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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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RAF1 is required for entry into mitosis. NRK cells were arrested in S-phase with aphidicolin. The cells were washed to remove aphidicolin and injected 5 h later with Raf-239 or GST. After an additional incubation for 2 h, the cells were stained with Golgi complex and DNA-specific markers. 400 cells were counted for each experimental condition to calculate the mitotic index. The mitotic index is the ratio of the percentage of mitotic cells in the pool of injected of cells compared with noninjected cells on the same coverslip. The mitotic index of cells injected with GST was found to be 91 ± 11 as compared with 41 ± 8 for the Raf-239–injected cells. The data are calculated from 16 independent experiments.
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fig3: RAF1 is required for entry into mitosis. NRK cells were arrested in S-phase with aphidicolin. The cells were washed to remove aphidicolin and injected 5 h later with Raf-239 or GST. After an additional incubation for 2 h, the cells were stained with Golgi complex and DNA-specific markers. 400 cells were counted for each experimental condition to calculate the mitotic index. The mitotic index is the ratio of the percentage of mitotic cells in the pool of injected of cells compared with noninjected cells on the same coverslip. The mitotic index of cells injected with GST was found to be 91 ± 11 as compared with 41 ± 8 for the Raf-239–injected cells. The data are calculated from 16 independent experiments.

Mentions: It has recently been suggested that RAF1 is specifically activated during mitosis (Laird et al., 1999; Zang et al., 2001, 2002). However, formal proof that RAF1 is required to enter mitosis is lacking. NRK cells were arrested in S-phase with aphidicolin. The cells were subsequently washed to remove aphidicolin, 5 h after aphidicolin removal, 400 cells were injected either with Raf-239 or GST. 2 h after injection, the cells were fixed and visualized by fluorescence microscopy to monitor the organization of DNA and Golgi apparatus. The mitotic index of the injected cells was determined relative to that of the noninjected cells in the same coverslip. The mitotic index of cells injected with GST was 91 ± 11. The mitotic index of cells injected with Raf-239 was 41 ± 8 (Fig. 3) . Thus, injection of Raf-239 inhibited entry into mitosis in >50% of cells. These results strengthen the proposal that a RAF1–MEK1-mediated process is necessary for entry into mitosis in mammalian cells.


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)

RAF1 is required for entry into mitosis. NRK cells were arrested in S-phase with aphidicolin. The cells were washed to remove aphidicolin and injected 5 h later with Raf-239 or GST. After an additional incubation for 2 h, the cells were stained with Golgi complex and DNA-specific markers. 400 cells were counted for each experimental condition to calculate the mitotic index. The mitotic index is the ratio of the percentage of mitotic cells in the pool of injected of cells compared with noninjected cells on the same coverslip. The mitotic index of cells injected with GST was found to be 91 ± 11 as compared with 41 ± 8 for the Raf-239–injected cells. The data are calculated from 16 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: RAF1 is required for entry into mitosis. NRK cells were arrested in S-phase with aphidicolin. The cells were washed to remove aphidicolin and injected 5 h later with Raf-239 or GST. After an additional incubation for 2 h, the cells were stained with Golgi complex and DNA-specific markers. 400 cells were counted for each experimental condition to calculate the mitotic index. The mitotic index is the ratio of the percentage of mitotic cells in the pool of injected of cells compared with noninjected cells on the same coverslip. The mitotic index of cells injected with GST was found to be 91 ± 11 as compared with 41 ± 8 for the Raf-239–injected cells. The data are calculated from 16 independent experiments.
Mentions: It has recently been suggested that RAF1 is specifically activated during mitosis (Laird et al., 1999; Zang et al., 2001, 2002). However, formal proof that RAF1 is required to enter mitosis is lacking. NRK cells were arrested in S-phase with aphidicolin. The cells were subsequently washed to remove aphidicolin, 5 h after aphidicolin removal, 400 cells were injected either with Raf-239 or GST. 2 h after injection, the cells were fixed and visualized by fluorescence microscopy to monitor the organization of DNA and Golgi apparatus. The mitotic index of the injected cells was determined relative to that of the noninjected cells in the same coverslip. The mitotic index of cells injected with GST was 91 ± 11. The mitotic index of cells injected with Raf-239 was 41 ± 8 (Fig. 3) . Thus, injection of Raf-239 inhibited entry into mitosis in >50% of cells. These results strengthen the proposal that a RAF1–MEK1-mediated process is necessary for entry into mitosis in mammalian cells.

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