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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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(A) Human B-type cyclins are expressed to similar levels in CHO cells. Serum-starved CHO cells were transfected with plasmids encoding human cyclin B1 or B2 and/or CDK1AF all tagged with one copy of the myc epitope and under the “tetracyclin-OFF” promoter. Cells were incubated in the presence or absence of tetracyclin to repress or induce protein expression, respectively. 16 h after induction, cells were lysed, and the extracts were run on one-dimensional SDS-PAGE and then immunoblotted with the 9E10 mAb to detect the proteins. Lane 1, mock-transfected cells; lanes 2–5, transfected cells: (lane 2) cyclin B1 plasmid plus tetracyclin; (lane 3) Cdk1AF; (lane 4) cyclin B1 minus tetracyclin for 16 h; (lane 5) cyclin B2 minus tetracyclin for 16 h. (B) Ectopic and endogenous cyclin B1 are expressed at similar levels in CHO cells. Approximately 980 serum-starved CHO cells were microinjected with plasmids encoding myc epitope–tagged human cyclin B1 with CDK1AF. 6 h after microinjection, cells were lysed and the samples were run on one-dimensional SDS-PAGE next to lysates from 250, 500, and 1,000 mitotic cells. Proteins were immunoblotted with an anticyclin B1 monoclonal antibody V152 that recognizes both human and rodent cyclin B1 to detect the proteins (a gift from J. Gannon and T. Hunt). Lane 1, uninjected cells; lane 2, cells injected with cyclin B1–CDK1AF; lanes 3–5, 250, 500, and 1,000 mitotic HeLa cells; lane 6, 1,000 mitotic CHO cells. M, molecular mass marker lane. Results shown are representative of three independent experiments. (C) There are no detectable endogenous mitotic cyclins in serum-starved CHO cells with or without ectopic human cyclin–CDKs. (a–d) CHO cells were serum starved for 24 h and then microinjected with NAGT–GFP as a Golgi apparatus and injection marker (green) and TOTO-3 to visualize the DNA (blue) together with CDK1AF and either cyclin B1 (a and b) or cyclin B2 (c and d). 6 h after microinjection, cells were fixed and stained with an anti–mouse cyclin A antibody (red) (a gift from Dr. M. Carrington, University of Cambridge, Cambridge, UK) (a and c) or with an anti–rodent cyclin B1 monoclonal antibody V143 (red) that recognizes rodent B-type cyclins but does not cross-react with human B-type cyclins (a gift from J. Gannon and T. Hunt) (b and d). (e and f) Uninjected asynchronous (e) and serum-starved (f) CHO cells were costained with anticyclin A (red), anticyclin B1 (green), and TOTO-3 to visualize the DNA (blue). Results shown are representative of two independent experiments. (D and E) Human B-type cyclins localize correctly in CHO cells. Human cyclin B1 or B2 was tagged with one copy of the myc epitope and microinjected as cDNA under the CMV promoter into serum-starved CHO cells. 3 h after microinjection, cells were treated or not with LMB. Cells were stained with the 9E10 mAb to detect the cyclins. (D) Cells were costained with an antimannosidase II antibody to detect the Golgi apparatus (red in the merged images) and cyclin B1 (top row) or cyclin B2 (bottom row) (green in the merged images). (E) Cells expressing human cyclin B1 or B2 were fixed before (a and b) or after (c and d) treatment with 20 nM LMB for 45 min and stained for ectopically expressed cyclin B1 (a and c) or cyclin B2 (b and d).
