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© The Rockefeller University Press,
0021-9525/2000//331 $5.00
The Journal of Cell Biology, Volume 149, Number 2,
, 2000 331-340
Original Article |
A Specific Activation of the Mitogen-Activated Protein Kinase Kinase 1 (Mek1) Is Required for Golgi Fragmentation during Mitosis
malhotra{at}biomail.ucsd.edu
Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal–regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.
Key Words: mitogen-activated protein kinase kinase 1 • extracellular signal–regulated kinases • mitosis • Golgi fragmentation • phosphorylation
© 2000 The Rockefeller University Press
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Introduction |
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To address these questions, we recently reconstituted Golgi fragmentation in permeabilized cells. In this system, normal rat kidney (NRK) cells are permeabilized, washed to remove cytosolic proteins, and then incubated with mitotic extracts prepared from NRK cells arrested in mitosis. The reaction mixture is incubated at 32°C for 60 min, and the organization of the Golgi membranes monitored by fluorescence microscopy using Golgi-specific antibodies. We have shown that mitotic cytosol is highly enriched in cdc2 kinase activity, but that the cdc2 kinase activity is not required for Golgi fragmentation and dispersal of the Golgi fragments from the pericentriolar region. On the other hand, inactivation of the mitogen-activated protein kinase kinase 1 (MEK1) by drugs, or depletion of MEK1 by affinity chromatography or fractionation of mitotic cytosol, results in a loss of Golgi fragmentation. Surprisingly, the cytosolic downstream targets of MEK1, i.e., extracellular signal–regulated kinase (ERK) 1 and ERK2 (MAPKs), are not required for the MEK1-dependent Golgi fragmentation (Acharya et al. 1998).
We now provide evidence that MEK1 undergoes specific activation by phosphorylation during mitosis. The mitosis-specific activation of MEK1 is required for the conversion of the Golgi into tubuloreticular elements, and this fragmentation step does not require cdc2 kinase.
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Materials and Methods |
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N7-MEK1 was a gift from Dr. Nick Duesbery. All EM reagents were purchased from the sources described before (Takizawa et al. 1993). Trypsin was purchased from Calbiochem.
All tissue culture cells are grown in complete medium consisting of 
-MEM (Gibco) with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2 incubator.
Preparation of Mitotic and Interphase Extracts
Cytosol from NRK cells arrested in mitosis or interphase was prepared as described before (Acharya et al. 1998). Cell permeabilization and assay for Golgi fragmentation by mitotic extracts were carried out as described before (Acharya et al. 1998).
Incubation of Permeabilized Cells with Lethal Factor–treated Cytosol
Mitotic extract was pretreated with lethal factor (30 ng/ml) for 10 min at 32°C. This mixture and an ATP regeneration system were then added to semi-intact cells and the incubation carried out for 1 h at 32°C. When PD and olomoucine were used, these compounds were added to cytosol pretreated with lethal factor at a final concentration of 75 and 200 µM, respectively. The mixture was incubated for 10 min at 32°C in the presence of an ATP regeneration system and then added to semi-intact cells for 60 min at 30°C to monitor effects on Golgi fragmentation using the permeabilized cell system.
Incubation of Recombinant MEK with Interphase and Mitotic Extract
In a typical preparation, 20 µg of recombinant His-MEK1 (wild-type or N7) was incubated in a total volume of 80 µl with interphase or mitotic extract at a final concentration of 4 mg/ml. The kinase buffer used was composed of 50 mM Hepes, pH 7.2, 10 mM MgCl2, 1 mM DTT, 500 µM ATP, and 30 µCi [32P]ATP. The reaction mixture was incubated for 30 min at 30°C. At the end of the incubation, His-MEK1 was adsorbed of nickel beads, resuspended in 50 mM Hepes, pH 7.2, 200 mM NaCl, 10 mM β-mercaptoethanol, 10 mM imidazole, 10% glycerol, and finally eluted with 200 mM imidazole.
Tryptic Digestion of MEK1
His-MEK1 incubated with mitotic or interphase extract as described above was incubated at room temperature with trypsin (1:100, enzyme/substrate in wt/wt). At the indicated times, aliquots were removed and the reaction terminated by boiling the samples in Laemmli sample buffer. The samples were analyzed by SDS-PAGE followed by autoradiography and immunoblotting with the indicated antibodies as described in the figure legends.
