Figure 1.
TPA-induced ERK activation, Thr 163 phosphorylation, and Mcl-1 stabilization occur rapidly and are subsequently downregulated. A:
The phosphorylation sites at Thr 163 and Ser 159 in the human Mcl-1 protein are diagrammed. Thr 163 is subject to phosphorylation by MAP kinases, as seen upon TPA-induced ERK activation in BL41-3 cells. Ser 159 is subject to phosphorylation by GSK3. Mcl-1 is a PEST protein subject to rapid turnover, the half-life of decay being ∼3 hours in BL41-3 cells (stippling of lesser versus greater density indicates poor PEST and potential PEST sequences; PESTFIND). However, Mcl-1 exhibits striking stabilization upon TPA-induced ERK activation and Thr 163 phosphorylation in these cells. Mcl-1 is also subject to another posttranslational modification involving truncation of the extreme N-terminus ([28] indicated with an arrowhead). This results in a closely spaced 42/40 kd doublet, where the lower 40 kd band lacks ∼16 amino acid residues and is the most abundant band present in BL41-3 cells. The doublet is best visualized on large format electrophoresis gels (Fig. S1B) but can sometimes be detected on standard gels (Fig. 1C). As Thr 163 phosphorylation does not prevent N-terminal truncation, Mcl-1 stabilization in the presence of this phosphorylation is seen as slowed decay of the 40 kd band. B: BL41-3 cells were either left untreated or exposed to TPA (5 nM) for the indicated times, and monitored for expression of Thr 163 phosphorylated Mcl-1 (Mcl-1 pT163), total Mcl-1, phosphorylated ERK (pERK), and GAPDH (ChemiDoc). All samples were run at the same time and subjected to the same autoradiographic exposure, where the black vertical line in this and subsequent figures indicates that lanes have been rearranged to facilitate comparison. Thr 163 phosphorylation was also induced by lower concentrations of TPA (e.g., 1 nM; Fig. S1C), and was inhibited by U0126 (Fig. S1D). m C: BL41-3 cells were either left untreated or exposed to TPA (1 nM). Some cells were immediately exposed to CHX to monitor Mcl-1 protein decay on Day 0. Additional TPA-treated cells were incubated for 24 hours (Day 1 after TPA Addition) and then exposed to CHX, where a portion of these cells was retreated with TPA at this time. The untreated cell sample (Time 0) is identical for each pair of Western blots. The blot at the bottom confirmed that pERK was reduced at 24 hours.
Figure 2.
Mcl-1 degradation is insensitive to the GSK3 inhibitor LiCl in BL41-3 cells.
A: BL41-3 cells were either left untreated or exposed to TPA (20 nM) and/or LiCl (20 mM, or NaCl as a control). After 18 hours, expression of Mcl-1, beta-catenin, and GAPDH and was assayed by Western blotting. Comparable results were observed when the cells were exposed to TPA for 30 minutes instead of 18 hours (not shown). B: BL41-3 cells were exposed to the indicated concentrations of LiCl (or KCl or NaCl as controls), and assayed for expression of Mcl-1 and beta-catenin after 24 hours. A large format gel that separates the Mcl-1 doublet bands was used. Slight inhibition of cell growth was seen at 10–20 mM LiCl (13 and 27%, respectively as compared to 5% with 20 mM NaCl). Results similar to those shown were obtained with an exposure time of 12 hours or a LiCl concentration of 40 mM (not shown). C: BL41-3 cells were incubated in the absence or presence of LiCl (20 mM) for 0.5 hours, at which time CHX was applied. Expression of Mcl-1, beta-catenin, and GAPDH was assayed after the indicated times. No substantial difference was seen in parallel cells exposed to NaCl (20 mM, not shown) instead of LiCl.
Figure 3.
Mcl-1 degradation is slowed by a T163E mutation (but not T163A) in transfected CHO cells.
