Report |
Address correspondence to Vivek Malhotra, Cell and Developmental Biology Department, B0347, University of California, San Diego, Pacific Hall, 2nd Floor, Rm. 2222A, La Jolla, CA 92093-0347. Tel.: (858) 534-8910. Fax: (858) 534-0555. E-mail: malhotra{at}biomail.ucsd.edu
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Abstract |
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Key Words: mitogen-activated protein kinase kinase 1; cell cycle; organelle inheritance; RKIP; phosphorylation
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Introduction |
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We have taken the following approach to identify components involved in the fragmentation of pericentriolar Golgi apparatus. Normal rat kidney (NRK)* cells grown on coverslips are permeabilized with ice-cold digitonin and washed with a buffer containing 1 M KCl to remove cytosolic and loosely attached peripheral membrane proteins. The permeabilized and salt-washed cells are incubated at 32°C for 60 min with cytosol prepared from mitotic NRK cells. The organization of Golgi membranes is monitored by fluorescence microscopy using antibodies to Golgi complexspecific proteins such as mannosidase II (ManII). As a result of this procedure, the Golgi apparatus is fragmented and dispersed in tubulo-reticular membranes throughout the cytoplasm (Acharya et al., 1998; Colanzi et al., 2000). We have previously shown that mitotically activated mitogen-activated protein kinase 1 (MEK1) and Polo-like kinase 1 are required for Golgi complex fragmentation (Acharya et al., 1998; Colanzi et al., 2000; Sutterlin et al., 2001). We now report that RAF1 is required to activate MEK1 for Golgi complex fragmentation during mitosis and that mitotically activated MEK1 is found on the Golgi membranes in late prophase.
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Results and discussion |
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We tested whether RAF1 is required for MEK1-dependent mitosis-specific Golgi complex fragmentation. There are no known RAF1-specific inhibitors and it is also difficult to deplete RAF1 from mitotic cytosol without using detergents. Therefore, we used the following strategy to specifically block RAF1 activation. We expressed a GST-tagged deletion mutant of RAF1 (corresponding to amino acids 1239) in Escherichia coli (Fig. 1 A). This peptide corresponds to the autoinhibitory domain of RAF1 and acts as dominant negative inhibitor of RAF1 when expressed in mammalian cells (Flory et al., 1998). Purified recombinant GST-RAF1/1-239 (Raf-239) was added to mitotic cytosol for 10 min at 32°C with ATP. The endogenous RAF1 was immunoprecipitated from this incubation mixture using an antibody against the COOH terminus of RAF1. The immunoprecipitated RAF1 (on beads) was incubated with recombinant MEK1 and ATP at 32°C. The sample was centrifuged to separate MEK1 (supernatant) from RAF1 (beads). RAF1 is known to phosphorylate MEK1 at serines 218 and 222 and the extent of this double phosphorylation is a reliable indicator of RAF1 activity (Bondzi et al., 2000). The supernatant containing MEK1 was analyzed by SDS-PAGE followed by Western blotting with an antibody that recognizes MEK1 phosphorylated at serines 218 and 222 (antiphospho-MEK [ppMEK] antibody). Incubation of mitotic cytosol with recombinant Raf-239 inhibited RAF1 activation (Fig. 1 B). Quantitation of the Western blot revealed that Raf-239 causes a 75% inhibition of RAF1 activity toward MEK1 (Fig. 1 B, bottom). Similar results were obtained with GST-RAF1/1-330 (Raf-330), which corresponds to the entire regulatory domain of RAF1 (Fig. 1 A). For the experiments described below, we used Raf-239 because it was easier to express and purify. Raf-239 most likely interferes with the complex network of proteins that regulates RAF1 function titrating out activating components (Bruder et al., 1992; Flory et al., 1998). Raf-239 does not titrate out MEK1 because the MEK1 binding sites are located in the COOH-terminal catalytic domain of RAF1 (Yeung et al., 2000).