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Figure 1: (A) Human B-type cyclins are expressed to similar levels in CHO cells. Serum-starved CHO cells were transfected with plasmids encoding human cyclin B1 or B2 and/or CDK1AF all tagged with one copy of the myc epitope and under the “tetracyclin-OFF” promoter. Cells were incubated in the presence or absence of tetracyclin to repress or induce protein expression, respectively. 16 h after induction, cells were lysed, and the extracts were run on one-dimensional SDS-PAGE and then immunoblotted with the 9E10 mAb to detect the proteins. Lane 1, mock-transfected cells; lanes 2–5, transfected cells: (lane 2) cyclin B1 plasmid plus tetracyclin; (lane 3) Cdk1AF; (lane 4) cyclin B1 minus tetracyclin for 16 h; (lane 5) cyclin B2 minus tetracyclin for 16 h. (B) Ectopic and endogenous cyclin B1 are expressed at similar levels in CHO cells. Approximately 980 serum-starved CHO cells were microinjected with plasmids encoding myc epitope–tagged human cyclin B1 with CDK1AF. 6 h after microinjection, cells were lysed and the samples were run on one-dimensional SDS-PAGE next to lysates from 250, 500, and 1,000 mitotic cells. Proteins were immunoblotted with an anticyclin B1 monoclonal antibody V152 that recognizes both human and rodent cyclin B1 to detect the proteins (a gift from J. Gannon and T. Hunt). Lane 1, uninjected cells; lane 2, cells injected with cyclin B1–CDK1AF; lanes 3–5, 250, 500, and 1,000 mitotic HeLa cells; lane 6, 1,000 mitotic CHO cells. M, molecular mass marker lane. Results shown are representative of three independent experiments. (C) There are no detectable endogenous mitotic cyclins in serum-starved CHO cells with or without ectopic human cyclin–CDKs. (a–d) CHO cells were serum starved for 24 h and then microinjected with NAGT–GFP as a Golgi apparatus and injection marker (green) and TOTO-3 to visualize the DNA (blue) together with CDK1AF and either cyclin B1 (a and b) or cyclin B2 (c and d). 6 h after microinjection, cells were fixed and stained with an anti–mouse cyclin A antibody (red) (a gift from Dr. M. Carrington, University of Cambridge, Cambridge, UK) (a and c) or with an anti–rodent cyclin B1 monoclonal antibody V143 (red) that recognizes rodent B-type cyclins but does not cross-react with human B-type cyclins (a gift from J. Gannon and T. Hunt) (b and d). (e and f) Uninjected asynchronous (e) and serum-starved (f) CHO cells were costained with anticyclin A (red), anticyclin B1 (green), and TOTO-3 to visualize the DNA (blue). Results shown are representative of two independent experiments. (D and E) Human B-type cyclins localize correctly in CHO cells. Human cyclin B1 or B2 was tagged with one copy of the myc epitope and microinjected as cDNA under the CMV promoter into serum-starved CHO cells. 3 h after microinjection, cells were treated or not with LMB. Cells were stained with the 9E10 mAb to detect the cyclins. (D) Cells were costained with an antimannosidase II antibody to detect the Golgi apparatus (red in the merged images) and cyclin B1 (top row) or cyclin B2 (bottom row) (green in the merged images). (E) Cells expressing human cyclin B1 or B2 were fixed before (a and b) or after (c and d) treatment with 20 nM LMB for 45 min and stained for ectopically expressed cyclin B1 (a and c) or cyclin B2 (b and d).

Mentions: To elucidate any differences in the biological properties of mammalian B-type cyclins, we sought to analyze their effects in cells lacking any other mitotic cyclins. Therefore, we chose to express B-type cyclins in cells just after release from serum starvation (G0/G1) when they lack endogenous mitotic cyclins (Brandeis and Hunt 1996). We used CHO cells because they have a well-defined Golgi apparatus, and we confirmed that serum-starved CHO cells do not have endogenous mitotic cyclins by both immunoblotting and immunofluorescence for cyclins A and B1 (Fig. 1a and Fig. c). To generate active cyclin B–CDK1 complexes, we coexpressed cyclin B1 or B2 with a mutant form of CDK1, CDK1T14A,Y15F (hereafter referred to as CDK1AF) that cannot be inactivated by either the Wee1 or Myt1 kinases (Morgan 1995). We coexpressed the proteins by one of two methods. In some experiments, we transfected the cDNAs under tetracycline-inducible promoters into a CHO cell line carrying a tetracycline repressor. Immunoblotting extracts of the cells showed that both cyclin B1 and B2 were expressed to similar levels (Fig. 1 A) with similar kinetics and that their expression paralleled an increase in histone H1 kinase activity (data not shown). In other experiments, we analyzed cells by time-lapse microscopy and immunofluorescence after microinjecting expression constructs, encoding the proteins under the control of the CMV promoter. We could detect the proteins by confocal immunofluorescence microscopy 3 h after microinjection. By immunoblotting these cells and quantifying the signals with NIH Image, we found that microinjected cells reproducibly expressed approximately the same amount of cyclin B1 as an equivalent number of mitotic cells (Fig. 1 B). (Note that this analysis underestimated the amount of cyclin B1 in the mitotic samples because they were collected by mitotic shake-off without nocodazole treatment, and therefore contained some anaphase and telophase cells without any B-type cyclins.)


