ERK1c regulates Golgi fragmentation during mitosis

Abstract

Extracellular signal-regulated kinase 1c (ERK1c) is an alternatively spliced form of ERK1 that is regulated differently than other ERK isoforms. We studied the Golgi functions of ERK1c and found that it plays a role in MEK-induced mitotic Golgi fragmentation. Thus, in late G2 and mitosis of synchronized cells, the expression and activity of ERK1c was increased and it colocalized mainly with Golgi markers. Small interfering RNA of ERK1c significantly attenuated, whereas ERK1c overexpression facilitated, mitotic Golgi fragmentation. These effects were also reflected in mitotic progression, indicating that ERK1c is involved in cell cycle regulation via modulation of Golgi fragmentation. Although ERK1 was activated in mitosis as well, it could not replace ERK1c in regulating Golgi fragmentation. Therefore, MEKs regulate mitosis via all three ERK isoforms, where ERK1c acts specifically in the Golgi, whereas ERK1 and 2 regulate other mitosis-related processes. Thus, ERK1c extends the specificity of the Ras-MEK cascade by activating ERK1/2-independent processes.

Figure 1.

Elevated ERK1c expression, phosphorylation, and activity in mitosis. HeLa cells were either synchronized by double thymidine arrest, which was followed by the removal of the block to enable cell cycle progression, or left untreated (NS, nonsynchronized) as a control. (A) Representative flow cytometry analysis of the synchronized HeLa cells. At the indicated times after the block release, the cells were fixed, stained with propidium iodide, and analyzed by flow cytometry to determine the amount of DNA per cell (2N and 4N). (B) Mitotic index determination. The tested 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 ± SEM of three experiments. n = 300. (C) ERK1c expression is elevated in the G2/M phase of the cell cycle. The tested cells were harvested and subjected to a Western blot with ERK1c, ERK1/2, and pERK antibodies. (D) ERK1c activity is enhanced in the G2/M phase of the cell cycle. The tested cells were harvested and ERK1 or ERK1c were immunoprecipitated using their respective antibodies. The immunoprecipitated proteins were either subjected to an in vitro kinase assay using MBP as a substrate (ERK1c and ERK1 act.) or detected with ERK1c and ERK1 antibodies. Coomassie blue staining of the phosphorylated MBP is shown to confirm equal amounts of substrate. (E) The diphosphorylation of ERK1c is elevated during mitosis. Synchronized HeLa at mitosis (M) or untreated cells (N) were harvested, followed by immunoprecipitation of their ERK1c using ERK1c antibody. Equal amounts of ERK1c (predetermined by Western blotting) were subjected to Western blots with pERK or ERK1c antibodies.

Figure 2.

Nocodazole treatment induces ERK1c expression and activation. (A) Nocodazole arrests HeLa cells in the G2/M phase of the cell cycle HeLa cells were either treated with 100 ng/ml nocodazole (M) for 24 h or left untreated as control (N). The cells were then fixed, stained with propidium iodide, and analyzed by flow cytometry to determine the amount of DNA per cell (2N and 4N, as indicated). (B) Nocodazole-dependent mitotic arrest induces ERK1c activation. HeLa cells were treated with nocodazole (M) or left untreated (N). ERK1c and ERK1 were immunoprecipitated from these cells using their respective COOH-terminal antibodies and either subjected to an in vitro kinase assay (Kinase act.) or Western blot with gERK antibody. In addition, the cell extracts were either subjected to Western blot (left, bottom middle, and bottom) or immunoprecipitated with ERK1c antibody followed by Western blot (right, bottom middle, and bottom) with pERK or ERK1c antibodies (right, bottom middle, and bottom). (C) Quantification of kinase activation. Fold activation of ERK1c and ERK1 was quantified by densitometry and presented as means ± SEM. n = 3.

Figure 3.

The subcellular localization of ERK1c during mitosis. Synchronized HeLa cells 11 h after the double thymidine block release were fixed and stained with ERK1c, GM130 or p58 antibodies, and DAPI. Cells from interphase, prophase, prometaphase, metaphase (A), and telophase (B) were selected by their DNA structure, which was observed using DAPI staining. Similar results were detected with at least 20 distinct cells for each condition. (C) ERK1 does not translocate to the Golgi during mitosis. HeLa cells were fixed and stained with ERK1, GM130 or p58 antibodies, and DAPI. Cells in prophase were selected by their DNA structure, which was observed using DAPI staining. Similar results were detected with at least 20 distinct cells for each condition. (D) Quantification of ERK1c colocalization in the Golgi during mitosis. The percentage of mitotic or interphase cells in which ERK1c was colocalized with the Golgi markers was determined by counting 200 cells in each slide. The results in the bar graph represent the mean ± SEM of three experiments. Bars, 10 μm.

Figure 4.