Assay of Enzymatic Activity of MEK1
The procedure was the same as described before (Acharya et al. 1998).
Immunoelectron Microscopy
NRK cells were grown in special petri dishes (Matteck Corporation; Takizawa et al. 1993), permeabilized as described above, and processed for immunoelectron microscopy as described by Polishchuk et al. 1999. For the analysis of intact mitotic cells, NRK cells were incubated for 12 h with 2.5 µg/ml aphidicolin to arrest cells in S phase. The cells were washed by repeated changes of medium and placed in fresh medium. After 6–8 h, the mitotic cells were collected by gentle shake-off and processed for immunoelectron microscopy using anti-ManII antibody followed by HRP-conjugated secondary antibody. Ultrathin sections (80 nm) were observed at 80 KeV with either a JEOL 100Cx or 2000Fx electron microscope, and semithin sections (0.5 µM) at 400 KeV with a JEOL 4000Ex intermediate voltage electron microscope. The mitotic stages were assigned as described by Lucocq et al. 1987.
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Results |
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Mitotically Activated MEK1 Is Differentially Proteolyzed by Trypsin with Respect to Its Interphase Counterpart
His-tagged MEK1 was incubated with either interphase or the mitotic extract in the presence of [32P]ATP for 30 min at 30°C. The tagged protein was isolated on Ni beads and subjected to limited tryptic digestion. At various times the reaction was analyzed by SDS-PAGE and autoradiography and Western blotting with either anti–COOH-terminal or NH2-terminal–specific MEK1 antibody or the phospho-MEK1 antibody. Interestingly, trypsin digestion of mitotically activated MEK, and not interphase MEK1, results in the production of a prominent phosphorylated polypeptide of 
20 kD (Fig. 2 A). This polypeptide is recognized by the COOH-terminal–specific antibody, but not by the phospho-MEK1–specific antibody or the NH2-terminal MEK1 antibody (Fig. 2 B). The mitotic form of MEK1 is less susceptible to digestion with trypsin compared with its interphase counterpart (Fig. 2 B). Thus, the overall susceptibility and pattern of trypsin digestion is different in MEK1 incubated with interphase vs. the mitotic cytosol. The 20-kD polypeptide of MEK1 is not generated when it is incubated with interphase extracts. This would explain the increase in phosphorylation of MEK1 during mitosis, without any increase in reactivity to the phospho-MEK1 antibody. We propose that MEK1, upon phosphorylation by the mitotic extracts, undergoes a change in conformation and this new conformation is differentially sensitive to proteolysis, rendering it active for the Golgi fragmentation process.
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N7-MEK1 was incubated with the mitotic or the interphase cytosol and [32P]ATP for 30 min at 32°C. The N7-MEK1 was isolated on Ni beads and then analyzed by SDS-PAGE followed by autoradiography and Western blotting with the anti–phospho-MEK antibody. Incubation of N7-MEK1 with mitotic extracts results in a fourfold increase in 32P incorporation, and the mutant protein is about fourfold less reactive with the phospho-MEK1 antibody compared with its counterpart incubated with the interphase cytosol (Fig. 3). Therefore, the wild-type MEK1 and the N7-MEK1 have the same properties with respect to phosphorylation and reaction with the phospho-MEK1 antibody upon incubation with mitotic extract.
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(a) The reaction mixture was analyzed by SDS-PAGE followed by Western blotting with the COOH-terminal–specific MEK1 antibody and the NH2-terminal–specific antibody (Fig. 4). Treatment of the cytosol with lethal factor cleaves MEK1. Western blotting with the COOH-terminal–specific antibody reveals a shift in the molecular weight of MEK1 (Fig. 4, top panel). As expected, the NH2-terminal–specific antibody fails to react with MEK1 in cytosol treated with lethal factor (Fig. 4, lower panel), indicating that this treatment is effective in cleaving MEK1.
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Is cdc2 Kinase Involved in the Fragmentation of Golgi Membranes with Mitotic Extracts Treated with Lethal Factor?
We have shown previously that cdc2 kinase is not required for conversion of the pericentriolar Golgi membranes into smaller fragments (punctate structures at the light microscopy level; Acharya et al. 1998). To confirm that this is also the case with lethal factor–treated mitotic extracts, they were incubated in the presence of the generic inhibitor of cdc2 kinase activity, olomoucine. This mixture was then applied to permeabilized cells (washed with 1 M KCl) and an ATP regenerating system at 32°C for 60 min. The samples were visualized by fluorescence microscopy. Results show that under conditions where cdc2 kinase is inactive (the cdc2 kinase activity measured in the histone H1 phosphorylation was completely inhibited under these conditions [data not shown]), lethal factor–treated mitotic cytosol can convert the pericentriolarly organized stacks of Golgi cisternae into smaller structures (Fig. 6a and Fig. b).