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)

(A) Human B-type cyclins are expressed to similar levels in CHO cells. Serum-starved CHO cells were transfected with plasmids encoding human cyclin B1 or B2 and/or CDK1AF all tagged with one copy of the myc epitope and under the “tetracyclin-OFF” promoter. Cells were incubated in the presence or absence of tetracyclin to repress or induce protein expression, respectively. 16 h after induction, cells were lysed, and the extracts were run on one-dimensional SDS-PAGE and then immunoblotted with the 9E10 mAb to detect the proteins. Lane 1, mock-transfected cells; lanes 2–5, transfected cells: (lane 2) cyclin B1 plasmid plus tetracyclin; (lane 3) Cdk1AF; (lane 4) cyclin B1 minus tetracyclin for 16 h; (lane 5) cyclin B2 minus tetracyclin for 16 h. (B) Ectopic and endogenous cyclin B1 are expressed at similar levels in CHO cells. Approximately 980 serum-starved CHO cells were microinjected with plasmids encoding myc epitope–tagged human cyclin B1 with CDK1AF. 6 h after microinjection, cells were lysed and the samples were run on one-dimensional SDS-PAGE next to lysates from 250, 500, and 1,000 mitotic cells. Proteins were immunoblotted with an anticyclin B1 monoclonal antibody V152 that recognizes both human and rodent cyclin B1 to detect the proteins (a gift from J. Gannon and T. Hunt). Lane 1, uninjected cells; lane 2, cells injected with cyclin B1–CDK1AF; lanes 3–5, 250, 500, and 1,000 mitotic HeLa cells; lane 6, 1,000 mitotic CHO cells. M, molecular mass marker lane. Results shown are representative of three independent experiments. (C) There are no detectable endogenous mitotic cyclins in serum-starved CHO cells with or without ectopic human cyclin–CDKs. (a–d) CHO cells were serum starved for 24 h and then microinjected with NAGT–GFP as a Golgi apparatus and injection marker (green) and TOTO-3 to visualize the DNA (blue) together with CDK1AF and either cyclin B1 (a and b) or cyclin B2 (c and d). 6 h after microinjection, cells were fixed and stained with an anti–mouse cyclin A antibody (red) (a gift from Dr. M. Carrington, University of Cambridge, Cambridge, UK) (a and c) or with an anti–rodent cyclin B1 monoclonal antibody V143 (red) that recognizes rodent B-type cyclins but does not cross-react with human B-type cyclins (a gift from J. Gannon and T. Hunt) (b and d). (e and f) Uninjected asynchronous (e) and serum-starved (f) CHO cells were costained with anticyclin A (red), anticyclin B1 (green), and TOTO-3 to visualize the DNA (blue). Results shown are representative of two independent experiments. (D and E) Human B-type cyclins localize correctly in CHO cells. Human cyclin B1 or B2 was tagged with one copy of the myc epitope and microinjected as cDNA under the CMV promoter into serum-starved CHO cells. 3 h after microinjection, cells were treated or not with LMB. Cells were stained with the 9E10 mAb to detect the cyclins. (D) Cells were costained with an antimannosidase II antibody to detect the Golgi apparatus (red in the merged images) and cyclin B1 (top row) or cyclin B2 (bottom row) (green in the merged images). (E) Cells expressing human cyclin B1 or B2 were fixed before (a and b) or after (c and d) treatment with 20 nM LMB for 45 min and stained for ectopically expressed cyclin B1 (a and c) or cyclin B2 (b and d).
