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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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Raf-239 inhibits endogenous RAF1 activation and Golgi complex fragmentation. (A) A schematic diagram of RAF1 domains. Full-length (FL) RAF1 is composed of a COOH-terminal catalytic domain (CD) and NH2-terminal regulatory domain containing a cysteine-rich domain (C1) and a serine/threonine rich-domain (ST). (B) Mitotic cytosol (350 μg) was incubated with buffer alone, 50 μg GST, or 15 μg Raf-239 for 10 min at 32°C. Endogenous RAF1 was immunoprecipitated with 2 μg anti-RAF1 (C-12) antibody complexed to beads. The samples containing immunoprecipitated RAF1 were incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of ATP. At the end of the incubation, MEK1 was recovered and immunoblotted with anti-ppMEK antibody. The quantitation of the results is shown in the lower panel. The extent of MEK1 phosphorylation is used as an indication of RAF1 activity. Raf-239 inhibits activation of the endogenous RAF1 by 75%. The quantification of the experiment shown in the figure is presented; similar results have been obtained in four different experiments. (C) Mitotic extract treated with Raf-239 as described above was applied to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (D) Quantitation of the effects of Raf-239 on Golgi complex fragmentation by mitotic cytosol. Raf-239 inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored Golgi complex fragmentation. The data represent the average of seven different experiments.
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fig1: Raf-239 inhibits endogenous RAF1 activation and Golgi complex fragmentation. (A) A schematic diagram of RAF1 domains. Full-length (FL) RAF1 is composed of a COOH-terminal catalytic domain (CD) and NH2-terminal regulatory domain containing a cysteine-rich domain (C1) and a serine/threonine rich-domain (ST). (B) Mitotic cytosol (350 μg) was incubated with buffer alone, 50 μg GST, or 15 μg Raf-239 for 10 min at 32°C. Endogenous RAF1 was immunoprecipitated with 2 μg anti-RAF1 (C-12) antibody complexed to beads. The samples containing immunoprecipitated RAF1 were incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of ATP. At the end of the incubation, MEK1 was recovered and immunoblotted with anti-ppMEK antibody. The quantitation of the results is shown in the lower panel. The extent of MEK1 phosphorylation is used as an indication of RAF1 activity. Raf-239 inhibits activation of the endogenous RAF1 by 75%. The quantification of the experiment shown in the figure is presented; similar results have been obtained in four different experiments. (C) Mitotic extract treated with Raf-239 as described above was applied to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (D) Quantitation of the effects of Raf-239 on Golgi complex fragmentation by mitotic cytosol. Raf-239 inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored Golgi complex fragmentation. The data represent the average of seven different experiments.

Mentions: 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 1–239) 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 (anti–phospho-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).


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)

Raf-239 inhibits endogenous RAF1 activation and Golgi complex fragmentation. (A) A schematic diagram of RAF1 domains. Full-length (FL) RAF1 is composed of a COOH-terminal catalytic domain (CD) and NH2-terminal regulatory domain containing a cysteine-rich domain (C1) and a serine/threonine rich-domain (ST). (B) Mitotic cytosol (350 μg) was incubated with buffer alone, 50 μg GST, or 15 μg Raf-239 for 10 min at 32°C. Endogenous RAF1 was immunoprecipitated with 2 μg anti-RAF1 (C-12) antibody complexed to beads. The samples containing immunoprecipitated RAF1 were incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of ATP. At the end of the incubation, MEK1 was recovered and immunoblotted with anti-ppMEK antibody. The quantitation of the results is shown in the lower panel. The extent of MEK1 phosphorylation is used as an indication of RAF1 activity. Raf-239 inhibits activation of the endogenous RAF1 by 75%. The quantification of the experiment shown in the figure is presented; similar results have been obtained in four different experiments. (C) Mitotic extract treated with Raf-239 as described above was applied to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (D) Quantitation of the effects of Raf-239 on Golgi complex fragmentation by mitotic cytosol. Raf-239 inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored Golgi complex fragmentation. The data represent the average of seven different experiments.
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Related In: Results  -  Collection

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fig1: Raf-239 inhibits endogenous RAF1 activation and Golgi complex fragmentation. (A) A schematic diagram of RAF1 domains. Full-length (FL) RAF1 is composed of a COOH-terminal catalytic domain (CD) and NH2-terminal regulatory domain containing a cysteine-rich domain (C1) and a serine/threonine rich-domain (ST). (B) Mitotic cytosol (350 μg) was incubated with buffer alone, 50 μg GST, or 15 μg Raf-239 for 10 min at 32°C. Endogenous RAF1 was immunoprecipitated with 2 μg anti-RAF1 (C-12) antibody complexed to beads. The samples containing immunoprecipitated RAF1 were incubated with 3 μg recombinant MEK1 for 10 min at 32°C in the presence of ATP. At the end of the incubation, MEK1 was recovered and immunoblotted with anti-ppMEK antibody. The quantitation of the results is shown in the lower panel. The extent of MEK1 phosphorylation is used as an indication of RAF1 activity. Raf-239 inhibits activation of the endogenous RAF1 by 75%. The quantification of the experiment shown in the figure is presented; similar results have been obtained in four different experiments. (C) Mitotic extract treated with Raf-239 as described above was applied to permeabilized cells. The organization of the Golgi membranes was analyzed by fluorescence microscopy using the anti-ManII antibody. (D) Quantitation of the effects of Raf-239 on Golgi complex fragmentation by mitotic cytosol. Raf-239 inhibited the mitotic- specific Golgi complex fragmentation. The addition of 3 μg of bacterially expressed constitutive active MEK1 (G1C) restored Golgi complex fragmentation. The data represent the average of seven different experiments.
Mentions: 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 1–239) 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 (anti–phospho-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).

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