MST kinases monitor actin cytoskeletal integrity and signal via c-Jun N-terminal kinase stress-activated kinase to regulate p21Waf1/Cip1 stability

Abstract

As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21(Waf1/Cip1) (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.

FIG. 1.

MST2 colocalizes with F-actin. (A) NIH 3T3 cells transfected with plasmid encoding FLAG epitope-tagged MST2 were fixed and stained with anti-FLAG antibody and Texas red phalloidin to visualize F-actin structures. TIRF microscopy was used to examine MST2 distribution proximal to the cell-substrate interface. While epifluorescence (EPI) microscopy showed that the MST2 distribution was a mixture of diffuse and organized, TIRF revealed that the MST2 distribution was strikingly similar to that of the actin filaments observed at the region of the cell that is enriched for these cytoskeletal structures. Scale bar = 10 μm. (B) To validate antibodies for immunofluorescence, MST2 was knocked down with siRNA in NIH 3T3 cells (left). MST2 levels were comparable in untransfected (Un), mock-transfected, or nontargeting control (NTC)-transfected cells, but the levels were reduced in cells transfected with MST2 siRNA. Untransfected (upper right) and MST2 siRNA-transfected (lower right) cells were stained with anti-MST2 antibody and analyzed by immunofluorescence. MST2 knockdown detected by Western blotting also was observed by immunofluorescence with one commercial antibody. Scale bars = 50 μm. (C) NIH 3T3 cells were fixed and stained with anti-MST2 antibody and Texas red phalloidin to visualize F-actin structures. TIRF microscopy was used to examine MST2 distribution proximal to the cell-substrate interface; two examples are shown. As with transfected MST2, TIRF revealed that the endogenous MST2 distribution was similar to that of the actin filaments observed at the region of the cell that is enriched for these cytoskeletal structures. Scale bar = 10 μm.

FIG. 2.

Actin cytoskeleton disruption activates MST kinases. (A) MST2 immunoprecipitated from NIH 3T3 cells treated with cell-permeable TAT-C3 exoenzyme (0.5 μM) had significantly increased activity compared to that from untreated control cells using an in-gel kinase assay (*, P < 0.05 by Student's t test; data are means ± standard errors of the means; n = 3). The lower band in the kinase assay likely represents immunoprecipitated (IP) MST1 due to the cross-reactivity of the antibody. The blotting of whole-cell extracts (WCE) showed equivalent levels of MST2 and ERK2 in each condition. Treatment with Tat-C3 produced a characteristic change in the mobility of a proportion of RhoA. (B) Disruption of the actin cytoskeleton in NIH 3T3 fibroblasts with CTD (200 nM), LTB (200 nM), or Tat-C3 (0.5 μM). Cells were fixed and stained with Texas red phalloidin to visualize F-actin structures. (C) Whole-cell extracts were separated by SDS-PAGE, and in-gel kinase assays were carried out with MBP embedded in the gel as the kinase substrate. The disruption of the actin cytoskeleton increased MBP phosphorylation at a position coincident with MST1 and MST2 mass (P-MBP). The positions of molecular size markers are indicated. The quantification of ³²P incorporation revealed that the increase (n-fold) in kinase activity relative to that of untreated cells was significantly increased in CTD- and Tat-C3-treated cells (**, P < 0.01; *, P < 0.05; both by Student's t test; data are means ± standard errors of the means; n = 6).

FIG. 3.