A: CHO cells were transfected with WT-Mcl-1 and incubated in the presence of LiCl (+LiCl; 20 mM) or in its absence (−LiCl) where 20 mM NaCl was added in the latter case. After 10 hours, CHX was applied and expression of the introduced WT-Mcl-1 gene product, and endogenous beta-catenin and GAPDH, was assayed after the indicated times (ChemiDoc). The half-life of Mcl-1 decay was estimated to be ∼4 hours in cells exposed to LiCl, and 3.7 hours in the controls exposed to NaCl. No substantial difference was seen in parallel cells not exposed to NaCl or LiCl. The blot shown is representative of three independent experiments. B–C: CHO cells were transfected with the indicated constructs, replated on the following day, and incubated for a 24-hour period to allow expression. CHX was then added and expression of the introduced Mcl-1 gene product and endogenous beta-tubulin was assayed after the indicated times by Western blotting. In Panel B, the decline in Mcl-1 expression at 3 hours in 2 independent experiments ranged from 53–66% with WT-Mcl-1, 25–54% with Mcl-1-S162A, and 25–50% with Mcl-1-T163A. While the decline in expression with WT-Mcl-1 appeared be slightly greater than that seen with Mcl-1-T163A, this was not a consistent finding. A short autoradiographic exposure is also shown because additional bands were detected at the high levels of expression obtained with Mcl-1-T163E. At the end of the 12-hour observation period, expression of Mcl-1-T163E was decreased by ∼15% in Panel B and ∼27% in Panel C, as estimated from short autoradiographic exposures. D: AKR-2B cells transfected with the indicated constructs were replated on the following day, at which time cell viability (trypan blue dye exclusion) was 88–92% in untransfected as well as transfected cultures. One day later, cells were exposed to 25 micrograms/ml CHX and assayed after the indicated times for expression of the introduced Mcl-1 gene product or endogenous actin by Western blotting. Duplicate plates are shown in adjacent lanes.
Figure 4.
Slowed degradation without increased synthesis increases Mcl-1 accumulation in the presence of the T163E mutation.
A: CHO cells were co-transfected with either WT-Mcl-1 or Mcl-1-T163E along with pEGFP, and assayed at the indicated times for expression of the introduced Mcl-1 gene product and EGFP by Western blotting. The rate of increase of the Mcl-1-T163E protein was estimated to be at least 10 times greater than that of WT-Mcl-1. Approximately equivalent expression of EGFP in WT-Mcl-1- and Mcl-1-T163E-transfected cultures was also seen upon assay by flow cytometry (not shown). B: CHO cells were transfected with WT-Mcl-1 or Mcl-1-T163E and subjected to a 2-hour pulse exposure to 35SMet, at which time a “time 0” sample was harvested. A chase with medium containing non-radioactive methionine was then performed and the 35SMet- labeled Mcl-1 proteins were assayed after the indicated times (upper photograph). A non-specific band of ∼32 kd is included on the figure (*) for comparative purposes and a separate aliquot of each sample was assayed for total Mcl-1 expression by Western blotting (lower photograph). The half-life of decay was estimated by PhosphorImager to be ,2.5-fold longer with 35S-Met- Mcl-1-T163E than with 35S-Met-WT-Mcl-1.
Figure 5.
Ara-C-induced Mcl-1 degradation and cell death are inhibited upon TPA-induced ERK activation in BL41-3 cells.
