Modulation of MEK1b expression affects Golgi fragmentation during mitosis. (A) MEK1b affects Golgi architecture during stages of mitosis. HeLa cells were transfected with GFP, GFP-MEK1b (MEK1b), or Sh-MEK1b with GFP (Sh-MEK1b). The cells were synchronized by double-thymidine block, and 9 h after the release from the block were stained with p58 Abs and DAPI. Cells from interphase, prophase, prometaphase, and metaphase were selected by their DNA structure. Bar, 5 μm. (B) Quantification of Golgi fragmentation in prophase prometaphase and metaphase. The cells in prophase and prometaphase were counted, and the percentage of cells with fragmented Golgi out of total transfected cells is presented as means ± SE of three experiments (n = 40). The cells in metaphase were counted, and the percentage of cells with partially intact Golgi per total transfected cells is presented as means ± SE of three experiments. A Student's t-test was used to determine statistical significance. (*) P < 0.05; (**) P < 0.01.

MEK1b induces Golgi fragmentation. (A) The effect of MEKs constructs on Golgi architecture. HeLa cells were transfected with GFP, MEK1b-GFP (MEK1b), MEK1-GFP (MEK1), ΔN-EE-MEK1b-GFP (ΔN-EE-MEK1b), or ΔN-EE-MEK1-GFP (ΔN-EE-MEK1-GFP). Fourteen hours after transfection, the cells were fixed, stained with anti-p58 Ab, and visualized by a fluorescent microscope. Arrows indicate fragmented Golgi. Bar, 8 μm. (B) ERK1c is required for MEK1b-induced Golgi fragmentation. HeLa cells were transfected with GFP alone as control or cotransfected with ΔN-EE-MEK1b-GFP together with GFP, ERK1c Sh-RNA (Sh-ERK1c), or ERK1 Sh-RNA (Sh-ERK1). The cells were then fixed, stained with anti-p58 Ab, and visualized by a fluorescent microscope. (Arrows indicate fragmented Golgi. Bar, 8 μm.) (C) Quantification of Golgi fragmentation. The HeLa cells in A and B were counted, and the percentage of fragmented Golgi per total transfected cells is presented. The results are means ± SE of three experiments, n = 200. A Student's t-test was used for statistical significance. (*) P < 0.05; (**) P < 0.01.

Specificity studies using constitutively active MEK1b and MEK1. (A) Time course of ERK1 and ERK1c phosphorylation by constitutively active MEK1b and MEK1. In vitro phosphorylation was preformed as described under Materials and Methods with recombinant His-ΔN-EE-MEK1b and His-ΔN-EE-MEK1 as kinases and GST-ERK1 and GST-ERK1c as substrates. The amounts of ERK1/1c and their phosphorylation levels were determined by Western blotting with anti-pERK, anti-gERK(CRS), and anti-gMEK(C-18) Abs. (B) Quantification of ERK1/1c phosphorylation. Known amounts of phosphorylated ERK were added to the blots to form a calibration curve, which enabled the determination of ERK1/1c phosphorylation levels (see text for details). The results in the graphs represent means ± SE of three experiments. (Squares) ERK1 phosphorylation; (triangles) ERK1c phosphorylation. (C) Side-by-side kinetics of ERK1/1c dual phosphorylation by MEK1/1b. In vitro phosphorylation was performed using amounts of recombinant proteins that were found optimal for each phosphorylation [His-ΔN-EE-MEK1 (CA-MEK1), 0.5 μg per reaction; His-ΔN-EE-MEK1b (CA-MEK1b), 0.75 μg; GST-ERK1, 0.5 μg or GST-ERK1c 0.5 μg] for 0, 2, and 10 min at 30°C as described in the Materials and Methods. The levels of ERK1/1c phosphorylation were detected by resolving the samples by one SDS-PAGE and Western blotting using anti-pERK, and anti-gERK(CRS) Abs.

ERK1c phosphorylation in mitosis is preferentially mediated by the alternative spliced MEK1b. (A) MEK1b undergoes phosphorylation during mitosis. HeLa cells were transfected with GFP, MEK1-GFP, MEK2-GFP, or MEK1b-GFP. Each of these transfected cells was treated as follows: untreated (−), nocodazole [100 ng/mL, 24 h (+)], or serum-starved; and then stimulated with EGF [50 ng/mL, 3 min (E)]. The cells were harvested and subjected to Western blotting with anti-pMEK, anti-GFP, and anti-gMEK(C-18) Abs. (B) MEK1b preferentially phosphorylates ERK1c during mitosis. HeLa cells were transfected as in A. The cells were treated with nocodazole [100 ng/mL, 24 h (+)] or left untreated (−). The cells were then harvested, and the GFP proteins were immunoprecipitated with anti-GFP Ab. The precipitated proteins were subjected to an in vitro phosphorylation system using GST-ERK1, 2 and 1c as substrates, as described in the Materials and Methods. The amounts of ERK1, 2 and 1c as well as their phosphorylation levels were determined by Western blotting using αpERK, and αgERK(CRS) Abs. The relative amounts of MEKs were determined by anti-gMEK(C-18) Ab, the MEKs used to phosphorylate ERK1/1c are in panel 5, and for ERK2 are in panel 8. (C) Differences between MEK1b and MEK1 stimulation and substrate determination. HeLa cells were transfected with MEK1b-GFP, MEK1-GFP, or GFP. The cells were treated as follows: untreated (−), nocodazole [100 ng/mL, 24 h (N)], serum-starved [16 h 0.1% FCS, (S)], or serum-starved and then stimulated with EGF [50 ng/mL, 3 min (E)]. The cells were harvested and the GFP-tagged proteins were immunoprecipitated by αGFP Ab, and each one of them was subjected to an in vitro phosphorylation using GST-ERK1/1c as substrates. The amounts of ERK1/1c as well as their phosphorylation levels were determined by Western blotting using anti-pERK, anti-gERK(CRS), and anti-GFP Abs.