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An additional strategy was used to interfere with the RAF1MEK1 pathway by using RAF1 kinase inhibitory protein (RKIP). In the presence of RKIP, RAF1 cannot bind MEK1 (Yeung et al., 2000). RKIP does not inhibit MEKK1-mediated phosphorylation and activation of MEK1 (Yeung et al., 1999). RAF1 and MEKK1 were immunoisolated from mitotic cytosol using anti-RAF1 and anti-MEKK1 antibodies conjugated to Sepharose beads, respectively. The beads containing immunoisolated RAF1 and MEKK1 were incubated with MEK1, RKIP, and ATP at 32°C for 10 min. The reaction mixture was centrifuged to separate beads from soluble MEK1. The supernatant containing MEK1 was analyzed by SDS-PAGE and Western blotted with anti-ppMEK antibody. Our results show that RKIP inhibited RAF1-mediated phosphorylation of MEK1, but did not affect MEKK1-mediated phosphorylation of MEK1 (Fig. 2 A). This is in agreement with the work of Yeung and colleagues (Yeung et al., 1999). Recombinant purified RKIP was incubated with mitotic cytosol and this sample added to the assay reconstituting Golgi complex fragmentation process. RKIP strongly inhibited mitotic cytosoldependent Golgi complex fragmentation (Fig. 2, B and C). Addition of recombinant constitutively activated MEK1 (G1C) significantly restored Golgi complex fragmentation activity of mitotic cytosol containing RKIP. The restoration of Golgi complex fragmentation activity was partial because RKIP is required in molar excess concentration to efficiently inhibit RAF1-dependent MEK1 activation (Yeung et al., 1999). The addition of higher concentration of G1C, sufficient to fully revert the inhibition, is not feasible in our assay because it dilutes the concentration of mitotic cytosol necessary for Golgi complex fragmentation. These results provide strong evidence that RAF1-mediated activation of MEK1 is required for Golgi complex fragmentation by mitotic cytosol. We have previously shown that mitotically activated MEK1 is conformationally different from its functional counterpart in interphase cells (Colanzi et al., 2000). Partial proteolysis of His-MEK1 incubated with mitotic cytosol reveals a 20-kD phosphopeptide, which is not observed upon proteolysis of His-MEK1 incubated with interphase cytosol. RAF1 was isolated from mitotic cytosol and incubated with His-MEK1. This preparation of His-MEK1 was subjected to partial proteolysis. The resulting proteolytic fragments did not contain the 20-kD phosphopeptide described above (unpublished data). Together, these results suggest that RAF1 is required, but is not sufficient for complete mitosis-specific activation of MEK1. The identity of additional components required for MEK1 activation in Golgi complex fragmentation is not known.
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Activated MEK1 showed nuclear localization during prophase (Fig. 4 A). This is in agreement with the fact that MEK1 translocates to the nucleus during this mitotic stage (Tolwinski et al., 1999). Interestingly, activated MEK1 was localized to the Golgi apparatus in late prophase (Fig. 4 A). To ensure that the Golgi complexspecific staining of ppMEK antibody was due to interaction with activated MEK1, cells were stained with ppMEK antibody that had been preincubated with either mitotically activated recombinant MEK1 (phosphorylated MEK1) or unphosphorylated recombinant MEK1. Preincubation of ppMEK antibody with mitotically phosphorylated MEK1 abolished the Golgi complex and nuclear staining. ppMEK antibody staining on the Golgi apparatus in late prophase, therefore, is due to its binding to the mitotically activated MEK1 (Fig. 4 B). The percentage of the ß-COP positive structures that contained mitotically activated MEK1 was 47 ± 6%. Similar results were obtained in HeLa, NIH, and COS-7 cells (not depicted).
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Conclusions
The timing of the appearance of activated MEK1 on the Golgi apparatus coincides with the initiation of Golgi complex fragmentation during mitosis. One of the components necessary for the activation of MEK1 in the Golgi complex fragmentation process is RAF1. Circumstantial evidence indicates that an ERK-like protein may be the downstream target of MEK1. An ERK-like protein or a mitotically activated ERK with novel modification has been found on the Golgi membranes (Acharya et al., 1998; Cha and Shapiro, 2001). It is also reported that ERK2 phosphorylates the Golgi complexassociated complex protein GRASP55 (Jesch et al., 2001). It is conceivable that a RAF1MEK1ERK/ERK-like pathway induces the phosphorylation of GRASP55 during mitosis and consequently destabilizes the Golgi stacks. This reaction, along with reactions catalyzed by Polo-like kinase 1 and the cdc2 kinase, may well explain the mechanism of Golgi complex fragmentation during mitosis (Lowe et al., 1998; Sutterlin et al., 2001).
