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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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Activated MEK1 is found on the Golgi apparatus in late prophase. NRK cells were blocked in S-phase with aphidicolin. The cells were washed to remove aphidicolin. 7 h after aphidicolin removal, cells were processed for fluorescence microscopy to visualize the Golgi apparatus, activated MEK1, and DNA with antibodies against β-COP, ppMEK, and the DNA-specific dye Hoechst, respectively. (A) Confocal microscopy image showing that activated-MEK1 is localized to the Golgi apparatus in late prophase (condensed DNA*) and not in interphase (uncondensed DNA). (B) Epifluorescence microscopy image showing cells stained with ppMEK antibody that had been preincubated with either mitotically activated MEK (phosphorylated MEK1) or unphosphorylated MEK1. Preincubation of ppMEK antibody with mitotically phosphorylated MEK1 abolished the Golgi complex–specific staining of cells in late prophase (condensed DNA*). Therefore, ppMEK antibody staining is due to the presence of activated MEK1 on the Golgi apparatus.
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fig4: Activated MEK1 is found on the Golgi apparatus in late prophase. NRK cells were blocked in S-phase with aphidicolin. The cells were washed to remove aphidicolin. 7 h after aphidicolin removal, cells were processed for fluorescence microscopy to visualize the Golgi apparatus, activated MEK1, and DNA with antibodies against β-COP, ppMEK, and the DNA-specific dye Hoechst, respectively. (A) Confocal microscopy image showing that activated-MEK1 is localized to the Golgi apparatus in late prophase (condensed DNA*) and not in interphase (uncondensed DNA). (B) Epifluorescence microscopy image showing cells stained with ppMEK antibody that had been preincubated with either mitotically activated MEK (phosphorylated MEK1) or unphosphorylated MEK1. Preincubation of ppMEK antibody with mitotically phosphorylated MEK1 abolished the Golgi complex–specific staining of cells in late prophase (condensed DNA*). Therefore, ppMEK antibody staining is due to the presence of activated MEK1 on the Golgi apparatus.

Mentions: 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 complex–specific 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 β-COP positive structures that contained mitotically activated MEK1 was 47 ± 6%. Similar results were obtained in HeLa, NIH, and COS-7 cells (not depicted).


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)

Activated MEK1 is found on the Golgi apparatus in late prophase. NRK cells were blocked in S-phase with aphidicolin. The cells were washed to remove aphidicolin. 7 h after aphidicolin removal, cells were processed for fluorescence microscopy to visualize the Golgi apparatus, activated MEK1, and DNA with antibodies against β-COP, ppMEK, and the DNA-specific dye Hoechst, respectively. (A) Confocal microscopy image showing that activated-MEK1 is localized to the Golgi apparatus in late prophase (condensed DNA*) and not in interphase (uncondensed DNA). (B) Epifluorescence microscopy image showing cells stained with ppMEK antibody that had been preincubated with either mitotically activated MEK (phosphorylated MEK1) or unphosphorylated MEK1. Preincubation of ppMEK antibody with mitotically phosphorylated MEK1 abolished the Golgi complex–specific staining of cells in late prophase (condensed DNA*). Therefore, ppMEK antibody staining is due to the presence of activated MEK1 on the Golgi apparatus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172875&req=5

fig4: Activated MEK1 is found on the Golgi apparatus in late prophase. NRK cells were blocked in S-phase with aphidicolin. The cells were washed to remove aphidicolin. 7 h after aphidicolin removal, cells were processed for fluorescence microscopy to visualize the Golgi apparatus, activated MEK1, and DNA with antibodies against β-COP, ppMEK, and the DNA-specific dye Hoechst, respectively. (A) Confocal microscopy image showing that activated-MEK1 is localized to the Golgi apparatus in late prophase (condensed DNA*) and not in interphase (uncondensed DNA). (B) Epifluorescence microscopy image showing cells stained with ppMEK antibody that had been preincubated with either mitotically activated MEK (phosphorylated MEK1) or unphosphorylated MEK1. Preincubation of ppMEK antibody with mitotically phosphorylated MEK1 abolished the Golgi complex–specific staining of cells in late prophase (condensed DNA*). Therefore, ppMEK antibody staining is due to the presence of activated MEK1 on the Golgi apparatus.
Mentions: 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 complex–specific 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 β-COP positive structures that contained mitotically activated MEK1 was 47 ± 6%. Similar results were obtained in HeLa, NIH, and COS-7 cells (not depicted).

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