Proapoptotic kinase MST2 coordinates signaling crosstalk between RASSF1A, Raf-1, and Akt

Abstract

Mammalian MST kinases function in stress-induced apoptosis to limit tumor progression. However, there is limited understanding about MST2 control by key regulators of cell division and survival. Raf-1 binds and inhibits MST2 kinase, whereas dissociation from Raf-1 and binding to tumor suppressor protein RASSF1A activates MST2. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras, or depletion of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10. We identified T117 and T384 as Akt phosphorylation sites in MST2. Mutation of these sites inhibited MST2 binding to Raf-1 kinase but enhanced binding to tumor suppressor RASSF1A, accentuating downstream c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase signaling and promoting apoptosis. We determined that MST2 phosphorylation by Akt limits MST2 activity in two ways: first, by blocking its binding to RASSF1A and by promoting its association into the Raf-1 inhibitory complex, and second, by preventing homodimerization of MST2, which is needed for its activation. Dissociation of the Raf-1-MST2 complex promoted mitogenic signaling and coordinately licensed apoptotic risk. Using Ras effector domain mutants, we found that Akt is essential to prevent MST2 activation after mitogenic stimulation. Our findings elucidate how MST2 serves as a hub to integrate biological outputs of the Raf-1 and Akt pathways.

Figure 1. PI3K/Akt regulates MST2-Raf1 interaction and MST2 activity

Serum starved HeLa cells (c) were treated with (a) LY294002 (LY, 5μM) or (b) Akt inhibitor (Akt I, 10μM) in the presence or absence of serum 10% (serum+LY or serum+Akt I) for the indicated periods of time. In the serum+LY and serum +Akt I conditions, cells were pre-incubated 30min with LY or Akt I, respectively, before stimulation with 10% serum. MST2 or Raf1 immunoprecipitates (IP) were analyzed by Western blots using antibodies for the indicated proteins. MST2 kinase activity was determined by in gel kinase assays as described in Materials and Methods. 10μg of cellular extracts were analyzed by Western blotting using phospho-S473Akt or phospho-S21GSK3β antibodies, and then re-probed with antibodies against total Akt and total GSK3β, respectively.

Figure 2. MST2 interacts with Akt and a target for Akt induced phosphorylation

(a) Serum starved HeLa and MCF7 cells were treated with serum 10% for 2hours in the presence or absence of LY294002 (LY; pre-incubated for 30min). MST2 immunoprecipitates (IP) were analyzed by Western blotting using phospho-serine/threonine substrate of Akt (P-Akt substrate) and total Akt antibodies. 10μg cellular extracts were Western blotted using a phospho-S473Akt antibody, and then re-probed with a total Akt antibody. (b) MCF7 cells were co-transfected with increasing amounts of gag-Akt (0, 0.1μg, 0.25μg, 0.5μg and 1μg) and HA-RASSF1A where indicated. MST2 IPs were analyzed by Western blotting with the indicated antibodies. MST2 kinase activity was measured by in gel kinase assays. 10μg of cellular extracts were analyzed by Western blot using HA antibody. (c) HeLa cells were transfected with HRasV12 (V12), HRasV12S35 (V12S35) or HRasV12C40 (V12C40). Raf-1 and MST2 IPs were analyzed by Western blot using antibodies as stated. MST2 kinase activity was determined as above. 10μg of cellular extracts were analyzed by Western blot using antibodies against the indicated proteins.

Figure 3. Regulation of endogenous MST2 activation by Akt, PTEN and Raf-1

(a) 10μg cellular extracts from MCF7, HeLa and HCC1937 cells were analyzed for Akt activation (phospho-S473Akt), expression of Akt, RASSF1A and PTEN. (b) HCC1937 cells were co-transfected with the indicated combinations of wild type PTEN, phosphatase dead PTEN C124S, and small interference RNAs for Raf-1 (siRNA Raf-1) or a control (siRNA C). MST2 immunoprecipitates were analyzed by Western blotting using antibodies as stated. MST2 kinase activity was tested by in gel kinase assays. 10μg cellular extracts were analyzed by Western blotting using the indicated antibodies. (c) Akt was downregulated by siRNA. Cell extracts, Raf-1 and MST2 IPs were analyzed by Western blotting with the indicated antibodies. (d) Cells were co-transfected with Myc- and Flag-tagged MST2 and either wild type PTEN (wt) or phosphatase-dead mutant PTEN (C124S). IP and Western blot analysis were performed as described above.

Figure 4. MST2 phosphorylation by Akt at T117 and T384 regulates MST2 kinase activity and Raf-1 interaction

(a) HCC1937 cells were transfected with empty vector (c), Flag-tagged MST2 wild type (wt), single mutants T117A and T384A (117A, 384A), the double mutant T117/384AA, (AA) or the inactive mutant T180A (180A). PTEN was co-expressed where indicated. Flag- IPs were analyzed by Western blot using specific antibodies as stated. MST2 kinase activity was determined by in gel kinase assays. (b) Cells were co-transfected with Flag-tagged MST2 mutants and control (siRNA control) or Raf-1 specific (siRNA Raf-1) siRNAs. MST2 IPs were analyzed for kinase activity. (c) Cells were co-transfected with GFP-MST2 or the GFP-MST2 double mutant T117/384AA and mRFP1-Raf1. The interaction between MST2 and Raf-1 was imaged by FLIM in cells treated with or without LY292002 (5μM, 2 hours). FRET between GFP and mRFP1 results in shortening of the fluorescent lifetime (τ) of GFP. The graph shows FRET efficiency calculated using the control lifetime (GFP alone) and effect of LY292002 (inset, 11 cells per group). Statistical analysis was performed using Student's t-test. (d) FLIM of cells co-expressing the GFP-MST2 double mutant T117/384AA and mRFP1-Raf1.

Figure 5. Akt phosphorylates Akt directly and prevents binding to RASSF1A

(a) HCC1937 cells were transfected with empty vector, Flag-tagged wild type MST2 (wt), the double mutant T117/384AA (AA) or the inactive mutant T180A (180A). PTEN wild type (wt) or the phosphatase-dead mutant C124S were co-expressed as indicated. RASSF1A and Raf-1 immunoprecipitates and 10μg cellular extracts were analyzed by Western blotting using the indicated antibodies. (b) Purified MST2 was incubated with purified Akt plus/minus ATP in a total volume of 100μl. 2μl aliquots were immunoblotted with the indicated antibodies. The remaining samples were divided in two and incubated with (c) Flag-Raf-1 or (d) Flag-RASSF1A immunoprecipitates. After washing MST2 proteins bound to the immunoprecipitates were examined with the indicated antibodies.

Figure 6. Akt protection against apoptosis involves MST2 inhibition

(a) HeLa cells were transfected with control or MST2 siRNA and treated or not (control) for 15hours with 5μM LY294002 (LY) or 5μM Akt inhibitor (Akt I). Apoptosis was quantified by assaying DNA fragmentation as described in Materials and Methods. Statistical analysis was performed using the Student's t-test (n=4). (b) 10μg cellular extracts were analyzed for full length MST2 expression by Western blot. (c) HeLa cells were transfected with empty vector (control), Flag-tagged wild type MST2 (wt), MST2 single mutants T117A and T384A (117A, 384A), the double mutant T117/384AA (AA), or the inactive mutant T180A (180A). Apoptosis was measured as above. Statistical analysis was performed using Student's t-test (n=5). There were no statistically significant differences between control and the T180A mutant, and wt MST2 and T180A. (d) HCC1937 cells were co-transfected with MST2 constructs as described above with or without (mock) PTEN. Apoptosis was measured as above.