Limits...
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.

Show MeSH
Expression of IAK1 causes microtubule defects in ipl1 mutant yeast cells. Isogenic wild-type IPL1 (CCY98-3D-1) and mutant ipl1-4 (CCY98-3D-1-1) cells carrying the indicated plasmids (see Fig. 10 for description) were grown to early log phase at 30°C in  supplemented minimal SD liquid medium (lacking uracil) that contained 2% raffinose as the sole carbon source (noninducing). At 0 h,  galactose was added to give a final concentration of 4% (inducing), and the cultures were incubated at 30°C for another 10 h. At the  time indicated, cells were fixed with formaldehyde, and the distribution of chromosomal DNA and microtubules in these cells were examined by indirect immunofluorescence microscopy. For each sample, 200 large-budded cells were scored, and the percentage of cells  belonging to each cytological class is shown here. The classes represent: (a) cells with unseparated chromatin mass and a short to medium bipolar mitotic spindle; (b) cells with chromatin mass that is not fully separated and with an elongated, bipolar mitotic spindle; (c)  cells with evenly separated chromatin masses and an elongated, bipolar mitotic spindle; (d) cells with evenly separated chromatin  masses and a partially disassembled mitotic spindle or no mitotic spindle; (e) cells with unseparated chromatin mass, a single unduplicated or unseparated MTOC (spindle pole body), and an apparently monopolar spindle or no mitotic spindle; (f) cells with unseparated  chromatin mass, a single unduplicated or unseparated MTOC, and no other microtubule structure; (g) cells with no MTOC or microtubule structure; and (h) cells clearly with unevenly separated chromatin masses. The boxed region highlights the abnormal phenotypes in  ipl-4 yeast expressing the wild-type and IAK1D287N mammalian proteins.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141637&req=5

Figure 11: Expression of IAK1 causes microtubule defects in ipl1 mutant yeast cells. Isogenic wild-type IPL1 (CCY98-3D-1) and mutant ipl1-4 (CCY98-3D-1-1) cells carrying the indicated plasmids (see Fig. 10 for description) were grown to early log phase at 30°C in supplemented minimal SD liquid medium (lacking uracil) that contained 2% raffinose as the sole carbon source (noninducing). At 0 h, galactose was added to give a final concentration of 4% (inducing), and the cultures were incubated at 30°C for another 10 h. At the time indicated, cells were fixed with formaldehyde, and the distribution of chromosomal DNA and microtubules in these cells were examined by indirect immunofluorescence microscopy. For each sample, 200 large-budded cells were scored, and the percentage of cells belonging to each cytological class is shown here. The classes represent: (a) cells with unseparated chromatin mass and a short to medium bipolar mitotic spindle; (b) cells with chromatin mass that is not fully separated and with an elongated, bipolar mitotic spindle; (c) cells with evenly separated chromatin masses and an elongated, bipolar mitotic spindle; (d) cells with evenly separated chromatin masses and a partially disassembled mitotic spindle or no mitotic spindle; (e) cells with unseparated chromatin mass, a single unduplicated or unseparated MTOC (spindle pole body), and an apparently monopolar spindle or no mitotic spindle; (f) cells with unseparated chromatin mass, a single unduplicated or unseparated MTOC, and no other microtubule structure; (g) cells with no MTOC or microtubule structure; and (h) cells clearly with unevenly separated chromatin masses. The boxed region highlights the abnormal phenotypes in ipl-4 yeast expressing the wild-type and IAK1D287N mammalian proteins.

Mentions: Given the sequence similarity between the mouse IAK1 and yeast Ipl1p protein kinases, we were interested in knowing whether these two kinases might be functionally related. In particular, we wanted to find out whether Ipl1p function could be substituted by the expression of IAK1 in yeast. For this purpose, we constructed plasmids expressing a wild-type or a mutant form (D287N) of IAK1 cDNA under the control of the glucose-repressible and galactose-inducible GAL10 promoter (Johnson and Kolodner, 1991). These same cDNAs were tested for their kinase activity by expressing flag-tagged versions in NIH 3T3 cells. The flag-tagged proteins were immunoprecipitated using an antiflag monoclonal antibody, and their kinase activity was measured, using MBP as the exogenous substrate. These data revealed that the D287N mutation in the metal binding site reduced the kinase activity by 2.5-fold compared to the wild-type kinase activity but did not totally abolish kinase activity (Fig. 9). The yeast expression plasmids (IAK1WT and IAK1D287N) were then introduced into wild-type and isogenic ipl1-4 temperature-sensitive mutant cells (Francisco et al., 1994). Under inducing conditions (on galactose medium), expression of IAK1 and the mutant IAK1D287N form had no effect on the growth of the wild-type yeast cells (Fig. 10). The wild-type construct also failed to suppress the temperature-sensitive phenotype caused by the recessive ipl-4 mutation. Thus, Ipl1p function cannot be substituted by the expression of IAK1. Surprisingly, expression of IAK1 actually resulted in the lethality of ipl1-4 but not wild-type cells at the otherwise permissive growth temperature of 30°C (Fig. 10), even though similar amounts of IAK1 were produced in both cell types (data not shown). Expression of a mutant form of IAK1 (IAK1D287N) having reduced kinase activity also resulted in lethality of ipl1-4 cells but not wild-type cells (Fig. 11). Thus, expression of IAK1 appeared to interfere with, rather than substitute for, Ipl1p function in yeast.