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Figure 1: (A) Human B-type cyclins are expressed to similar levels in CHO cells. Serum-starved CHO cells were transfected with plasmids encoding human cyclin B1 or B2 and/or CDK1AF all tagged with one copy of the myc epitope and under the “tetracyclin-OFF” promoter. Cells were incubated in the presence or absence of tetracyclin to repress or induce protein expression, respectively. 16 h after induction, cells were lysed, and the extracts were run on one-dimensional SDS-PAGE and then immunoblotted with the 9E10 mAb to detect the proteins. Lane 1, mock-transfected cells; lanes 2–5, transfected cells: (lane 2) cyclin B1 plasmid plus tetracyclin; (lane 3) Cdk1AF; (lane 4) cyclin B1 minus tetracyclin for 16 h; (lane 5) cyclin B2 minus tetracyclin for 16 h. (B) Ectopic and endogenous cyclin B1 are expressed at similar levels in CHO cells. Approximately 980 serum-starved CHO cells were microinjected with plasmids encoding myc epitope–tagged human cyclin B1 with CDK1AF. 6 h after microinjection, cells were lysed and the samples were run on one-dimensional SDS-PAGE next to lysates from 250, 500, and 1,000 mitotic cells. Proteins were immunoblotted with an anticyclin B1 monoclonal antibody V152 that recognizes both human and rodent cyclin B1 to detect the proteins (a gift from J. Gannon and T. Hunt). Lane 1, uninjected cells; lane 2, cells injected with cyclin B1–CDK1AF; lanes 3–5, 250, 500, and 1,000 mitotic HeLa cells; lane 6, 1,000 mitotic CHO cells. M, molecular mass marker lane. Results shown are representative of three independent experiments. (C) There are no detectable endogenous mitotic cyclins in serum-starved CHO cells with or without ectopic human cyclin–CDKs. (a–d) CHO cells were serum starved for 24 h and then microinjected with NAGT–GFP as a Golgi apparatus and injection marker (green) and TOTO-3 to visualize the DNA (blue) together with CDK1AF and either cyclin B1 (a and b) or cyclin B2 (c and d). 6 h after microinjection, cells were fixed and stained with an anti–mouse cyclin A antibody (red) (a gift from Dr. M. Carrington, University of Cambridge, Cambridge, UK) (a and c) or with an anti–rodent cyclin B1 monoclonal antibody V143 (red) that recognizes rodent B-type cyclins but does not cross-react with human B-type cyclins (a gift from J. Gannon and T. Hunt) (b and d). (e and f) Uninjected asynchronous (e) and serum-starved (f) CHO cells were costained with anticyclin A (red), anticyclin B1 (green), and TOTO-3 to visualize the DNA (blue). Results shown are representative of two independent experiments. (D and E) Human B-type cyclins localize correctly in CHO cells. Human cyclin B1 or B2 was tagged with one copy of the myc epitope and microinjected as cDNA under the CMV promoter into serum-starved CHO cells. 3 h after microinjection, cells were treated or not with LMB. Cells were stained with the 9E10 mAb to detect the cyclins. (D) Cells were costained with an antimannosidase II antibody to detect the Golgi apparatus (red in the merged images) and cyclin B1 (top row) or cyclin B2 (bottom row) (green in the merged images). (E) Cells expressing human cyclin B1 or B2 were fixed before (a and b) or after (c and d) treatment with 20 nM LMB for 45 min and stained for ectopically expressed cyclin B1 (a and c) or cyclin B2 (b and d).
Mentions: To elucidate any differences in the biological properties of mammalian B-type cyclins, we sought to analyze their effects in cells lacking any other mitotic cyclins. Therefore, we chose to express B-type cyclins in cells just after release from serum starvation (G0/G1) when they lack endogenous mitotic cyclins (Brandeis and Hunt 1996). We used CHO cells because they have a well-defined Golgi apparatus, and we confirmed that serum-starved CHO cells do not have endogenous mitotic cyclins by both immunoblotting and immunofluorescence for cyclins A and B1 (Fig. 1a and Fig. c). To generate active cyclin B–CDK1 complexes, we coexpressed cyclin B1 or B2 with a mutant form of CDK1, CDK1T14A,Y15F (hereafter referred to as CDK1AF) that cannot be inactivated by either the Wee1 or Myt1 kinases (Morgan 1995). We coexpressed the proteins by one of two methods. In some experiments, we transfected the cDNAs under tetracycline-inducible promoters into a CHO cell line carrying a tetracycline repressor. Immunoblotting extracts of the cells showed that both cyclin B1 and B2 were expressed to similar levels (Fig. 1 A) with similar kinetics and that their expression paralleled an increase in histone H1 kinase activity (data not shown). In other experiments, we analyzed cells by time-lapse microscopy and immunofluorescence after microinjecting expression constructs, encoding the proteins under the control of the CMV promoter. We could detect the proteins by confocal immunofluorescence microscopy 3 h after microinjection. By immunoblotting these cells and quantifying the signals with NIH Image, we found that microinjected cells reproducibly expressed approximately the same amount of cyclin B1 as an equivalent number of mitotic cells (Fig. 1 B). (Note that this analysis underestimated the amount of cyclin B1 in the mitotic samples because they were collected by mitotic shake-off without nocodazole treatment, and therefore contained some anaphase and telophase cells without any B-type cyclins.)

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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Related in: MedlinePlus