MEK-ERK is involved in SUMO-1 foci formation on apoptosis

Abstract

Small ubiquitin-related modifier (SUMO) modification appears to regulate the activity, intracellular localization, and stability of the targeted proteins. To explore the relationship among sumoylation, antitumor reagent, and apoptosis, we treated green fluorescence protein (GFP)-SUMO-1-overexpressed K562 cells (K562/GFP-SUMO-1) with mitoxantrone (MIT) as an antitumor reagent. By the treatment with MIT, GFP-SUMO-1 formed foci in nuclei. While by the treatment with a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), GFP-SUMO-1 located homogeneously in nuclei. When K562/GFP-SUMO-1 cells were treated with TPA plus MIT, GFP-SUMO-1 foci became larger and apoptosis was induced more than with MIT alone. The apoptosis induced by TPA plus MIT was prevented by blockage of GFP-SUMO-1 foci by small interfering RNA (siRNA) against SUMO-1. The formation of GFP-SUMO-1 foci was reduced by a MEK inhibitor U0126 or a nuclear export inhibitor leptomycin B, and endogenous SUMO-1 foci were reduced in K562 cells expressing the dominant-negative MEK1 mutant. These results suggest that the formation of SUMO-1 foci is regulated by the MEK-ERK pathway and may induce apoptosis.

Figure 1

Relationship between the characteristics of expression of GFP‐SUMO‐1 and the sensitivity to apoptosis in the treatment of mitoxantrone (MIT) with or without 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). (a) panels a–d, K562/GFP‐SUMO‐1 cells were treated with or without TPA (2 nM), MIT (0.5 µg/mL), and TPA plus MIT for 24 h, followed by TdT‐mediated dUTP nick end labeling (TUNEL) staining. Data were analyzed by laser scanning confocal microscopy. Green and red signals indicate GFP‐SUMO‐1 and TUNEL‐positive cells, respectively. Bar, 20 µm. (b) Wild‐type K562 cells and K562/GFP‐SUMO‐1 cells were treated as described in (a). TUNEL‐positive wild‐type K562 cells (white bar) and K562/GFP‐SUMO‐1 cells (black bar) were counted on three different fields counting 200 cells per field. Means ± SD from triplicate determination are shown. *P < 0.01.

Figure 2

Blockage of the formation of small ubiquitin‐related modifier (SUMO)‐1 foci by siRNA decreases apoptosis. (a) The transfection of siRNA for SUMO‐1 to K562/GFP‐SUMO‐1 cells for 48 h, the cells were treated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM) plus mitoxantrone (MIT) (0.5 µg/mL) for 24 h, and lysed with sample buffer. Western blot analysis was performed with anti‐SUMO‐1 and anti‐actin antibodies. (b) The cells were treated as described in (a) followed by TdT‐mediated dUTP nick end labeling (TUNEL) staining. Data were analyzed by laser scanning confocal microscopy. Green, red, and blue signals indicate GFP‐SUMO‐1, TUNEL, and Hoechst 33258, respectively. Bar, 50 µm. (c) TUNEL‐positive K562/GFP‐SUMO‐1 cells were counted on three different fields counting 200 cells per field. Means ± SD from triplicate determination are shown. *P < 0.01.

Figure 3

Inhibition of GFP‐SUMO‐1 foci formation by U0126. (a) Schedules for treating K562/GFP‐SUMO‐1 cells with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM), phorbol 12, 13‐dibutyrate (PDBu) (2 nM), mitoxantrone (MIT) (0.5 µg/mL), and U0126 (10 µM). (b) Panels a‐l, the cells were treated as described in (a), followed by TdT‐mediated dUTP nick end labeling (TUNEL) staining, and analyzed by laser scanning confocal microscopy. Green and red signals indicate GFP‐SUMO‐1 and TUNEL‐positive cells, respectively. Bar, 20 µm. (c) The cells were treated as described in (a) and lysed with sample buffer. Western blot analysis was performed with anti‐phospho‐ERK1/2 and anti‐ERK1/2 antibodies.

Figure 4

Inhibition of GFP‐SUMO‐1 foci formation by leptomycin B (LMB). (a) Schedules for treating K562/GFP‐SUMO‐1 cells with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM), phorbol 12, 13‐dibutyrate (PDBu) (2 nm), mitoxantrone (MIT) (0.5 µg/mL), and LMB (10 nM). (b) The cells were treated as described in (a). Data were analyzed by laser scanning confocal microscopy. Green and red signals indicate GFP‐SUMO‐1 and TdT‐mediated dUTP nick end labeling (TUNEL)‐positive cells, respectively. Bar, 20 µm. (c) The cells were treated as described in (a) and lyzed with sample buffer. Western blot analysis was performed with antiphospho‐ERK1/2 and anti‐ERK1/2 antibodies.

Figure 5

Inhibition of endogenous small ubiquitin‐related modifier (SUMO) foci formation in MEK1 mutant K97A. (a) Panels a‐h, K562 cells were transfected with mock (K562/mock) and MEK1 mutant K97A (K562/MEK1 K97A), and treated with or without 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM), mitoxantrone (MIT) (0.5 µg/mL), and TPA plus MIT for 48 h. The cells were fixed and immunostained with anti‐SUMO‐1 antibody followed by Alexa Fluor 488 conjugated antisecondary antibody, and then the nuclei were counter‐stained with TOPRO‐3. Data were analyzed by laser scanning confocal microscopy. Green and red signals indicate endogenous SUMO‐1 and TOPRO‐3, respectively. Bar, 20 µm. (b) The cells were treated with TPA (2 nM), MIT (0.5 µg/mL), and TPA plus MIT for 6 h and lysed with sample buffer. Western blot analysis was performed with anti‐phospho‐ERK1/2 and anti‐ERK1/2 antibodies. (c) The cells were treated with TPA plus MIT for 0 h, 24 h, 48 h, or 72 h followed by TdT‐mediated dUTP nick end labeling (TUNEL) staining. TUNEL‐positive K562/mock cells (white bar) and K562/MEK1 K97A cells (black bar) were counted on three different fields counting 200 cells per field. Means ± SD from triplicate determination are shown. *P < 0.01.

Figure 6

The transition of GFP‐SUMO‐1 expression by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and mitoxantrone (MIT). (a) Before treatment: there are some GFP‐SUMO‐1 particles in the nucleus (black circles). (b) TPA treatment: the expression of GFP‐SUMO‐1 particles is increased dramatically and homogeneously in the nucleus. (c) MIT treatment: more than two GFP‐SUMO‐1 particles accumulate and form small foci in the nucleus. (d) TPA plus MIT treatment: the GFP‐SUMO‐1 focus becomes larger than with MIT alone. Furthermore, some particles and small foci (MIT type) are also mixed in the nucleus.