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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) Characterization of an IAK1-specific  COOH-terminal peptide antiserum. A COOH-terminal  peptide antiserum was characterized by Western blot  analysis against a bacterially  produced recombinant IAK1  protein (lanes B) and extracts of mitotic NIH 3T3  cells (lanes 3T3). For peptide  competition, the antiserum  was preincubated with 5 μl of  a 1 mg/ml solution of the  peptide immunogen or an  unrelated peptide for 1 h at  room temperature before incubation with the blot. The  arrowhead indicates bacterially expressed recombinant  IAK1, and the arrow indicates the endogenous IAK1  in NIH 3T3 cells. (B) In vitro  translation of IAK1. Full-length IAK1 cDNA cloned  in pcDNA3 was used for in  vitro translation using the  TNT T7 Quick Coupled Transcription/Translation system.  An aliquot of the translated  products was analyzed along  with cell extract from nocodazole-blocked cells on 10% polyacrylamide gel. A similar plasmid containing luciferase gene in the place of  IAK1 cDNA was used for the control. The gel was blotted and probed with IAK1-specific peptide antiserum as described in the Materials and Methods section. Lane 1, Control luciferase cDNA translated in vitro; lane 2, IAK1 cDNA translated in vitro; lane 3, nocodazole-treated NIH 3T3 cell lysate.
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Figure 5: (A) Characterization of an IAK1-specific COOH-terminal peptide antiserum. A COOH-terminal peptide antiserum was characterized by Western blot analysis against a bacterially produced recombinant IAK1 protein (lanes B) and extracts of mitotic NIH 3T3 cells (lanes 3T3). For peptide competition, the antiserum was preincubated with 5 μl of a 1 mg/ml solution of the peptide immunogen or an unrelated peptide for 1 h at room temperature before incubation with the blot. The arrowhead indicates bacterially expressed recombinant IAK1, and the arrow indicates the endogenous IAK1 in NIH 3T3 cells. (B) In vitro translation of IAK1. Full-length IAK1 cDNA cloned in pcDNA3 was used for in vitro translation using the TNT T7 Quick Coupled Transcription/Translation system. An aliquot of the translated products was analyzed along with cell extract from nocodazole-blocked cells on 10% polyacrylamide gel. A similar plasmid containing luciferase gene in the place of IAK1 cDNA was used for the control. The gel was blotted and probed with IAK1-specific peptide antiserum as described in the Materials and Methods section. Lane 1, Control luciferase cDNA translated in vitro; lane 2, IAK1 cDNA translated in vitro; lane 3, nocodazole-treated NIH 3T3 cell lysate.

Mentions: To study the expression pattern of the IAK1 protein and its subcellular localization, we raised a polyclonal antiserum to the 12 COOH-terminal amino acids of the IAK1 protein. This peptide sequence is distinct from that of other members of this family including STK1 and the germ cell–specific kinase related to IAK1. In Western blot analysis of nocodazole-treated NIH 3T3 cells, this antiserum recognized a major protein of ∼46 kD, as well as some additional minor bands (Fig. 5 A, lane 3). Antiserum binding to the 46-kD band (but not the other minor bands) was completely competed with the immunogenic peptide (Fig. 5 A, lane 5). A recombinant, histidine-tagged IAK1 protein produced in bacteria was also recognized by this antiserum (Fig. 5 A, lane 4), and again the binding could be completely competed out with the immunogenic peptide (Fig. 5 A, lane 6). Antibody binding was not inhibited by another, unrelated peptide, suggesting that peptide competition was specific (Fig. 5 A, lanes 7 and 8). In vitro translation of the full-length IAK1 cDNA in a reticulocyte lysate resulted in two protein bands (Fig. 5 B, lane 2), in which the faster migrating one is predicted to result from the translational initiation from the downstream methio-nine at position 23 of IAK1 protein. Interestingly, the endogenous IAK1 protein (Fig. 5 B, lane 3) corresponds to the faster migrating band, and we do not detect any endogenous protein corresponding to our full-length IAK1 cDNA. This suggests that the cDNA we isolated could be a splice variant of IAK1 and that the protein product corresponding to this variant is less abundant or less stable in NIH 3T3 cells. Taken together, these data suggest that this antiserum specifically recognizes the IAK1 protein in NIH 3T3 cells.


