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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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Different effects of cyclin B1– and B2–CDKs on subcellular architecture. Serum-starved CHO cells were unperturbed (bottom row) or microinjected with expression vectors coding for a Golgi marker NAGT–GFP (d, j, and o; green in e, k, and p) and CDK1AF, alone (third row), with cyclin B1 (top row), or with cyclin B2 (second row). After 6 h, the cells were fixed and stained with TOTO-3 to visualize the DNA (a, g, l, and q) and with an antilamin antibody (b, h, m, and r) or an anti–β-tubulin antibody (c, i, n, and s; red in e, k, p, and u). Antimannosidase II was used to stain the Golgi in uninjected cells (t; green in u). Note that cyclin B1 + CDK1AF caused the nuclear lamina to disassemble, and the solubilized lamin protein was washed out of the cell during fixation. The Golgi apparatus fragmented in both cyclin B1–CDK1AF and cyclin B2–CDK1AF injected cells, but only cyclin B1 + CDK1AF caused the microtubules to become much shorter and the centrosome to nucleate more microtubules. Cells are representative of more than 120 cells analyzed in more than three separate experiments. Bars, 10 μm.
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Figure 2: Different effects of cyclin B1– and B2–CDKs on subcellular architecture. Serum-starved CHO cells were unperturbed (bottom row) or microinjected with expression vectors coding for a Golgi marker NAGT–GFP (d, j, and o; green in e, k, and p) and CDK1AF, alone (third row), with cyclin B1 (top row), or with cyclin B2 (second row). After 6 h, the cells were fixed and stained with TOTO-3 to visualize the DNA (a, g, l, and q) and with an antilamin antibody (b, h, m, and r) or an anti–β-tubulin antibody (c, i, n, and s; red in e, k, p, and u). Antimannosidase II was used to stain the Golgi in uninjected cells (t; green in u). Note that cyclin B1 + CDK1AF caused the nuclear lamina to disassemble, and the solubilized lamin protein was washed out of the cell during fixation. The Golgi apparatus fragmented in both cyclin B1–CDK1AF and cyclin B2–CDK1AF injected cells, but only cyclin B1 + CDK1AF caused the microtubules to become much shorter and the centrosome to nucleate more microtubules. Cells are representative of more than 120 cells analyzed in more than three separate experiments. Bars, 10 μm.

Mentions: The striking difference in the localization of the two B-type cyclins raised the possibility that a cyclin B1–CDK1 complex might have a different substrate specificity compared with cyclin B2–CDK1. Therefore, we analyzed changes in the subcellular architecture as a consequence of expressing CDK1AF with or without B-type cyclins. CDK1AF alone had no effect on any component of the cells that we analyzed, further evidence that G0/G1 phase CHO cells did not contain any endogenous mitotic cyclins (Fig. 2, compare l–p and q–u). When we coexpressed CDK1AF with cyclin B1, we found that the nuclear lamina was rapidly solubilized (within 3 h) in almost all the cells (Fig. 2 b; Table ). After 4 h, ∼30% of cells expressing cyclin B1–CDK1AF had rounded up, and in these cells the microtubules had reorganized to form a large aster and the chromosomes had prematurely condensed (Fig. 2, a and c; Table ). After 6.5 h, almost all cells expressing cyclin B1–CDK1AF had rounded up. These changes were characteristic of mitotic cells, indicating that cyclin B1–CDK1AF was able, directly or indirectly, to induce mitotic changes in the nucleus and the cytoplasm. Time-lapse fluorescence studies also showed that cyclin B1–GFP translocated into the nucleus of those cells that rounded up and condensed their DNA (data not shown).


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)

Different effects of cyclin B1– and B2–CDKs on subcellular architecture. Serum-starved CHO cells were unperturbed (bottom row) or microinjected with expression vectors coding for a Golgi marker NAGT–GFP (d, j, and o; green in e, k, and p) and CDK1AF, alone (third row), with cyclin B1 (top row), or with cyclin B2 (second row). After 6 h, the cells were fixed and stained with TOTO-3 to visualize the DNA (a, g, l, and q) and with an antilamin antibody (b, h, m, and r) or an anti–β-tubulin antibody (c, i, n, and s; red in e, k, p, and u). Antimannosidase II was used to stain the Golgi in uninjected cells (t; green in u). Note that cyclin B1 + CDK1AF caused the nuclear lamina to disassemble, and the solubilized lamin protein was washed out of the cell during fixation. The Golgi apparatus fragmented in both cyclin B1–CDK1AF and cyclin B2–CDK1AF injected cells, but only cyclin B1 + CDK1AF caused the microtubules to become much shorter and the centrosome to nucleate more microtubules. Cells are representative of more than 120 cells analyzed in more than three separate experiments. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Different effects of cyclin B1– and B2–CDKs on subcellular architecture. Serum-starved CHO cells were unperturbed (bottom row) or microinjected with expression vectors coding for a Golgi marker NAGT–GFP (d, j, and o; green in e, k, and p) and CDK1AF, alone (third row), with cyclin B1 (top row), or with cyclin B2 (second row). After 6 h, the cells were fixed and stained with TOTO-3 to visualize the DNA (a, g, l, and q) and with an antilamin antibody (b, h, m, and r) or an anti–β-tubulin antibody (c, i, n, and s; red in e, k, p, and u). Antimannosidase II was used to stain the Golgi in uninjected cells (t; green in u). Note that cyclin B1 + CDK1AF caused the nuclear lamina to disassemble, and the solubilized lamin protein was washed out of the cell during fixation. The Golgi apparatus fragmented in both cyclin B1–CDK1AF and cyclin B2–CDK1AF injected cells, but only cyclin B1 + CDK1AF caused the microtubules to become much shorter and the centrosome to nucleate more microtubules. Cells are representative of more than 120 cells analyzed in more than three separate experiments. Bars, 10 μm.
Mentions: The striking difference in the localization of the two B-type cyclins raised the possibility that a cyclin B1–CDK1 complex might have a different substrate specificity compared with cyclin B2–CDK1. Therefore, we analyzed changes in the subcellular architecture as a consequence of expressing CDK1AF with or without B-type cyclins. CDK1AF alone had no effect on any component of the cells that we analyzed, further evidence that G0/G1 phase CHO cells did not contain any endogenous mitotic cyclins (Fig. 2, compare l–p and q–u). When we coexpressed CDK1AF with cyclin B1, we found that the nuclear lamina was rapidly solubilized (within 3 h) in almost all the cells (Fig. 2 b; Table ). After 4 h, ∼30% of cells expressing cyclin B1–CDK1AF had rounded up, and in these cells the microtubules had reorganized to form a large aster and the chromosomes had prematurely condensed (Fig. 2, a and c; Table ). After 6.5 h, almost all cells expressing cyclin B1–CDK1AF had rounded up. These changes were characteristic of mitotic cells, indicating that cyclin B1–CDK1AF was able, directly or indirectly, to induce mitotic changes in the nucleus and the cytoplasm. Time-lapse fluorescence studies also showed that cyclin B1–GFP translocated into the nucleus of those cells that rounded up and condensed their DNA (data not shown).

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