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RAF1-activated MEK1 is found on the Golgi apparatus in late prophase and is required for Golgi complex fragmentation in mitosis.

Colanzi A, Sutterlin C, Malhotra V - J. Cell Biol. (2003)

Bottom Line: Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells.We show that activated MEK1 is found on the Golgi apparatus in late prophase.The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1.

View Article: PubMed Central - PubMed

Affiliation: Cell and Developmental Biology Biology, University of California, San Diego, La Jolla, CA 92093-0347, USA.

ABSTRACT
Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells. We show that activated MEK1 is found on the Golgi apparatus in late prophase. The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1. MEK1-dependent Golgi complex fragmentation is through activation by RAF1 and not MEK1 kinase 1. We propose that a RAF1-dependent activation of MEK1 and its presence on the Golgi apparatus in late prophase is required for Golgi complex fragmentation.

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RKIP does not block MEKK1-dependent MEK1 activation, but inhibits RAF1-dependent MEK1 activation and Golgi complex fragmentation. (A) RAF1 and MEKK1 were immunoprecipitated from 250 μg mitotic cytosol using 2 μg of C-12 and 43-Y antibodies, respectively. The beads were subsequently incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of buffer alone, 50 μg BSA, or 15 μg RKIP. MEK1 was recovered by centrifugation and immunoblotted with anti-ppMEK antibody. Quantitation of the results is shown in the lower panel. RKIP inhibits RAF1-dependent MEK1 activation. The quantification of the experiment shown in the figure is presented; similar results have been obtained in three different experiments. (B) Mitotic extract treated with RKIP as described above was added to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (C) Quantification of the effects of RKIP on Golgi complex fragmentation by mitotic cytosol. Inhibition of the RAF1-dependent MEK1 activation inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored the Golgi complex fragmentation. The data represent the average of six different experiments.
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fig2: RKIP does not block MEKK1-dependent MEK1 activation, but inhibits RAF1-dependent MEK1 activation and Golgi complex fragmentation. (A) RAF1 and MEKK1 were immunoprecipitated from 250 μg mitotic cytosol using 2 μg of C-12 and 43-Y antibodies, respectively. The beads were subsequently incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of buffer alone, 50 μg BSA, or 15 μg RKIP. MEK1 was recovered by centrifugation and immunoblotted with anti-ppMEK antibody. Quantitation of the results is shown in the lower panel. RKIP inhibits RAF1-dependent MEK1 activation. The quantification of the experiment shown in the figure is presented; similar results have been obtained in three different experiments. (B) Mitotic extract treated with RKIP as described above was added to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (C) Quantification of the effects of RKIP on Golgi complex fragmentation by mitotic cytosol. Inhibition of the RAF1-dependent MEK1 activation inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored the Golgi complex fragmentation. The data represent the average of six different experiments.

Mentions: An additional strategy was used to interfere with the RAF1–MEK1 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 cytosol–dependent 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.


RAF1-activated MEK1 is found on the Golgi apparatus in late prophase and is required for Golgi complex fragmentation in mitosis.

Colanzi A, Sutterlin C, Malhotra V - J. Cell Biol. (2003)

RKIP does not block MEKK1-dependent MEK1 activation, but inhibits RAF1-dependent MEK1 activation and Golgi complex fragmentation. (A) RAF1 and MEKK1 were immunoprecipitated from 250 μg mitotic cytosol using 2 μg of C-12 and 43-Y antibodies, respectively. The beads were subsequently incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of buffer alone, 50 μg BSA, or 15 μg RKIP. MEK1 was recovered by centrifugation and immunoblotted with anti-ppMEK antibody. Quantitation of the results is shown in the lower panel. RKIP inhibits RAF1-dependent MEK1 activation. The quantification of the experiment shown in the figure is presented; similar results have been obtained in three different experiments. (B) Mitotic extract treated with RKIP as described above was added to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (C) Quantification of the effects of RKIP on Golgi complex fragmentation by mitotic cytosol. Inhibition of the RAF1-dependent MEK1 activation inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored the Golgi complex fragmentation. The data represent the average of six different experiments.
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Related In: Results  -  Collection

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fig2: RKIP does not block MEKK1-dependent MEK1 activation, but inhibits RAF1-dependent MEK1 activation and Golgi complex fragmentation. (A) RAF1 and MEKK1 were immunoprecipitated from 250 μg mitotic cytosol using 2 μg of C-12 and 43-Y antibodies, respectively. The beads were subsequently incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of buffer alone, 50 μg BSA, or 15 μg RKIP. MEK1 was recovered by centrifugation and immunoblotted with anti-ppMEK antibody. Quantitation of the results is shown in the lower panel. RKIP inhibits RAF1-dependent MEK1 activation. The quantification of the experiment shown in the figure is presented; similar results have been obtained in three different experiments. (B) Mitotic extract treated with RKIP as described above was added to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (C) Quantification of the effects of RKIP on Golgi complex fragmentation by mitotic cytosol. Inhibition of the RAF1-dependent MEK1 activation inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored the Golgi complex fragmentation. The data represent the average of six different experiments.
Mentions: An additional strategy was used to interfere with the RAF1–MEK1 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 cytosol–dependent 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.

Bottom Line: Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells.We show that activated MEK1 is found on the Golgi apparatus in late prophase.The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1.

View Article: PubMed Central - PubMed

Affiliation: Cell and Developmental Biology Biology, University of California, San Diego, La Jolla, CA 92093-0347, USA.

ABSTRACT
Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells. We show that activated MEK1 is found on the Golgi apparatus in late prophase. The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1. MEK1-dependent Golgi complex fragmentation is through activation by RAF1 and not MEK1 kinase 1. We propose that a RAF1-dependent activation of MEK1 and its presence on the Golgi apparatus in late prophase is required for Golgi complex fragmentation.

Show MeSH
Related in: MedlinePlus