JNK activation by actin disruption via MST. (A) Activation of JNK and sensitivity to JNK inhibitor SP600125 were determined by Western blotting for c-Jun phosphorylation (left). The ratios of phospho-c-Jun/total c-Jun relative to those of untreated cells are shown in right panel. Treatment with CTD significantly induced c-Jun phosphorylation that could be significantly reduced by SP600125, similarly to the effects of MST1 or MST2 expression. (*, P < 0.05 by Student's t test compared to results for untreated cells or cells treated with SP600125 as indicated; data are means ± standard errors of the means; n = 3). (B) Expression of FLAG-MST1 or FLAG-MST2 increased MBP kinase activity above basal levels, while a kinase-dead version of MST2 (MST2KD) inhibited basal kinase activity, indicating that it acts in a dominant-negative manner. WCE, whole-cell extract. (C) Expression of FLAG-MST1 or FLAG-MST2 increased endogenous JNK1 kinase activity. JNK1 was immunoprecipitated (IP) from transfected cells and assayed for the phosphorylation of recombinant c-Jun in vitro. (D) Dominant-negative kinase-dead MST2 (MST2KD) inhibited JNK1 kinase activity induced by TatC3. (E) Dominant-negative kinase-dead MST2 (MST2KD) inhibited JNK1 kinase activity induced by actin cytoskeleton disruption (**, P < 0.01; *, P < 0.05; both by Student's t test; data are means ± standard errors of the means; n = 13). (F) Knockdown of MST2 by siRNA significantly reduced the CTD-induced c-Jun phosphorylation (*, P < 0.01 by Student's t test; data are means ± standard errors of the means; n = 3).

FIG. 4.

Turnover of p21 is inhibited by MST activation of the JNK pathway. (A) Inhibition of protein translation with emetine for 2 h revealed that FLAG-p21 levels decreased while RhoA and ERK2 levels remained constant, indicating that p21 is relatively unstable. The inhibition of RhoA with Tat-C3 maintained p21 levels remaining after emetine treatment, indicating increased protein stability. (B) Disruption of the actin cytoskeleton increased the proportion of p21 remaining after emetine treatment, which is indicative of increased protein stability (*, P < 0.05 by Student's t test; data are means ± standard errors of the means; n = 6). (C) Increased levels of p21 following 2 h of emetine treatment in cells expressing FLAG-tagged MST1 or MST2, which is indicative of increased p21 stability. (D) The increased p21 stability induced by MST2 expression could be reversed by the JNK inhibitor SP600125 (SP; 30 μM) (*, P < 0.05 by Student's t test; data are means ± standard errors of the means; n = 4). (E) Stabilization of p21 induced by Tat-C3 can be reversed by kinase-dead MST2.

FIG. 5.

Thr57 is required for p21 stabilization by actin disruption, MST kinases, or JNK. (A) Phosphorylation sites reported to regulate p21 stability were mutated individually to nonphosphorylatable alanine residues. Mutants were expressed and protein levels determined following treatment with or without Tat-C3 and the protein synthesis inhibitor emetine as indicated. While Tat-C3 elevated levels of wild-type p21 in emetine-treated cells, indicating increased protein stability, the nonphosphorylatable T57A mutant was not stabilized in response to Tat-C3. S98A and T145A mutants were stabilized by Tat-C3 compared to the stabilization of untreated cells, while the S130A and S146A mutants appeared to increase basal stability. Therefore, T57 appeared to be the critical site for stabilization in response to Tat-C3. (B) Stabilization of p21 by Tat-C3 or direct activation of the JNK pathway by the MAP3K MEKK1. (C) Stabilization of p21 by Tat-C3 or MEKK1 was reduced in the T57A p21 mutant. (D) The p21 T57A mutant no longer was stabilized by MST1 or MST2.

FIG. 6.

Association of JNK with p21 and phosphorylation on Thr57. (A) Immunoprecipitated (IP) FLAG-tagged p21 copurifies with endogenous JNK kinases. Treatment with Tat-C3 did not affect the levels of copurified JNK protein. WCE, whole-cell extract. (B) The ability of recombinant JNK1 to phosphorylate p21 was greatly reduced by the mutation of T57 to alanine. WB, Western blotting.

FIG. 7.

Model of role played by MST kinases linking the integrity of the actin cytoskeleton via the JNK SAPK pathway to the phosphorylation and stability of p21. (A) Under conditions of actin cytoskeletal integrity, MST kinases have low activity and do not significantly activate the JNK SAPK pathway. As a consequence, p21 is not phosphorylated on Thr57 and is relatively unstable. (B) Following the disruption of cytoskeletal integrity, MST becomes activated, leading to increased JNK SAPK activity and increased p21 phosphorylation on Thr57, resulting in protein stabilization.