Modulation of MEK1b expression affects nocodazole-dependent ERK1c activity in vivo. (A) HeLa cells were transfected as follows: GFP, MEK1b-GFP, MEK1-GFP, Sh-MEK1b, and Sh-MEK1. The cells were treated with nocodazole [100 ng/mL, 24 h, (+)]. The MEK inhibitor U0126 (5 μM, 1 h) was added prior to the cell harvesting. The cells were harvested, and endogenous ERK1 or ERK1c was immunoprecipitated. The precipitated proteins were subjected to in vitro phosphorylation using MBP as a substrate (ERK1c and ERK1 act.). The amounts of immunopreipitated endogenous ERKs (immunoprecipitated endoERK1 and ERK1c) were determined by Western blotting using anti-gERK(CRS) Ab. (B) Quantification of kinase activation. Fold activations of ERK1c and ERK1 were quantified by densitometry and presented as means ± SE of three experiments. All bar-graphs, except of the first two, represent stimulation with nocodazole. A Student's t-test was used to determine statistical significance ([*] P < 0.05). (C) Specific knockdown of MEK1b and MEK1 by their corresponding Sh-RNAs. HeLa cells were transfected with a plasmid containing Sh-RNA of MEK1b (Sh-MEK1b, +), MEK1 (Sh-MEK1, +), or a control plasmid (−). Forty-eight hours after transfection, the cells were harvested and subjected to SDS-PAGE and Western blotting with anti-gMEK (H-8) Abs.

The expression levels and activity of MEK1b are elevated during mitosis. HeLa cells were synchronized by a double-thymidine block, followed by the removal of the block to enable cell cycle progression (Syn), or left untreated (nonsynchronized, N.S.) as a control. (A) Mitotic index determination. The cells were fixed and stained with DAPI. The percentage of mitotic cells out of the total number of nuclei counted is presented as the means ± SE of three experiments; N = 300. (B) MEK1b expression and phosphorylation are elevated in the G₂/M phase of the cell cycle. The cells were harvested and subjected to Western blotting with anti-gMEK(H-8) and anti-pMEK Abs. For a better resolution of the MEK1b, SDS-PAGE was longer than in previous experiments. Exposure times for gMEK1/2 and gMEK1b were different. (C) Specific MEK1b phsphorylation is elevated during mitosis. Equal amounts of MEK1b (judged by a calibrating Western blot) from each time after block release were subjected to Western blotting with anti-pMEK Ab. (D) MEK1b activity is enhanced in the G₂/M phase of the cell cycle. HeLa cells were transfected with MEK1b-GFP or MEK1-GFP. The cells were synchronized by double-thymidine block, and times after release are indicated. In addition, one plate was serum-starved and stimulated with EGF (50 ng/mL, 3 min; E). The cells were harvested, and MEK1b or MEK1 was immunoprecipitated using anti-GFP Ab. The immunoprecipitated proteins were used as kinases for in vitro phosphorylation, where GST-ERK1c served as a substrate for MEK1b and GST-ERK1 for MEK1. The amounts of ERK1/1c as well as their phosphorylation levels were determined by Western blotting using anti-pERK, anti-gMEK(H-8), and anti-gERK(CRS) Abs.

Fractionation of endogenous MEK1b verifies its specificity toward ERK1c. (A) Fractionation of MEK1b on a Mono-Q FPLC column. HeLa cells (10 plates of 10 cm) were treated with nocodazole (100 ng/mL, 24 h) and harvested in Buffer A. Cell extracts were loaded onto a Mono-Q FPLC column, which was washed with Buffer A and developed using an increasing NaCl gradient (dashed line), to give rise to the protein profile shown by the solid line. Aliquots (40 μL, boiled in sample buffer) of every third fraction were subjected to Western blotting with anti-gMEK(H-8) Abs. Identical fractionation of MEK1/2 and MEK1b was observed with extracts from HeLa cells that were harvested 9 h after release from double-thymidine block and from untreated cells (data not shown). (B,C) Endogenous MEK1b from G₂/M cells is phosphorylated on its regulatory residues. Fractions containing MEK1/2 (#4, #5, #9) or MEK1b (#41, #42, #43) from cells that underwent treatment with nocodazole as described above (B), 9 h after release from double-thymidine block as described above (C), or untreated cells (B,C, indicated fractions) were subjected to Western blotting with anti-pMEK (top panel) and anti-gMEK(H-8) Ab (bottom panel). Similar results were obtained with Ab from other companies (data not shown). (D,E) The endogenous activated MEK1b from G₂/M cells phosphorylates mainly ERK1c in vitro. The indicated aliquots of MEK1/2- or MEK1b-containing fractions from nocodazole-treated cells (D), from cells that were released from double-thymidine block (E), or from untreated cells (E,D, indicated fractions) were used to phosphorylate GST-ERK1c (0.5 μg) or GST-ERK1 (0.5 μg), as described in Material and Methods. The phosphorylated proteins were analyzed by autoradiography (top panels) or by Western blotting with anti-pERK and anti-gERK(CRS) Abs (bottom panels) as indicated.