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The Organization of Golgi Membranes in Permeabilized Cells Treated with the Mitotic Extracts
We have shown before that mitotic cytosol causes extensive fragmentation of the pericentriolar Golgi membranes (Acharya et al. 1998). However, the analysis was performed only at the light microscopy level and the organization or the morphology of the fragmented Golgi membranes was not reported because of technical limitations, which we have now resolved. NRK cells were permeabilized, washed with 1 M KCl, and then incubated with mitotic cytosol and an ATP regenerating system. A light microscopic analysis reveals the conversion of the pericentriolar Golgi apparatus (Fig. 7 A) into small punctate structures distributed throughout the cells. A similar preparation was processed for immunoelectron microscopy with an antibody to the cis/medial specific Golgi protein ManII. Thin sections of the cells revealed the presence of ManII in small structures composed of small cisternae and tubuloreticular elements (Fig. 7B and C–E). Fig. 7 F shows the control Golgi membranes by immunofluorescence and immunoelectron microscopy with anti-ManII antibody in intact nonmitotic NRK cells. A calculation of the size of the Golgi fragments stained with ManII antibody reveals that the stacks of Golgi cisternae of 1.5 µm average size are converted into tubuloreticular fragments of 0.60 µm in size by the action of mitotic cytosol. Incubation of the permeabilized cells with cytosol made from interphase cells and an ATP regeneration system at 32°C for 60 min does not affect the pericentriolar organization of the Golgi membranes (data not shown).
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0.60 µm. The Golgi fragments in mitotic cells, permeabilized cells incubated with mitotic extracts that are treated with or without the lethal factor, appear morphologically similar and are of similar size and distribution.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mitosis-specific Activation of MEK1
Our results show that MEK1 is hyperphosphorylated in mitosis. Interestingly, this hyperphosphorylation results in a decreased affinity towards the generic phospho-MEK1 antibody. The mitosis-specific activation of MEK1 is inhibited by pretreatment with PD but not with the lethal factor of anthrax toxin. Thus, the fragmentation of the pericentriolar Golgi stacks into tubuloreticular elements is due to a PD-sensitive and lethal factor–insensitive activity of MEK1, which occurs during mitosis. We believe that this mitosis-specific activation by phosphorylation is responsible for a change in the substrate specificity of MEK1. The 20-kD phosphopeptide of MEK1 is generated only as a result of mitosis-specific phosphorylation. It is also interesting to note that Golgi fragmentation by MEK1 does not require the only known substrates, ERK1 and ERK2. Thus, specific activation by phosphorylation changes the substrate specificity of MEK1 so that it can cause Golgi fragmentation without using the ERKs. How a change in the substrate specificity is achieved based on the phosphorylation state of MEK1 is currently not known.
Warren and colleagues have argued that MEK1 is not required for entry into mitosis and Golgi fragmentation (Lowe et al. 1998). This interpretation is based on experiments in which they loaded cells with the lethal factor and found that this resulted in inactivation of ERK1 and ERK2. The cells entered mitosis and the Golgi membranes were found as clusters of small vesicles (Lowe et al. 1998). However, we can explain their findings based on our results that cleavage with lethal factor prevents activation of ERK1 and ERK2, but this form of MEK is still activated by its upstream kinase and can convert Golgi membranes into tubuloreticular elements (Fig. 5, Fig. 7, and Fig. 8).
It has also been argued that MEK activity is lower in mitosis and that this is consistent with the inhibition of MEK1 activity by cdc2 kinase. Lowe et al. 1998 suggested that the low MEK1 activity is consistent with MEK1 not being a key mitotic regulator of Golgi fragmentation. We argue that MEK1 activity is low as measured by the activation of ERK1 and ERK2 phosphorylation. However, its activity towards other potential substrates, such as those involved in Golgi fragmentation, spindle dynamics, and checkpoint control may be regulated by an alternative mechanism (Wang et al. 1997; Acharya et al. 1998; Shapiro et al. 1998; Zecevic et al. 1998).