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Materials and methods |
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Preparation of mitotic extract, Raf-239, and RKIP
Preparation of cytosol from NRK cells arrested in mitosis and the assay for Golgi complex fragmentation have been described before (Acharya et al., 1998). Raf-239 and RKIP were expressed and purified by the published procedure (Pumiglia et al., 1995; Yeung et al., 1999). The recombinant proteins were dialyzed against KHM buffer containing 5% glycerol and stored at 80°C.
Assay of endogenous RAF activity
The assay was performed as described previously (Bondzi et al., 2000) with the following modifications. 15 µg of Raf-239 RAF1 was added to 350 µg of mitotic cytosol. After a 10-min incubation at 32°C in the presence of ATP, the endogenous RAF1 was immunoprecipitated from the incubation mixture using 2 µg of anti-RAF1 antibody raised against the COOH terminus of the protein. The immunoprecipitated RAF1 (still on beads) was then incubated with 3 µg of recombinant MEK1 and ATP at 32°C. The sample was centrifuged to separate MEK1 (in the supernatant) from RAF1 (on the beads). The supernatant was analyzed by SDS-PAGE followed by Western blotting with anti-ppMEK antibody. The film was scanned and the bands quantitated using the Kodak 1D 3.0 software.
Assay for MEK1 activation
RAF1 and MEKK1 were immunoisolated from 250 µg of mitotic cytosol using 2 µg of anti-RAF1 and anti-MEKK1 antibodies conjugated to Sepharose beads. The beads containing immunoisolated RAF1 and MEKK1 were incubated as described (Bondzi et al., 2000) with MEK1, RKIP, and ATP at 32°C for 10 min. The reaction was then centrifuged to separate beads from the soluble MEK1. The supernatant containing MEK1 was analyzed by SDS-PAGE and Western blotted with anti-ppMEK antibody and analyzed as reported above.
Microinjections
Purified GST and Raf-239 were injected into NRK cells by the same procedures as described previously for GRAPS65-related reagents (Sutterlin et al., 2002).
Effect of Raf-239 and RKIP on Golgi complex fragmentation
350 µg of mitotic extract was pretreated with Raf-239 or RKIP (15 µg/sample) and an ATP regeneration system for 10 min at 32°C. This mixture was added to semi-intact cells and the incubation was performed for 1 h at 32°C. G1C was used at a final concentration of 3 µg/sample. 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 32°C.
Localization of activated MEK1
NRK cells were plated and treated as described before (Sutterlin et al., 2002). Cells were fixed with 4% formaldehyde at 37°C and blocked with PBS containing 0.1% TX-100 and 5% BSA. To reveal the localization of activated MEK1, cells were incubated for 2 h at room temperature in blocking buffer containing 2 µg/ml of ppMEK1 antibody followed by a 1-h incubation with goat antirabbit Alexa-546 (1:3,000 dilution). All the other staining procedures were as described (Sutterlin et al., 2002). Epifluorescence microscopy images were acquired with a Nikon microphot-FXA connected to an Olympus CCD camera. Confocal microscope images were captured on a Leika microscope using 488-nm laser excitation for secondary Alexa486, 568 nm for Alexa546, and 351 nm for Hoechst dye. Images for illustrations were captured with a 60 NA objective. Microsoft PowerPoint and Adobe Photoshop 7.0 were used to assemble the figures. To calculate the extent of active MEK1 colocalization with ß-COP, five images showing double labeling for active MEK1 and ß-COP were selected, the respective Golgi complex areas were selected and cut using Adobe Photoshop. Finally, the NIH Image software (version 1.62) was used to measure the percentage of the ß-COP containing pixels and active MEK1. To evaluate the specificity of ppMEK antibody, immunofluorescence blocking buffer containing 0.2 µg/ml of ppMEK antibody were incubated with a 50-M excess of recombinant MEK1 that had been previously phosphorylated as described above. Control sample consisted of ppMEK antibody incubated with nonphosphorylated recombinant MEK1.
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Acknowledgments |
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Work in the Malhotra Lab is supported by grants from the National Institutes of Health and Human Frontiers Science program.
Submitted: 16 August 2002
Revised: 5 March 2003
Accepted: 5 March 2003
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References |
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