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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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The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi and restricts its activity. (A) Schematic diagram of the chimera constructed between cyclin B2 and cyclin B1. Cyclin B2 is represented by a solid line and cyclin B1 by an open rectangle. The cyclin B2–B1 mutant exchanges at the sequence LCS (S130 in cyclin B2, and S177 in cyclin B1) in both cyclins. The hydrophobic patch is represented by the filled oval. (B) The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi apparatus. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). One uninjected cell and one cell expressing the chimera are shown. (C) The NH2 terminus of cyclin B2 confers the properties of cyclin B2 on cyclin B1. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3 to visualize the DNA. Cells are representative of >100 cells analyzed in three separate experiments. (D) Cyclin B chimeras bind and activate CDK1 to a similar extent as wild-type cyclins. Human 293T cells were mock transfected with an empty vector or transfected with untagged CDK1AF and myc epitope–tagged cyclins B1, B2, or the B1–B2 or B2–B1 chimera. 12 h after transfection, cells were lysed with NP-40 lysis buffer and the transfected cyclins were immunoprecipitated with an anti-myc epitope antibody. Immunoprecipitates were processed for H1 kinase assays and the amount of phosphate incorporated was quantitated and normalized to the H1 kinase activity in the cyclin B1–CDK sample. In parallel, immunoprecipitates were immunoblotted with an anti-CDK1 monoclonal (inset) and an anti-myc epitope antibody (not shown) to demonstrate that equivalent amounts of cyclins were immunoprecipitated. Results shown are representative of two independent experiments. Bars, 10 μm.
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Figure 4: The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi and restricts its activity. (A) Schematic diagram of the chimera constructed between cyclin B2 and cyclin B1. Cyclin B2 is represented by a solid line and cyclin B1 by an open rectangle. The cyclin B2–B1 mutant exchanges at the sequence LCS (S130 in cyclin B2, and S177 in cyclin B1) in both cyclins. The hydrophobic patch is represented by the filled oval. (B) The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi apparatus. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). One uninjected cell and one cell expressing the chimera are shown. (C) The NH2 terminus of cyclin B2 confers the properties of cyclin B2 on cyclin B1. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3 to visualize the DNA. Cells are representative of >100 cells analyzed in three separate experiments. (D) Cyclin B chimeras bind and activate CDK1 to a similar extent as wild-type cyclins. Human 293T cells were mock transfected with an empty vector or transfected with untagged CDK1AF and myc epitope–tagged cyclins B1, B2, or the B1–B2 or B2–B1 chimera. 12 h after transfection, cells were lysed with NP-40 lysis buffer and the transfected cyclins were immunoprecipitated with an anti-myc epitope antibody. Immunoprecipitates were processed for H1 kinase assays and the amount of phosphate incorporated was quantitated and normalized to the H1 kinase activity in the cyclin B1–CDK sample. In parallel, immunoprecipitates were immunoblotted with an anti-CDK1 monoclonal (inset) and an anti-myc epitope antibody (not shown) to demonstrate that equivalent amounts of cyclins were immunoprecipitated. Results shown are representative of two independent experiments. Bars, 10 μm.

Mentions: Our results indicated that cyclin B2 restricted the substrate specificity of CDK1 to Golgi-associated proteins by targeting it to the Golgi apparatus. However, an alternative explanation could be that the hydrophobic patch region of cyclin B2 was only able to bind a restricted set of substrates, although this appeared unlikely because there were only a few changes between cyclin B1 and B2 in this region. Nevertheless, we constructed a series of chimeras between cyclin B1 and B2 to identify the region that targeted cyclin B2 to the Golgi apparatus. We generated chimeras in which the exchange between the cyclins was made at conserved motifs in the two cyclins. This showed that amino acids 1–127 were required to target cyclin B2 to the Golgi apparatus (Fig. 4 A). Replacing the NH2 terminus of cyclin B1 with this region of cyclin B2 (Fig. 4 A) targeted cyclin B1 to the Golgi apparatus (Fig. 4 B). Importantly, this chimera contained the whole of the cyclin box and second cyclin fold of cyclin B1, including the hydrophobic patch of cyclin B1 and was able to bind and activate CDK1 to a similar extent as wild-type cyclin B1 (Fig. 4 D). When we coexpressed the B2–B1 chimera with CDK1AF, it was now only able to cause the Golgi apparatus to disassemble (Fig. 4 C). It did not cause chromosome condensation or nuclear lamina breakdown (Fig. 4 C), nor did it alter the microtubule array or translocate to the nucleus in any of the cells that we assayed.