ERK1c is activated by MEKs and is mainly mono-Tyr204 phosphorylated. (A) Mitotic ERK1c is either mono-Tyr phosphorylated or diphosphorylated and is activated downstream of MEKs. HeLa cells were transfected with CA-MEK (ΔN-EE-MEK1, marked as MEK) or with GFP control. The cells were then synchronized by double thymidine block and released for 11 h. 25 μM PD98059 was added to one of the GFP plates (PD) 9 h after release, and the other GFP plate was left untreated (Con). After harvesting, ERK1c was immunoprecipitated and either subjected to an in vitro kinase assay (ERK1c act.) or to a Western blot with pERK, monophosphorylated ERK, and ERK1c antibodies. Coomassie blue staining of the MBP is shown as a control for equal substrate amounts. (B) ERK1c colocalizes with monophosphorylated ERK during mitosis. The synchronized HeLa cells were fixed and stained with ERK1c, ERK1, pY-ERK or pERK antibodies, and DAPI. Cells from interphase and prophase were selected by their DNA structure, which was observed using DAPI staining. Similar results were detected with at least 20 distinct cells for each condition. Bar, 5 μm. (C) ERK1c undergoes preferential phosphorylation on its Tyr204. HeLa cells were transfected with MEK1-GFP. The cells were serum starved for 16 h and then stimulated with 50 ng/ml EGF for 3 min. MEK1-GFP was immunoprecipitated and used to phosphorylate purified GST-ERK1c or GST-ERK1 for the indicated times. The amount of ERK1c and ERK1, as well as their phosphorylation, were determined by Western blotting using pY-ERK, pERK, and ERK1 antibodies. The results in the graphs represent the mean and SEM of three experiments. Squares represent diphosphorylation and circles represents monophosphorylated ERK1 or ERK1c.

Figure 5.

Modulation of ERK1c expression and activity with siRNA and GFP-ERK1c. (A) Specific knockdown of ERK1c and ERK1 by their corresponding siRNAs. HeLa cells were transfected with GFP together with either a plasmid containing si-ERK1c, a plasmid containing si-ERK1, or additional GFP (Con). 72 h after transfection, the cells were harvested and subjected to a Western blot with ERK1c and ERK1/2 (αgERK) antibodies. (B) si-ERK1c reduces the amount of ERK1c in transfected cells. HeLa cells were cotransfected with si-ERK1c and GFP, and 72 h after transfection the cells were fixed and stained with αERK1c antibody and DAPI. Bar, 30 μM. (C) Modulation in ERK1c expression correlates with its MBP phosphorylation. HeLa cells were transfected with pEGFP (Vector), si-ERK1c, or GFP-ERK1c (ERK1c) synchronized by double thymidine block and release for 11 h. After harvesting, ERK1c was immunoprecipitated from the cells and was either subjected to an in vitro kinase assay (ERK1c act.) or Western blot using ERK1c antibody. Coomassie blue staining of the MBP confirmed equal substrate.

Figure 6.

Modulation of ERK1c expression affect Golgi fragmentation during mitosis. HeLa cells were transfected with GFP, GFP-ERK1c (ERK1c), or si-ERK1c with GFP (si-ERK1c). The cells were synchronized by double thymidine block, and 11 h after the release were stained with GM130 or p58 antibodies and DAPI. (A) si-ERK1c and GFP-ERK1c affect Golgi architecture during prophase and prometaphase. Cells from interphase, prophase, and prometaphase were selected by their DNA structure. The percentage of cells with fragmented Golgi apparatus is presented as means ± SEM of three experiments. n = 40. (B) Knockdown of ERK1c attenuates the formation of Golgi haze in metaphase. Cells in metaphase were selected by their DNA structure. The percentage of cells with big vesicles out of total metaphase cells is presented as means ± SEM of three experiments. n = 40 cells. (C) Knockdown of ERK1c results in unequal Golgi division. Telophase cells were selected by their DNA structure. The percentage of cells with unequal Golgi division from total telophase cells is presented as means ± SEM of three experiments. n = 40. Bars: (A and C) 10 μm; (B) 5 μm.

Figure 7.

Effects of ERK1c on mitotic progression. (A) Modulation of ERK1c expression affects cell cycle progression. HeLa cells were transfected with GFP, GFP-ERK1c, (ERK1c), si-ERK1c together with GFP (si-ERK1c), GFP-ERK1 (ERK1), and si-ERK1 together with GFP (si-ERK1). The cells were synchronized by double thymidine block as in Fig. 6. At the indicated times, the cells were fixed, stained with propidium iodide, and analyzed by flow cytometry for DNA content (2N or 4N). (B) Effect of MEK activity on cell cycle progression. HeLa cells were transfected with either GFP (GFP and PD98059) or with CA-MEK1 (CA-MEK). The cells were synchronized by double-thymidine, as in Fig. 6. 25 μM PD98059 was added to one of the GFP plates (PD98059) 2 h before fixation, whereas the other GFP plate was left untreated as control. At the indicated times, the cells were fixed, stained with propidium iodide, and analyzed by flow cytometry for their DNA content (2N or 4N). (C) Quantification of the effect of the various treatments. The ratio between G1/G2 was compared with that of the control cells. The results are means ± SEM of three experiments. A t test was used for statistical analysis (*, P < 0.01). (D) Determination of mitotic index and percentage of cells in telophase. HeLa cells were transfected and synchronized as in A, and stained with DAPI. The percentage of mitotic cells (left, Mitosis) and cells at telophase out of the mitotic cells (right, Telophase) is presented in the bar graph as the means ± SEM of three experiments. n = 200 cells each. A t test was used for analysis. *, P < 0.05; **, P < 0.01.