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The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus.

Draviam VM, Orrechia S, Lowe M, Pardi R, Pines J - J. Cell Biol. (2001)

Bottom Line: We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2.Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1.Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.

View Article: PubMed Central - PubMed

Affiliation: Wellcome/Cancer Research Campaign Institute and Department of Zoology, Cambridge CB2 1QR, United Kingdom.

ABSTRACT
In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin-CDKs underlies the ability of cyclin B1-CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2-CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.

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MEK activity is not required for Golgi disassembly in normal mitosis. (A and B) CHO cells were serum starved for 36 h and then stimulated with DME plus 10% serum. At 18 h, the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of cells. Samples were taken for flow cytometry analysis and for immunoblotting at 45 min and 2, 3, and 6 h after adding MEK inhibitors while cells were progressing through mitosis. Flow cytometry showed that the peak of mitosis was at 3 h after addition of inhibitors (data not shown). Samples were processed to detect phophorylated ERK1 and ERK2 (A) and total ERK1 and ERK2 (B) as in the legend to Fig. 6. Results shown are representative of three independent experiments. (C) CHO cells were serum starved and refed and the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of G2 phase cells as in A and B. DMSO was added to the other set. 3 h later, cells were processed for immunofluorescence with antigiantin antibodies (green) and TOTO-3 to stain the DNA (blue). Cells at various stages of mitosis are shown and are representative of >100 cells examined in three independent experiments.
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Figure 7: MEK activity is not required for Golgi disassembly in normal mitosis. (A and B) CHO cells were serum starved for 36 h and then stimulated with DME plus 10% serum. At 18 h, the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of cells. Samples were taken for flow cytometry analysis and for immunoblotting at 45 min and 2, 3, and 6 h after adding MEK inhibitors while cells were progressing through mitosis. Flow cytometry showed that the peak of mitosis was at 3 h after addition of inhibitors (data not shown). Samples were processed to detect phophorylated ERK1 and ERK2 (A) and total ERK1 and ERK2 (B) as in the legend to Fig. 6. Results shown are representative of three independent experiments. (C) CHO cells were serum starved and refed and the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of G2 phase cells as in A and B. DMSO was added to the other set. 3 h later, cells were processed for immunofluorescence with antigiantin antibodies (green) and TOTO-3 to stain the DNA (blue). Cells at various stages of mitosis are shown and are representative of >100 cells examined in three independent experiments.

Mentions: We also excluded the possibility that MEK was required to break down the Golgi apparatus during normal mitosis, that is, in cells with endogenous levels of B-type cyclin–CDK activity. We treated G2 phase CHO cells with the same concentrations of U0126 and PD98059 that we used for serum-stimulated cells and found that this inhibited MEK in G2 phase cells (Fig. 7A and Fig. B). These cells proceeded through mitosis at the same rate as untreated cells (Draviam, V.M., and J. Pines, manuscript in preparation), and there was no difference between control cells and MEK-inhibited cells in disassembly of the Golgi apparatus as analyzed by confocal immunofluorescence microscopy (Fig. 7 C). These data showed that neither MEK, ERK1, nor ERK2 was required for the disassembly of the Golgi apparatus in normal mitosis.


The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus.

Draviam VM, Orrechia S, Lowe M, Pardi R, Pines J - J. Cell Biol. (2001)

MEK activity is not required for Golgi disassembly in normal mitosis. (A and B) CHO cells were serum starved for 36 h and then stimulated with DME plus 10% serum. At 18 h, the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of cells. Samples were taken for flow cytometry analysis and for immunoblotting at 45 min and 2, 3, and 6 h after adding MEK inhibitors while cells were progressing through mitosis. Flow cytometry showed that the peak of mitosis was at 3 h after addition of inhibitors (data not shown). Samples were processed to detect phophorylated ERK1 and ERK2 (A) and total ERK1 and ERK2 (B) as in the legend to Fig. 6. Results shown are representative of three independent experiments. (C) CHO cells were serum starved and refed and the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of G2 phase cells as in A and B. DMSO was added to the other set. 3 h later, cells were processed for immunofluorescence with antigiantin antibodies (green) and TOTO-3 to stain the DNA (blue). Cells at various stages of mitosis are shown and are representative of >100 cells examined in three independent experiments.
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Related In: Results  -  Collection

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Figure 7: MEK activity is not required for Golgi disassembly in normal mitosis. (A and B) CHO cells were serum starved for 36 h and then stimulated with DME plus 10% serum. At 18 h, the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of cells. Samples were taken for flow cytometry analysis and for immunoblotting at 45 min and 2, 3, and 6 h after adding MEK inhibitors while cells were progressing through mitosis. Flow cytometry showed that the peak of mitosis was at 3 h after addition of inhibitors (data not shown). Samples were processed to detect phophorylated ERK1 and ERK2 (A) and total ERK1 and ERK2 (B) as in the legend to Fig. 6. Results shown are representative of three independent experiments. (C) CHO cells were serum starved and refed and the MEK inhibitors U0126 (25 μM) and PD98059 (20 μM) were added to one set of G2 phase cells as in A and B. DMSO was added to the other set. 3 h later, cells were processed for immunofluorescence with antigiantin antibodies (green) and TOTO-3 to stain the DNA (blue). Cells at various stages of mitosis are shown and are representative of >100 cells examined in three independent experiments.
Mentions: We also excluded the possibility that MEK was required to break down the Golgi apparatus during normal mitosis, that is, in cells with endogenous levels of B-type cyclin–CDK activity. We treated G2 phase CHO cells with the same concentrations of U0126 and PD98059 that we used for serum-stimulated cells and found that this inhibited MEK in G2 phase cells (Fig. 7A and Fig. B). These cells proceeded through mitosis at the same rate as untreated cells (Draviam, V.M., and J. Pines, manuscript in preparation), and there was no difference between control cells and MEK-inhibited cells in disassembly of the Golgi apparatus as analyzed by confocal immunofluorescence microscopy (Fig. 7 C). These data showed that neither MEK, ERK1, nor ERK2 was required for the disassembly of the Golgi apparatus in normal mitosis.

Bottom Line: We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2.Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1.Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.

View Article: PubMed Central - PubMed

Affiliation: Wellcome/Cancer Research Campaign Institute and Department of Zoology, Cambridge CB2 1QR, United Kingdom.

ABSTRACT
In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin-CDKs underlies the ability of cyclin B1-CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2-CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.

Show MeSH
Related in: MedlinePlus