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) Characterization of an IAK1-specific  COOH-terminal peptide antiserum. A COOH-terminal  peptide antiserum was characterized by Western blot  analysis against a bacterially  produced recombinant IAK1  protein (lanes B) and extracts of mitotic NIH 3T3  cells (lanes 3T3). For peptide  competition, the antiserum  was preincubated with 5 μl of  a 1 mg/ml solution of the  peptide immunogen or an  unrelated peptide for 1 h at  room temperature before incubation with the blot. The  arrowhead indicates bacterially expressed recombinant  IAK1, and the arrow indicates the endogenous IAK1  in NIH 3T3 cells. (B) In vitro  translation of IAK1. Full-length IAK1 cDNA cloned  in pcDNA3 was used for in  vitro translation using the  TNT T7 Quick Coupled Transcription/Translation system.  An aliquot of the translated  products was analyzed along  with cell extract from nocodazole-blocked cells on 10% polyacrylamide gel. A similar plasmid containing luciferase gene in the place of  IAK1 cDNA was used for the control. The gel was blotted and probed with IAK1-specific peptide antiserum as described in the Materials and Methods section. Lane 1, Control luciferase cDNA translated in vitro; lane 2, IAK1 cDNA translated in vitro; lane 3, nocodazole-treated NIH 3T3 cell lysate.
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Figure 5: (A) Characterization of an IAK1-specific COOH-terminal peptide antiserum. A COOH-terminal peptide antiserum was characterized by Western blot analysis against a bacterially produced recombinant IAK1 protein (lanes B) and extracts of mitotic NIH 3T3 cells (lanes 3T3). For peptide competition, the antiserum was preincubated with 5 μl of a 1 mg/ml solution of the peptide immunogen or an unrelated peptide for 1 h at room temperature before incubation with the blot. The arrowhead indicates bacterially expressed recombinant IAK1, and the arrow indicates the endogenous IAK1 in NIH 3T3 cells. (B) In vitro translation of IAK1. Full-length IAK1 cDNA cloned in pcDNA3 was used for in vitro translation using the TNT T7 Quick Coupled Transcription/Translation system. An aliquot of the translated products was analyzed along with cell extract from nocodazole-blocked cells on 10% polyacrylamide gel. A similar plasmid containing luciferase gene in the place of IAK1 cDNA was used for the control. The gel was blotted and probed with IAK1-specific peptide antiserum as described in the Materials and Methods section. Lane 1, Control luciferase cDNA translated in vitro; lane 2, IAK1 cDNA translated in vitro; lane 3, nocodazole-treated NIH 3T3 cell lysate.
Mentions: To study the expression pattern of the IAK1 protein and its subcellular localization, we raised a polyclonal antiserum to the 12 COOH-terminal amino acids of the IAK1 protein. This peptide sequence is distinct from that of other members of this family including STK1 and the germ cell–specific kinase related to IAK1. In Western blot analysis of nocodazole-treated NIH 3T3 cells, this antiserum recognized a major protein of ∼46 kD, as well as some additional minor bands (Fig. 5 A, lane 3). Antiserum binding to the 46-kD band (but not the other minor bands) was completely competed with the immunogenic peptide (Fig. 5 A, lane 5). A recombinant, histidine-tagged IAK1 protein produced in bacteria was also recognized by this antiserum (Fig. 5 A, lane 4), and again the binding could be completely competed out with the immunogenic peptide (Fig. 5 A, lane 6). Antibody binding was not inhibited by another, unrelated peptide, suggesting that peptide competition was specific (Fig. 5 A, lanes 7 and 8). In vitro translation of the full-length IAK1 cDNA in a reticulocyte lysate resulted in two protein bands (Fig. 5 B, lane 2), in which the faster migrating one is predicted to result from the translational initiation from the downstream methio-nine at position 23 of IAK1 protein. Interestingly, the endogenous IAK1 protein (Fig. 5 B, lane 3) corresponds to the faster migrating band, and we do not detect any endogenous protein corresponding to our full-length IAK1 cDNA. This suggests that the cDNA we isolated could be a splice variant of IAK1 and that the protein product corresponding to this variant is less abundant or less stable in NIH 3T3 cells. Taken together, these data suggest that this antiserum specifically recognizes the IAK1 protein in NIH 3T3 cells.

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