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 B2 targets cyclin B1 to the Golgi and restricts its activity. (A) Schematic diagram of the chimera constructed between cyclin B2 and cyclin B1. Cyclin B2 is represented by a solid line and cyclin B1 by an open rectangle. The cyclin B2–B1 mutant exchanges at the sequence LCS (S130 in cyclin B2, and S177 in cyclin B1) in both cyclins. The hydrophobic patch is represented by the filled oval. (B) The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi apparatus. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). One uninjected cell and one cell expressing the chimera are shown. (C) The NH2 terminus of cyclin B2 confers the properties of cyclin B2 on cyclin B1. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3 to visualize the DNA. Cells are representative of >100 cells analyzed in three separate experiments. (D) Cyclin B chimeras bind and activate CDK1 to a similar extent as wild-type cyclins. Human 293T cells were mock transfected with an empty vector or transfected with untagged CDK1AF and myc epitope–tagged cyclins B1, B2, or the B1–B2 or B2–B1 chimera. 12 h after transfection, cells were lysed with NP-40 lysis buffer and the transfected cyclins were immunoprecipitated with an anti-myc epitope antibody. Immunoprecipitates were processed for H1 kinase assays and the amount of phosphate incorporated was quantitated and normalized to the H1 kinase activity in the cyclin B1–CDK sample. In parallel, immunoprecipitates were immunoblotted with an anti-CDK1 monoclonal (inset) and an anti-myc epitope antibody (not shown) to demonstrate that equivalent amounts of cyclins were immunoprecipitated. Results shown are representative of two independent experiments. Bars, 10 μm.
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Figure 4: The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi and restricts its activity. (A) Schematic diagram of the chimera constructed between cyclin B2 and cyclin B1. Cyclin B2 is represented by a solid line and cyclin B1 by an open rectangle. The cyclin B2–B1 mutant exchanges at the sequence LCS (S130 in cyclin B2, and S177 in cyclin B1) in both cyclins. The hydrophobic patch is represented by the filled oval. (B) The NH2 terminus of cyclin B2 targets cyclin B1 to the Golgi apparatus. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP (green) and with a myc epitope–tagged cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti-myc epitope antibody (red). One uninjected cell and one cell expressing the chimera are shown. (C) The NH2 terminus of cyclin B2 confers the properties of cyclin B2 on cyclin B1. Serum-starved CHO cells were microinjected with expression vectors coding for a Golgi marker NAGT–GFP and CDK1AF with a cyclin B2–B1 chimera. After 6 h, the cells were fixed and stained with an anti–β-tubulin antibody or with an antilamin antibody and TOTO-3 to visualize the DNA. Cells are representative of >100 cells analyzed in three separate experiments. (D) Cyclin B chimeras bind and activate CDK1 to a similar extent as wild-type cyclins. Human 293T cells were mock transfected with an empty vector or transfected with untagged CDK1AF and myc epitope–tagged cyclins B1, B2, or the B1–B2 or B2–B1 chimera. 12 h after transfection, cells were lysed with NP-40 lysis buffer and the transfected cyclins were immunoprecipitated with an anti-myc epitope antibody. Immunoprecipitates were processed for H1 kinase assays and the amount of phosphate incorporated was quantitated and normalized to the H1 kinase activity in the cyclin B1–CDK sample. In parallel, immunoprecipitates were immunoblotted with an anti-CDK1 monoclonal (inset) and an anti-myc epitope antibody (not shown) to demonstrate that equivalent amounts of cyclins were immunoprecipitated. Results shown are representative of two independent experiments. Bars, 10 μm.
Mentions: Our results indicated that cyclin B2 restricted the substrate specificity of CDK1 to Golgi-associated proteins by targeting it to the Golgi apparatus. However, an alternative explanation could be that the hydrophobic patch region of cyclin B2 was only able to bind a restricted set of substrates, although this appeared unlikely because there were only a few changes between cyclin B1 and B2 in this region. Nevertheless, we constructed a series of chimeras between cyclin B1 and B2 to identify the region that targeted cyclin B2 to the Golgi apparatus. We generated chimeras in which the exchange between the cyclins was made at conserved motifs in the two cyclins. This showed that amino acids 1–127 were required to target cyclin B2 to the Golgi apparatus (Fig. 4 A). Replacing the NH2 terminus of cyclin B1 with this region of cyclin B2 (Fig. 4 A) targeted cyclin B1 to the Golgi apparatus (Fig. 4 B). Importantly, this chimera contained the whole of the cyclin box and second cyclin fold of cyclin B1, including the hydrophobic patch of cyclin B1 and was able to bind and activate CDK1 to a similar extent as wild-type cyclin B1 (Fig. 4 D). When we coexpressed the B2–B1 chimera with CDK1AF, it was now only able to cause the Golgi apparatus to disassemble (Fig. 4 C). It did not cause chromosome condensation or nuclear lamina breakdown (Fig. 4 C), nor did it alter the microtubule array or translocate to the nucleus in any of the cells that we assayed.

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