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)

Expression of IAK1 causes microtubule defects in ipl1 mutant yeast cells. Isogenic wild-type IPL1 (CCY98-3D-1) and mutant ipl1-4 (CCY98-3D-1-1) cells carrying the indicated plasmids (see Fig. 10 for description) were grown to early log phase at 30°C in  supplemented minimal SD liquid medium (lacking uracil) that contained 2% raffinose as the sole carbon source (noninducing). At 0 h,  galactose was added to give a final concentration of 4% (inducing), and the cultures were incubated at 30°C for another 10 h. At the  time indicated, cells were fixed with formaldehyde, and the distribution of chromosomal DNA and microtubules in these cells were examined by indirect immunofluorescence microscopy. For each sample, 200 large-budded cells were scored, and the percentage of cells  belonging to each cytological class is shown here. The classes represent: (a) cells with unseparated chromatin mass and a short to medium bipolar mitotic spindle; (b) cells with chromatin mass that is not fully separated and with an elongated, bipolar mitotic spindle; (c)  cells with evenly separated chromatin masses and an elongated, bipolar mitotic spindle; (d) cells with evenly separated chromatin  masses and a partially disassembled mitotic spindle or no mitotic spindle; (e) cells with unseparated chromatin mass, a single unduplicated or unseparated MTOC (spindle pole body), and an apparently monopolar spindle or no mitotic spindle; (f) cells with unseparated  chromatin mass, a single unduplicated or unseparated MTOC, and no other microtubule structure; (g) cells with no MTOC or microtubule structure; and (h) cells clearly with unevenly separated chromatin masses. The boxed region highlights the abnormal phenotypes in  ipl-4 yeast expressing the wild-type and IAK1D287N mammalian proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141637&req=5

Figure 11: Expression of IAK1 causes microtubule defects in ipl1 mutant yeast cells. Isogenic wild-type IPL1 (CCY98-3D-1) and mutant ipl1-4 (CCY98-3D-1-1) cells carrying the indicated plasmids (see Fig. 10 for description) were grown to early log phase at 30°C in supplemented minimal SD liquid medium (lacking uracil) that contained 2% raffinose as the sole carbon source (noninducing). At 0 h, galactose was added to give a final concentration of 4% (inducing), and the cultures were incubated at 30°C for another 10 h. At the time indicated, cells were fixed with formaldehyde, and the distribution of chromosomal DNA and microtubules in these cells were examined by indirect immunofluorescence microscopy. For each sample, 200 large-budded cells were scored, and the percentage of cells belonging to each cytological class is shown here. The classes represent: (a) cells with unseparated chromatin mass and a short to medium bipolar mitotic spindle; (b) cells with chromatin mass that is not fully separated and with an elongated, bipolar mitotic spindle; (c) cells with evenly separated chromatin masses and an elongated, bipolar mitotic spindle; (d) cells with evenly separated chromatin masses and a partially disassembled mitotic spindle or no mitotic spindle; (e) cells with unseparated chromatin mass, a single unduplicated or unseparated MTOC (spindle pole body), and an apparently monopolar spindle or no mitotic spindle; (f) cells with unseparated chromatin mass, a single unduplicated or unseparated MTOC, and no other microtubule structure; (g) cells with no MTOC or microtubule structure; and (h) cells clearly with unevenly separated chromatin masses. The boxed region highlights the abnormal phenotypes in ipl-4 yeast expressing the wild-type and IAK1D287N mammalian proteins.
Mentions: Given the sequence similarity between the mouse IAK1 and yeast Ipl1p protein kinases, we were interested in knowing whether these two kinases might be functionally related. In particular, we wanted to find out whether Ipl1p function could be substituted by the expression of IAK1 in yeast. For this purpose, we constructed plasmids expressing a wild-type or a mutant form (D287N) of IAK1 cDNA under the control of the glucose-repressible and galactose-inducible GAL10 promoter (Johnson and Kolodner, 1991). These same cDNAs were tested for their kinase activity by expressing flag-tagged versions in NIH 3T3 cells. The flag-tagged proteins were immunoprecipitated using an antiflag monoclonal antibody, and their kinase activity was measured, using MBP as the exogenous substrate. These data revealed that the D287N mutation in the metal binding site reduced the kinase activity by 2.5-fold compared to the wild-type kinase activity but did not totally abolish kinase activity (Fig. 9). The yeast expression plasmids (IAK1WT and IAK1D287N) were then introduced into wild-type and isogenic ipl1-4 temperature-sensitive mutant cells (Francisco et al., 1994). Under inducing conditions (on galactose medium), expression of IAK1 and the mutant IAK1D287N form had no effect on the growth of the wild-type yeast cells (Fig. 10). The wild-type construct also failed to suppress the temperature-sensitive phenotype caused by the recessive ipl-4 mutation. Thus, Ipl1p function cannot be substituted by the expression of IAK1. Surprisingly, expression of IAK1 actually resulted in the lethality of ipl1-4 but not wild-type cells at the otherwise permissive growth temperature of 30°C (Fig. 10), even though similar amounts of IAK1 were produced in both cell types (data not shown). Expression of a mutant form of IAK1 (IAK1D287N) having reduced kinase activity also resulted in lethality of ipl1-4 cells but not wild-type cells (Fig. 11). Thus, expression of IAK1 appeared to interfere with, rather than substitute for, Ipl1p function in yeast.

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