 inhibits Akt1 in zebrafish development. Injection of MST1, MST1-KD (K59R), MST1-N, MST1-C, or MST1-N (K59R) cRNA alone or together with MYR-AKT1 cRNA into single-cell stage zebrafish embryos. Data were collected 24 h post-fertilization. Data are representative of multiple injections. Results are presented in Table I (experiments A and B).
Figure 8
Figure 8
TMA analysis of Mst1 and Akt1 expression in human prostate tissues. (A, B) High magnification of Mst1 (A) and Akt1 (B) immunostaining, showing similar subcellular localization. (C, D) Selected images of TMA cores showing immunohistochemical staining of Mst1 (A) and Akt1 (B) in prostate cancer progression. The micrographs are representative of quantitative data obtained by ChromaVision analysis. (E) Selected TMA cores showing immunhistochemical staining with p-Akt (S473) antibody. MET, metastasis; PCA, organ confined prostate cancer. (F) Relative intensities of Mst1 and p-Akt as assessed by ChromaVision. Benign tissue (N, n=18), prostate adenocarcinoma (PCA, n=36), hormone naive (HN) metastases (n=19), hormone refractory (HR) metastases (n=17). *P⩽10−6, **P⩽10−3. Error bars denote 95% confidence intervals.
Figure 9
Figure 9
A model illustrating Mst1/2 inhibition of Akt. Mst1/2 and Akt reside in an endogenous complex in cholesterol-rich, lipid raft membranes. Following activation of an apoptotic signal, cytosolic and possibly lipid raft-residing Mst1/2 inhibit Akt. Mst1 caspase cleavage products bind to and inhibit Akt in the cytoplasm and nucleus. Association of Mst1/2 with activated Akt may provide a rapid means of inhibiting the PI3K/Akt pathway during apoptosis and/or providing negative feedback when Akt is activated.
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