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A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators.

Gopalan G, Chan CS, Donovan PJ - J. Cell Biol. (1997)

Bottom Line: In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers.We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily.The members of this subfamily may be important regulators of chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology of Development and Differentiation Group, ABL Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.

ABSTRACT
We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases-Ipl1 and aurora, respectively-mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.

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(A) Localization of IAK1 in nocodazole-treated cells. NIH 3T3 cells were treated with nocodazole (5 μg/ml) for 4 h, washed in  fresh medium, and then fixed in ice-cold methanol at various times after release from nocodazole block. Cells were stained with a monoclonal anti–β-tubulin antibody or an anti-IAK1 antiserum followed by the appropriate secondary antibodies and visualized by laser  scanning confocal microscopy as described in Materials and Methods. Shown are cells at time zero (0), 10 min after release from nocodazole (10), 15 min after release (15), and 30 min after release (30). Bar, 5 μm. (B) Localization of IAK1 in taxol-treated cells. NIH 3T3  cells were treated with taxol (10 μM) for 5 h and then fixed in ice-cold methanol. Cells were stained with antibodies as above for β-tubulin and IAK1. The two lower cells represent M-phase cells, while the upper cell is in interphase. Bar, 5 μm.
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Figure 8: (A) Localization of IAK1 in nocodazole-treated cells. NIH 3T3 cells were treated with nocodazole (5 μg/ml) for 4 h, washed in fresh medium, and then fixed in ice-cold methanol at various times after release from nocodazole block. Cells were stained with a monoclonal anti–β-tubulin antibody or an anti-IAK1 antiserum followed by the appropriate secondary antibodies and visualized by laser scanning confocal microscopy as described in Materials and Methods. Shown are cells at time zero (0), 10 min after release from nocodazole (10), 15 min after release (15), and 30 min after release (30). Bar, 5 μm. (B) Localization of IAK1 in taxol-treated cells. NIH 3T3 cells were treated with taxol (10 μM) for 5 h and then fixed in ice-cold methanol. Cells were stained with antibodies as above for β-tubulin and IAK1. The two lower cells represent M-phase cells, while the upper cell is in interphase. Bar, 5 μm.

Mentions: To further investigate the association of IAK1 protein with centrosomes and spindle microtubules, we analyzed IAK1 localization in cells treated with the microtubule-destabilizing drug nocodazole. When log phase cultures of NIH 3T3 cells are treated with nocodazole, cells arrest and accumulate at metaphase. Nocodazole-arrested cells were stained with a monoclonal anti–β-tubulin antibody and the anti-IAK1 antiserum. In nocodazole-treated cells, β-tubulin staining was observed throughout the cytoplasm, and two weak foci were also observed. These foci are likely to be the centrioles, which are known to be resistant to the effects of nocodazole (De Brabander et al., 1980). Fig. 8 A shows such a cell in which only one of these foci is in the focal plane. IAK1 staining in these cells was also observed weakly throughout the cytoplasm and in bright dots coincident with the foci of β-tubulin staining (Fig. 8 A). These data strongly suggest that most of the IAK1 protein in dividing cells is associated with pericentriolar material and tubulin of the mitotic spindle, which is consistent with the observed IAK1 staining pattern in dividing cells. They also suggest that a pool of IAK1 is associated with the centrioles independently of microtubule polymerization. In cells recovering from nocodazole block, microtubules reform from multiple MTOCs, which coalesce to form a bipolar spindle. Under these conditions, IAK1 is found to be associated with each MTOC as well as the mitotic spindle (Fig. 8 A).


A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators.

Gopalan G, Chan CS, Donovan PJ - J. Cell Biol. (1997)

(A) Localization of IAK1 in nocodazole-treated cells. NIH 3T3 cells were treated with nocodazole (5 μg/ml) for 4 h, washed in  fresh medium, and then fixed in ice-cold methanol at various times after release from nocodazole block. Cells were stained with a monoclonal anti–β-tubulin antibody or an anti-IAK1 antiserum followed by the appropriate secondary antibodies and visualized by laser  scanning confocal microscopy as described in Materials and Methods. Shown are cells at time zero (0), 10 min after release from nocodazole (10), 15 min after release (15), and 30 min after release (30). Bar, 5 μm. (B) Localization of IAK1 in taxol-treated cells. NIH 3T3  cells were treated with taxol (10 μM) for 5 h and then fixed in ice-cold methanol. Cells were stained with antibodies as above for β-tubulin and IAK1. The two lower cells represent M-phase cells, while the upper cell is in interphase. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 8: (A) Localization of IAK1 in nocodazole-treated cells. NIH 3T3 cells were treated with nocodazole (5 μg/ml) for 4 h, washed in fresh medium, and then fixed in ice-cold methanol at various times after release from nocodazole block. Cells were stained with a monoclonal anti–β-tubulin antibody or an anti-IAK1 antiserum followed by the appropriate secondary antibodies and visualized by laser scanning confocal microscopy as described in Materials and Methods. Shown are cells at time zero (0), 10 min after release from nocodazole (10), 15 min after release (15), and 30 min after release (30). Bar, 5 μm. (B) Localization of IAK1 in taxol-treated cells. NIH 3T3 cells were treated with taxol (10 μM) for 5 h and then fixed in ice-cold methanol. Cells were stained with antibodies as above for β-tubulin and IAK1. The two lower cells represent M-phase cells, while the upper cell is in interphase. Bar, 5 μm.
Mentions: To further investigate the association of IAK1 protein with centrosomes and spindle microtubules, we analyzed IAK1 localization in cells treated with the microtubule-destabilizing drug nocodazole. When log phase cultures of NIH 3T3 cells are treated with nocodazole, cells arrest and accumulate at metaphase. Nocodazole-arrested cells were stained with a monoclonal anti–β-tubulin antibody and the anti-IAK1 antiserum. In nocodazole-treated cells, β-tubulin staining was observed throughout the cytoplasm, and two weak foci were also observed. These foci are likely to be the centrioles, which are known to be resistant to the effects of nocodazole (De Brabander et al., 1980). Fig. 8 A shows such a cell in which only one of these foci is in the focal plane. IAK1 staining in these cells was also observed weakly throughout the cytoplasm and in bright dots coincident with the foci of β-tubulin staining (Fig. 8 A). These data strongly suggest that most of the IAK1 protein in dividing cells is associated with pericentriolar material and tubulin of the mitotic spindle, which is consistent with the observed IAK1 staining pattern in dividing cells. They also suggest that a pool of IAK1 is associated with the centrioles independently of microtubule polymerization. In cells recovering from nocodazole block, microtubules reform from multiple MTOCs, which coalesce to form a bipolar spindle. Under these conditions, IAK1 is found to be associated with each MTOC as well as the mitotic spindle (Fig. 8 A).

Bottom Line: In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers.We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily.The members of this subfamily may be important regulators of chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology of Development and Differentiation Group, ABL Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.

ABSTRACT
We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases-Ipl1 and aurora, respectively-mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.

Show MeSH
Related in: MedlinePlus