A: BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours, followed by the addition of the indicated concentrations of Ara-C. After 24 hours, PARP cleavage and Mcl-1 expression were assayed (ChemiDoc). A representative experiment is included (upper photograph) along with the average (± SE) of 5 independent experiments. Also shown for comparative purposes is the percentage of cells exhibiting morphological apoptosis, assayed with a concentration of 1 nM TPA applied 1 hour before the addition of Ara-C (mean of 3 independent experiments ± SE run in parallel with previous studies; some of the BL41-3 cell samples not treated with TPA were included in a previous publication comparing these to BL41 parental cells (Fig. 4 in reference [23]). *significant difference in the presence of Ara-C as compared to its absence (p<0.05; ANOVA, post-hoc Holm-Sidak test). B: BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours, followed by the addition of the indicated concentrations of Ara-C. After 6 hours, PARP cleavage and Mcl-1 expression were assayed as in Panel A. The average of 4 experiments is shown below representative blots, where the values for Ara-C concentrations that had similar effects are shown together and overlined. For PARP Cleavage, the SE of the values shown in successive lanes was 1, 8, 7, 3, and 3, respectively. The Decrease in Mcl-1 Expression was calculated relative to untreated control cells; the SE of the values shown (other than the untreated control value) was 11, 8, 8, and 7. Linear regression analysis of the Decrease in Mcl-1 Expression versus PARP Cleavage for 0–10 micromolar Ara-C in the absence of TPA demonstrated an average slope of 1.2 [±0.49 (SE) for the 4 experiments)], with an average r2 value of 0.87.
Figure 6.
TPA-induced Mcl-1 stabilization is associated with increased resistance to various chemotherapeutic agents in BL41-3 cells.
A: BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours, followed by the addition of the indicated concentrations of etoposide. After 6 hours, PARP cleavage, Mcl-1 expression, and Thr 163 phosphorylation were assayed (ChemiDoc), where the values for PARP Cleavage (%) and the Decrease in Mcl-1 Expression (%) are indicated below the respective blots. B: BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours prior to the addition of the indicated concentrations of vinblastine. After 6 hours, PARP cleavage and Mcl-1 expression were assayed as in Panel A. C: BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours prior to the addition of the indicated concentrations of cisplatin. After 24 hours, PARP cleavage and Mcl-1 expression were assayed as in Panel A. The blot shown is representative of 3 independent experiments. The SE of the values shown in successive lanes for PARP Cleavage was 2, 2, 5, 11, 17, 0.2, 4, 2, 3, 4, 6, and 13, respectively. The Decrease in Mcl-1 Expression was calculated relative to untreated control cells; the SE of the other values shown was 10, 12, 18, 12, 7, 9, and 11.
Figure 7.
Inhibition of TPA-induced ERK activation partially restores chemotherapy-induced Mcl-1 degradation and cell death.
A: BL41-3 cells were exposed to U0126 (25 micromolar) for 0.5 hours, followed by the addition of TPA (5 nM) for 0.5 hours and then addition of the indicated concentrations of Ara-C. After 24 hours, PARP cleavage and Mcl-1 expression were assayed (ChemiDoc). The blot shown is representative of 3 independent experiments, where similar effects were seen with the various Ara-C concentrations as above (Fig. 5A). In the graph below the blot, the Decrease in Mcl-1 Expression (±SE) is plotted against PARP Cleavage (±SE), where points representing the average of cells treated with 1–10 mM Ara-C are labeled and the unlabeled points represent cells not treated with Ara-C. B: BL41-3 cells were exposed to U0126 (25 micromolar) for 0.5 hours, followed by the addition of TPA (5 nM) for 0.5 hours and then addition of the indicated concentrations of etoposide. After 6 hours, PARP cleavage and Mcl-1 expression were assayed. The blot shown is representative of 3 independent experiments. The lanes that are overlined on the Mcl-1 blot represent concentrations of etoposide where U0126 resulted in partial inhibition of Mcl-1 stabilization. In the graph, the average Decrease in Mcl-1 Expression for each concentration of etoposide (±SE) is plotted against average PARP Cleavage (±SE). The lines shown for No TPA and U0126+ TPA were determined by linear regression, where the points representing 10 and 100 micromolar etoposide are labeled. The slope of this line averaged 0.85 in the absence of TPA [±0.1 (SE); average r2 of 0.92], and 1.0 in the presence of U0126+ TPA (±0.19; average r2 of 0.7).