The fact that cdc2 kinase inhibits MEK1 activation is based on in vitro analysis. Whether this also happens in vivo is not known (Rossomondo et al. 1994). Although cdc2 kinase is required for entry into mitosis and controls the fate of many cellular process, this does not mean that all the mitotic-specific events are regulated by cdc2 kinase or that all phosphorylations by cdc2 kinase (in vitro and in vivo) have a physiological significance (Moreno and Nurse 1990). For example, in the case of lamin disassembly, a recent report shows that this involves sequential phosphorylation first by PKC, which is then followed by cdc2 kinase (Collas 1999).
Warren and colleagues reported that 50 µM completely inactivated MEK1 in vivo in cells grown in the presence of serum without affecting the ability of cells to enter mitosis or undergo Golgi fragmentation (Lowe et al. 1998). We found that 80 µM PD is required for MEK1 inactivation and inhibits the entry of NRK cells into mitosis (data not shown). We carried out this experiment as follows: NRK cells were treated with aphidicolin (2.5 µg/ml) in complete medium (containing 10% FBS) for 12 h at 37°C. Cells were washed extensively to remove aphidicolin and incubated with fresh medium containing serum. The cells were then washed and incubated with complete medium containing either PD (80 µM) or DMSO (the solvent for PD) at 37°C. After 5 h, the cells were analyzed by fluorescence microscopy with the DNA-specific fluorescent dye HOECHST 33258 to quantitate the mitotic index. In DMSO-treated cells, 30% of the cells were in mitosis, whereas only 5% of the cells were in mitosis in incubations with PD. Our observations are supported by those of Wright et al. 1999, who have recently shown that when PD is applied to cells grown in defined conditions (and not in complete medium containing serum), MEK1 is inhibited and the cells are arrested in G2 (Wright et al. 1999).
What Is the Ultimate Fate of Golgi Membranes during Mitosis?
Our data show that MEK1 is involved in the breakdown of the pericentriolarly organized Golgi membranes into numerous smaller tubuloreticular elements (Acharya et al. 1998; this work). Similar fragments have also been found in cells undergoing mitosis naturally and in cells induced to enter mitosis by okadaic acid treatment (Lucocq 1992; Lucocq et al. 1995). But do these fragments undergo further processing into clusters of vesicles or fuse with the ER (Thyberg and Moskalewski 1992; Zaal et al. 1999)? If the Golgi membranes are converted into numerous smaller fragments (resembling tubuloreticular elements), this should be sufficient for their partitioning into the daughter cells. But there is a large volume of data showing that Golgi membranes are converted into clusters of vesicles in mitotic cells (Lucocq et al. 1987; Warren 1993). Jesch and Linstedt 1998 also find that Golgi membranes are converted into vesicles of 60 nm diameter and can be separated from the ER in HeLa cells treated with nocadazole for 24 h. It is possible, however, that these cells are apoptotic not mitotic, and Sesso et al. 1999 have shown that in apoptotic cells, Golgi membranes are found distributed throughout the cell in the form of clusters of 40–60-nm vesicles. Is Golgi vesiculation a property of cells undergoing mitosis, apoptosis, or both?
In summary there is a general agreement in the field that the pericentriolarly organized Golgi apparatus is converted into smaller elements (tubuloreticular elements and smaller stacks) during mitosis. It remains to be determined whether these fragments undergo further processing to form vesicles or fuse with the ER. A comprehensive understanding of Golgi partitioning will require a resolution of this issue, as the process by which Golgi membranes are rebuilt into stacks (postmitosis) will differ depending on the final domicile of the Golgi during mitosis.
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Acknowledgments |
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Some of the work reported was conducted at the National Center for Microscopy and Imaging Research, a National Institutes of Health (NIH) Research Resource supported under NIH grant RR04050 to M.H. Ellisman. The work in the Malhotra lab is supported by the NIH grants to V. Malhotra. V. Malhotra is an established investigator of the American Heart Association. A. Colanzi is supported by a fellowship from the Consiglio Nazionale Delle Ricerche, Italy.
Submitted: 21 December 1999
Revised: 3 February 2000
Accepted: 7 February 2000
Abbreviations used in this paper: ERK, extracellular signal–regulated kinase; ManII, mannosidase II; MBP, myelin basic protein; MEK1, mitogen-activated protein kinase kinase 1; NRK, normal rat kidney.
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