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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) Western blot analysis of IAK1 levels during the cell cycle. NIH 3T3 cells were blocked at various stages of the cell cycle by  serum starvation or treatment with aphidicolin or nocodazole. After release from block, cells were harvested at intervals to collect cells  at different stages of the cell cycle. Cell lysates prepared from 400,000 cells were then separated by SDS-PAGE and analyzed by Western blot analysis with the IAK1 antiserum. Cells were harvested 7 h after release from serum starvation (G1), 0, 3, 5, 7, 8, 9, and 10 h after release from aphidicolin block (A0–A10), and 0, 15, 30, 60, and 120 min after release from nocodazole block (N0–N120). The percentage of cells at G2/M phase of the cell cycle, as judged by propidium iodide staining and FACS® analysis, is shown below each lane.  (B) Activity profile of IAK1 kinase through cell cycle. NIH 3T3 cells at different stages of the cell cycle were collected as described in  Fig. 5 C. Cells were lysed, and in vitro kinase activity of IAK1 was determined in cell lysates using myelin basic protein as the exogenous  substrate as detailed in Materials and Methods. The percentage of cells in G2/M phase was determined for each sample by flow cytometry and presented for comparison.
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Figure 6: (A) Western blot analysis of IAK1 levels during the cell cycle. NIH 3T3 cells were blocked at various stages of the cell cycle by serum starvation or treatment with aphidicolin or nocodazole. After release from block, cells were harvested at intervals to collect cells at different stages of the cell cycle. Cell lysates prepared from 400,000 cells were then separated by SDS-PAGE and analyzed by Western blot analysis with the IAK1 antiserum. Cells were harvested 7 h after release from serum starvation (G1), 0, 3, 5, 7, 8, 9, and 10 h after release from aphidicolin block (A0–A10), and 0, 15, 30, 60, and 120 min after release from nocodazole block (N0–N120). The percentage of cells at G2/M phase of the cell cycle, as judged by propidium iodide staining and FACS® analysis, is shown below each lane. (B) Activity profile of IAK1 kinase through cell cycle. NIH 3T3 cells at different stages of the cell cycle were collected as described in Fig. 5 C. Cells were lysed, and in vitro kinase activity of IAK1 was determined in cell lysates using myelin basic protein as the exogenous substrate as detailed in Materials and Methods. The percentage of cells in G2/M phase was determined for each sample by flow cytometry and presented for comparison.

Mentions: Using this antiserum, we analyzed IAK1 protein levels by Western blot analysis in cells at different stages of the cell cycle (Fig. 6 A). In cells isolated 7 h after release from serum-starvation (G1 phase), we were unable to detect IAK1 protein, suggesting that the protein is not present during this stage of the cell cycle. Cells were also blocked with aphidicolin at the G1/S phase boundary, and after release of cells from this block, we gradually saw accumulation of IAK1 protein. The peak of IAK1 expression occurred ∼7 h after release from aphidicolin block, at which time a large percentage of cells were in G2/M-phase as judged by FACS® analysis. We also blocked cells at M-phase with nocodazole and followed IAK1 protein levels after release from the nocodazole block. Consistent with data from the aphidicolin block and release experiments, we detected high levels of IAK1 protein in M-phase cells. As cells released from nocodazole block reentered and progressed through the cell cycle into the G1 phase, IAK1 protein levels rapidly declined. These data demonstrate that IAK1 protein levels, like IAK1 transcripts, are regulated in a cell cycle–dependent manner.


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) Western blot analysis of IAK1 levels during the cell cycle. NIH 3T3 cells were blocked at various stages of the cell cycle by  serum starvation or treatment with aphidicolin or nocodazole. After release from block, cells were harvested at intervals to collect cells  at different stages of the cell cycle. Cell lysates prepared from 400,000 cells were then separated by SDS-PAGE and analyzed by Western blot analysis with the IAK1 antiserum. Cells were harvested 7 h after release from serum starvation (G1), 0, 3, 5, 7, 8, 9, and 10 h after release from aphidicolin block (A0–A10), and 0, 15, 30, 60, and 120 min after release from nocodazole block (N0–N120). The percentage of cells at G2/M phase of the cell cycle, as judged by propidium iodide staining and FACS® analysis, is shown below each lane.  (B) Activity profile of IAK1 kinase through cell cycle. NIH 3T3 cells at different stages of the cell cycle were collected as described in  Fig. 5 C. Cells were lysed, and in vitro kinase activity of IAK1 was determined in cell lysates using myelin basic protein as the exogenous  substrate as detailed in Materials and Methods. The percentage of cells in G2/M phase was determined for each sample by flow cytometry and presented for comparison.
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Related In: Results  -  Collection

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Figure 6: (A) Western blot analysis of IAK1 levels during the cell cycle. NIH 3T3 cells were blocked at various stages of the cell cycle by serum starvation or treatment with aphidicolin or nocodazole. After release from block, cells were harvested at intervals to collect cells at different stages of the cell cycle. Cell lysates prepared from 400,000 cells were then separated by SDS-PAGE and analyzed by Western blot analysis with the IAK1 antiserum. Cells were harvested 7 h after release from serum starvation (G1), 0, 3, 5, 7, 8, 9, and 10 h after release from aphidicolin block (A0–A10), and 0, 15, 30, 60, and 120 min after release from nocodazole block (N0–N120). The percentage of cells at G2/M phase of the cell cycle, as judged by propidium iodide staining and FACS® analysis, is shown below each lane. (B) Activity profile of IAK1 kinase through cell cycle. NIH 3T3 cells at different stages of the cell cycle were collected as described in Fig. 5 C. Cells were lysed, and in vitro kinase activity of IAK1 was determined in cell lysates using myelin basic protein as the exogenous substrate as detailed in Materials and Methods. The percentage of cells in G2/M phase was determined for each sample by flow cytometry and presented for comparison.
Mentions: Using this antiserum, we analyzed IAK1 protein levels by Western blot analysis in cells at different stages of the cell cycle (Fig. 6 A). In cells isolated 7 h after release from serum-starvation (G1 phase), we were unable to detect IAK1 protein, suggesting that the protein is not present during this stage of the cell cycle. Cells were also blocked with aphidicolin at the G1/S phase boundary, and after release of cells from this block, we gradually saw accumulation of IAK1 protein. The peak of IAK1 expression occurred ∼7 h after release from aphidicolin block, at which time a large percentage of cells were in G2/M-phase as judged by FACS® analysis. We also blocked cells at M-phase with nocodazole and followed IAK1 protein levels after release from the nocodazole block. Consistent with data from the aphidicolin block and release experiments, we detected high levels of IAK1 protein in M-phase cells. As cells released from nocodazole block reentered and progressed through the cell cycle into the G1 phase, IAK1 protein levels rapidly declined. These data demonstrate that IAK1 protein levels, like IAK1 transcripts, are regulated in a cell cycle–dependent manner.

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