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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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Cyclin B1–CDK1AF and cyclin B2–CDK1AF have different effects on the Golgi apparatus. Serum- starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (c and d) and cyclin B1 with CDK1AF (a, c, e, and g) or cyclin B2 with CDK1AF (b, d, f, and h). Cells were fixed and stained with antimannosidase II (not shown) and antigiantin (a and b, arrows denote uninjected cells) or with an anti–β-tubulin antibody (e and f). The merge shown (g and h) is between antitubulin and NAGT–GFP. Note that cyclin B1–CDK1AF causes the Golgi to break down into smaller and more numerous vesicles than does cyclin B2–CDK1AF in cells before observable changes in the cytoskeleton. Cells are representative of >250 cells analyzed in >10 separate experiments. Bars, 10 μm.
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Figure 3: Cyclin B1–CDK1AF and cyclin B2–CDK1AF have different effects on the Golgi apparatus. Serum- starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (c and d) and cyclin B1 with CDK1AF (a, c, e, and g) or cyclin B2 with CDK1AF (b, d, f, and h). Cells were fixed and stained with antimannosidase II (not shown) and antigiantin (a and b, arrows denote uninjected cells) or with an anti–β-tubulin antibody (e and f). The merge shown (g and h) is between antitubulin and NAGT–GFP. Note that cyclin B1–CDK1AF causes the Golgi to break down into smaller and more numerous vesicles than does cyclin B2–CDK1AF in cells before observable changes in the cytoskeleton. Cells are representative of >250 cells analyzed in >10 separate experiments. Bars, 10 μm.

Mentions: Expressing CDK1AF alone had no effect on the integrity of the Golgi apparatus in G0/G1 phase CHO cells as judged by either antimannosidase II or NAGT–GFP staining (Fig. 2o). When we coexpressed cyclin B1 with CDK1AF, the Golgi broke down and took on an appearance similar to that seen in early metaphase (Fig. 2d and Fig. e; Table ). In marked contrast to its lack of effect on the nucleus and the microtubules, coexpressing cyclin B2 with CDK1AF also caused the Golgi to disperse (Fig. 2j and Fig. k; Table ). Using three different Golgi markers, we noticed that cyclin B2–CDK1AF tended to cause the Golgi to disperse into fewer and larger vesicles than cyclin B1–CDK1AF (NAGT–GFP and antigiantin shown in Fig. 3; antimannosidase, not shown). This could not be ascribed to changes in microtubule organization instigated by cyclin B1–CDK1AF because it was also seen in cells expressing cyclin B1–CDK1AF in which the microtubule array was unchanged (Fig. 3c, Fig. e, and Fig. g).


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)

Cyclin B1–CDK1AF and cyclin B2–CDK1AF have different effects on the Golgi apparatus. Serum- starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (c and d) and cyclin B1 with CDK1AF (a, c, e, and g) or cyclin B2 with CDK1AF (b, d, f, and h). Cells were fixed and stained with antimannosidase II (not shown) and antigiantin (a and b, arrows denote uninjected cells) or with an anti–β-tubulin antibody (e and f). The merge shown (g and h) is between antitubulin and NAGT–GFP. Note that cyclin B1–CDK1AF causes the Golgi to break down into smaller and more numerous vesicles than does cyclin B2–CDK1AF in cells before observable changes in the cytoskeleton. Cells are representative of >250 cells analyzed in >10 separate experiments. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198800&req=5

Figure 3: Cyclin B1–CDK1AF and cyclin B2–CDK1AF have different effects on the Golgi apparatus. Serum- starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (c and d) and cyclin B1 with CDK1AF (a, c, e, and g) or cyclin B2 with CDK1AF (b, d, f, and h). Cells were fixed and stained with antimannosidase II (not shown) and antigiantin (a and b, arrows denote uninjected cells) or with an anti–β-tubulin antibody (e and f). The merge shown (g and h) is between antitubulin and NAGT–GFP. Note that cyclin B1–CDK1AF causes the Golgi to break down into smaller and more numerous vesicles than does cyclin B2–CDK1AF in cells before observable changes in the cytoskeleton. Cells are representative of >250 cells analyzed in >10 separate experiments. Bars, 10 μm.
Mentions: Expressing CDK1AF alone had no effect on the integrity of the Golgi apparatus in G0/G1 phase CHO cells as judged by either antimannosidase II or NAGT–GFP staining (Fig. 2o). When we coexpressed cyclin B1 with CDK1AF, the Golgi broke down and took on an appearance similar to that seen in early metaphase (Fig. 2d and Fig. e; Table ). In marked contrast to its lack of effect on the nucleus and the microtubules, coexpressing cyclin B2 with CDK1AF also caused the Golgi to disperse (Fig. 2j and Fig. k; Table ). Using three different Golgi markers, we noticed that cyclin B2–CDK1AF tended to cause the Golgi to disperse into fewer and larger vesicles than cyclin B1–CDK1AF (NAGT–GFP and antigiantin shown in Fig. 3; antimannosidase, not shown). This could not be ascribed to changes in microtubule organization instigated by cyclin B1–CDK1AF because it was also seen in cells expressing cyclin B1–CDK1AF in which the microtubule array was unchanged (Fig. 3c, Fig. e, and Fig. g).

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