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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 B–CDK1 complexes will disassemble the Golgi apparatus in a physiologically relevant manner in the absence of MEK activity. (A and B) CHO cells were incubated in DME containing 0.1% serum for 3 d (lanes 3 and 7), after which DME with 2% FCS was added back in the presence (lanes 1 and 5) or absence (lanes 4 and 8) of the MEK inhibitors, U0126 (25 μM) and PD98059 (20 μM). MEK inhibitors were also added to cells in the absence of serum (lanes 2 and 6). Cells were lysed after 10 min (lanes 1–4) or 3 h (lanes 5–8), and whole cell extracts were probed with an anti–phospho-MAP kinase antibody ( A) and subsequently reprobed with an antibody that recognizes all forms of MAP kinase (B) as a loading control. Results shown are representative of three independent experiments. (C) CHO cells were incubated in DME containing 0.1% serum for 3 d and then microinjected with expression constructs for NAGT–GFP (left; and green, in right panels) with either cyclin B1 and CDK1AF (top panels), cyclin B2 and CDK1AF (middle panels), or CDK1AF (bottom panels) in the presence of 2% serum and MEK inhibitors as in A and B. After 3 h, cells were fixed and stained with an antibody that recognizes the phosphorylated form of GM130 (center; and red, in right panels) and an anti–β-tubulin antibody (blue in right panels). Results shown are representative of two independent experiments. Bars, 10 μm.
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Figure 6: Cyclin B–CDK1 complexes will disassemble the Golgi apparatus in a physiologically relevant manner in the absence of MEK activity. (A and B) CHO cells were incubated in DME containing 0.1% serum for 3 d (lanes 3 and 7), after which DME with 2% FCS was added back in the presence (lanes 1 and 5) or absence (lanes 4 and 8) of the MEK inhibitors, U0126 (25 μM) and PD98059 (20 μM). MEK inhibitors were also added to cells in the absence of serum (lanes 2 and 6). Cells were lysed after 10 min (lanes 1–4) or 3 h (lanes 5–8), and whole cell extracts were probed with an anti–phospho-MAP kinase antibody ( A) and subsequently reprobed with an antibody that recognizes all forms of MAP kinase (B) as a loading control. Results shown are representative of three independent experiments. (C) CHO cells were incubated in DME containing 0.1% serum for 3 d and then microinjected with expression constructs for NAGT–GFP (left; and green, in right panels) with either cyclin B1 and CDK1AF (top panels), cyclin B2 and CDK1AF (middle panels), or CDK1AF (bottom panels) in the presence of 2% serum and MEK inhibitors as in A and B. After 3 h, cells were fixed and stained with an antibody that recognizes the phosphorylated form of GM130 (center; and red, in right panels) and an anti–β-tubulin antibody (blue in right panels). Results shown are representative of two independent experiments. Bars, 10 μm.

Mentions: Time-lapse fluorescence microscopy using NAGT–GFP showed that both cyclin B1– and cyclin B2–CDK1 complexes caused the Golgi to begin to disassemble at approximately the same time after microinjection (data not shown). This raised the question of whether B-type cyclin–CDK complexes required MEK to cause Golgi disassembly. In our microinjection regime, we used serum-starved cells to which we added 2% serum to aid recovery from the microinjection before analyzing them 3 h later. Although this treatment did not generate any endogenous mitotic cyclins over the time course of our experiments (Fig. 1 C), it did activate MEK (Fig. 6A and Fig. B). However, none of the cells disassembled their Golgi apparatus in the absence of mitotic cyclin–CDKs (Fig. 2o and Fig. t).


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 B–CDK1 complexes will disassemble the Golgi apparatus in a physiologically relevant manner in the absence of MEK activity. (A and B) CHO cells were incubated in DME containing 0.1% serum for 3 d (lanes 3 and 7), after which DME with 2% FCS was added back in the presence (lanes 1 and 5) or absence (lanes 4 and 8) of the MEK inhibitors, U0126 (25 μM) and PD98059 (20 μM). MEK inhibitors were also added to cells in the absence of serum (lanes 2 and 6). Cells were lysed after 10 min (lanes 1–4) or 3 h (lanes 5–8), and whole cell extracts were probed with an anti–phospho-MAP kinase antibody ( A) and subsequently reprobed with an antibody that recognizes all forms of MAP kinase (B) as a loading control. Results shown are representative of three independent experiments. (C) CHO cells were incubated in DME containing 0.1% serum for 3 d and then microinjected with expression constructs for NAGT–GFP (left; and green, in right panels) with either cyclin B1 and CDK1AF (top panels), cyclin B2 and CDK1AF (middle panels), or CDK1AF (bottom panels) in the presence of 2% serum and MEK inhibitors as in A and B. After 3 h, cells were fixed and stained with an antibody that recognizes the phosphorylated form of GM130 (center; and red, in right panels) and an anti–β-tubulin antibody (blue in right panels). Results shown are representative of two independent experiments. Bars, 10 μm.
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Figure 6: Cyclin B–CDK1 complexes will disassemble the Golgi apparatus in a physiologically relevant manner in the absence of MEK activity. (A and B) CHO cells were incubated in DME containing 0.1% serum for 3 d (lanes 3 and 7), after which DME with 2% FCS was added back in the presence (lanes 1 and 5) or absence (lanes 4 and 8) of the MEK inhibitors, U0126 (25 μM) and PD98059 (20 μM). MEK inhibitors were also added to cells in the absence of serum (lanes 2 and 6). Cells were lysed after 10 min (lanes 1–4) or 3 h (lanes 5–8), and whole cell extracts were probed with an anti–phospho-MAP kinase antibody ( A) and subsequently reprobed with an antibody that recognizes all forms of MAP kinase (B) as a loading control. Results shown are representative of three independent experiments. (C) CHO cells were incubated in DME containing 0.1% serum for 3 d and then microinjected with expression constructs for NAGT–GFP (left; and green, in right panels) with either cyclin B1 and CDK1AF (top panels), cyclin B2 and CDK1AF (middle panels), or CDK1AF (bottom panels) in the presence of 2% serum and MEK inhibitors as in A and B. After 3 h, cells were fixed and stained with an antibody that recognizes the phosphorylated form of GM130 (center; and red, in right panels) and an anti–β-tubulin antibody (blue in right panels). Results shown are representative of two independent experiments. Bars, 10 μm.
Mentions: Time-lapse fluorescence microscopy using NAGT–GFP showed that both cyclin B1– and cyclin B2–CDK1 complexes caused the Golgi to begin to disassemble at approximately the same time after microinjection (data not shown). This raised the question of whether B-type cyclin–CDK complexes required MEK to cause Golgi disassembly. In our microinjection regime, we used serum-starved cells to which we added 2% serum to aid recovery from the microinjection before analyzing them 3 h later. Although this treatment did not generate any endogenous mitotic cyclins over the time course of our experiments (Fig. 1 C), it did activate MEK (Fig. 6A and Fig. B). However, none of the cells disassembled their Golgi apparatus in the absence of mitotic cyclin–CDKs (Fig. 2o and Fig. t).

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