Limits...
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.

Show MeSH

Related in: MedlinePlus

The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm and broadens its activity. (A) Schematic diagram of the chimera constructed between cyclin B1 and cyclin B2. Cyclin B1 is represented by an open rectangle and cyclin B2 by a solid line. The cyclin B1–B2 mutant exchanges at the sequence LCQ in both cyclins (Q106 in B1, and Q144 in B2). (B) The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). (C) The NH2 terminus of cyclin B1 confers some of the properties of cyclin B1 on cyclin B2. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3. Cells are representative of >150 cells analyzed in three separate experiments. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198800&req=5

Figure 5: The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm and broadens its activity. (A) Schematic diagram of the chimera constructed between cyclin B1 and cyclin B2. Cyclin B1 is represented by an open rectangle and cyclin B2 by a solid line. The cyclin B1–B2 mutant exchanges at the sequence LCQ in both cyclins (Q106 in B1, and Q144 in B2). (B) The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). (C) The NH2 terminus of cyclin B1 confers some of the properties of cyclin B1 on cyclin B2. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3. Cells are representative of >150 cells analyzed in three separate experiments. Bars, 10 μm.

Mentions: From these results, we predicted that the converse chimera, one where the cyclin box and second cyclin fold of cyclin B2 was targeted to the cytoplasm by the NH2 terminus of cyclin B1, would confer on cyclin B2 the broader substrate specificity of cyclin B1. This prediction was confirmed when we coexpressed a B1–B2 chimera (Fig. 5 A) with CDK1AF and found that it was localized throughout the cytoplasm and not restricted to the Golgi apparatus (Fig. 5 B). This chimera also bound and activated CDK1 to a similar extent as the wild-type protein (Fig. 4 D). Although the chimera had the hydrophobic patch and cyclin folds of cyclin B2, it was able to cause the microtubules to reorganize in addition to causing Golgi disassembly (Fig. 5 C). However, the chimera was not able to shuttle into the nucleus in the presence of LMB (data not shown). In agreement with its restricted cytoplasmic localization, it had no effect on either the condensation state of the DNA or the integrity of the nuclear lamina (Fig. 5 C).


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)

The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm and broadens its activity. (A) Schematic diagram of the chimera constructed between cyclin B1 and cyclin B2. Cyclin B1 is represented by an open rectangle and cyclin B2 by a solid line. The cyclin B1–B2 mutant exchanges at the sequence LCQ in both cyclins (Q106 in B1, and Q144 in B2). (B) The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). (C) The NH2 terminus of cyclin B1 confers some of the properties of cyclin B1 on cyclin B2. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3. Cells are representative of >150 cells analyzed in three separate experiments. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm and broadens its activity. (A) Schematic diagram of the chimera constructed between cyclin B1 and cyclin B2. Cyclin B1 is represented by an open rectangle and cyclin B2 by a solid line. The cyclin B1–B2 mutant exchanges at the sequence LCQ in both cyclins (Q106 in B1, and Q144 in B2). (B) The NH2 terminus of cyclin B1 targets cyclin B2 to the cytoplasm. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). (C) The NH2 terminus of cyclin B1 confers some of the properties of cyclin B1 on cyclin B2. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B1–B2 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3. Cells are representative of >150 cells analyzed in three separate experiments. Bars, 10 μm.
Mentions: From these results, we predicted that the converse chimera, one where the cyclin box and second cyclin fold of cyclin B2 was targeted to the cytoplasm by the NH2 terminus of cyclin B1, would confer on cyclin B2 the broader substrate specificity of cyclin B1. This prediction was confirmed when we coexpressed a B1–B2 chimera (Fig. 5 A) with CDK1AF and found that it was localized throughout the cytoplasm and not restricted to the Golgi apparatus (Fig. 5 B). This chimera also bound and activated CDK1 to a similar extent as the wild-type protein (Fig. 4 D). Although the chimera had the hydrophobic patch and cyclin folds of cyclin B2, it was able to cause the microtubules to reorganize in addition to causing Golgi disassembly (Fig. 5 C). However, the chimera was not able to shuttle into the nucleus in the presence of LMB (data not shown). In agreement with its restricted cytoplasmic localization, it had no effect on either the condensation state of the DNA or the integrity of the nuclear lamina (Fig. 5 C).

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