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Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

Kurihara D, Matsunaga S, Omura T, Higashiyama T, Fukui K - BMC Plant Biol. (2011)

Bottom Line: Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana.Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT

Background: Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.

Results: Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.

Conclusions: Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

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GST-AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vitro. (A) GST-AtHaspin and GST-AtHaspin-KD were incubated with or without ATP, and phosphorylated proteins were stained with ProQ Diamond Phosphoprotein stain. (B) GST-AtHaspin and GST-AtAUR3 were incubated with GST-H3 tail (left and right lanes). Negative control: GST-H3 tail only (middle lane). Phosphorylated GST-H3 tail was immunostained using anti-H3T3ph, H3T11ph, H3S10ph, and H3S28ph antibodies. (C) GST-AtHaspin and GST-AtHaspin-KD were incubated with GST-H3 tails or mutants as substrates. Phosphorylated GST-H3 tails were immunostained with anti-H3T3ph and anti-H3T11ph antibodies.
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Figure 2: GST-AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vitro. (A) GST-AtHaspin and GST-AtHaspin-KD were incubated with or without ATP, and phosphorylated proteins were stained with ProQ Diamond Phosphoprotein stain. (B) GST-AtHaspin and GST-AtAUR3 were incubated with GST-H3 tail (left and right lanes). Negative control: GST-H3 tail only (middle lane). Phosphorylated GST-H3 tail was immunostained using anti-H3T3ph, H3T11ph, H3S10ph, and H3S28ph antibodies. (C) GST-AtHaspin and GST-AtHaspin-KD were incubated with GST-H3 tails or mutants as substrates. Phosphorylated GST-H3 tails were immunostained with anti-H3T3ph and anti-H3T11ph antibodies.

Mentions: The human Haspin protein K511A, which contains a mutation of a single conserved lysine residue that is important for ATP binding, has no kinase activity [13]. To examine whether purified GST-AtHaspin has kinase activity, an in vitro kinase assay was performed using purified GST-AtHaspin and GST-AtHaspin KD (kinase dead) (K309A) with or without ATP. Phosphorylated proteins were detected by ProQ Diamond Phosphoprotein Stain. As expected, GST-AtHaspin-KD was not autophosphorylated even in the presence of ATP (Figure 2A, second lane). However, GST-AtHaspin was autophosphorylated in the presence or absence of ATP (Figure 2A, first and third lanes). This result indicated that autophosphorylation of AtHaspin was not dependent on addition of ATP, and that this lysine residue is also required for autophosphorylation of AtHaspin. This result also suggested that GST-AtHaspin was autophosphorylated during production in Escherichia coli.


Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

Kurihara D, Matsunaga S, Omura T, Higashiyama T, Fukui K - BMC Plant Biol. (2011)

GST-AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vitro. (A) GST-AtHaspin and GST-AtHaspin-KD were incubated with or without ATP, and phosphorylated proteins were stained with ProQ Diamond Phosphoprotein stain. (B) GST-AtHaspin and GST-AtAUR3 were incubated with GST-H3 tail (left and right lanes). Negative control: GST-H3 tail only (middle lane). Phosphorylated GST-H3 tail was immunostained using anti-H3T3ph, H3T11ph, H3S10ph, and H3S28ph antibodies. (C) GST-AtHaspin and GST-AtHaspin-KD were incubated with GST-H3 tails or mutants as substrates. Phosphorylated GST-H3 tails were immunostained with anti-H3T3ph and anti-H3T11ph antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113928&req=5

Figure 2: GST-AtHaspin phosphorylates histone H3 at Thr3 and Thr11 in vitro. (A) GST-AtHaspin and GST-AtHaspin-KD were incubated with or without ATP, and phosphorylated proteins were stained with ProQ Diamond Phosphoprotein stain. (B) GST-AtHaspin and GST-AtAUR3 were incubated with GST-H3 tail (left and right lanes). Negative control: GST-H3 tail only (middle lane). Phosphorylated GST-H3 tail was immunostained using anti-H3T3ph, H3T11ph, H3S10ph, and H3S28ph antibodies. (C) GST-AtHaspin and GST-AtHaspin-KD were incubated with GST-H3 tails or mutants as substrates. Phosphorylated GST-H3 tails were immunostained with anti-H3T3ph and anti-H3T11ph antibodies.
Mentions: The human Haspin protein K511A, which contains a mutation of a single conserved lysine residue that is important for ATP binding, has no kinase activity [13]. To examine whether purified GST-AtHaspin has kinase activity, an in vitro kinase assay was performed using purified GST-AtHaspin and GST-AtHaspin KD (kinase dead) (K309A) with or without ATP. Phosphorylated proteins were detected by ProQ Diamond Phosphoprotein Stain. As expected, GST-AtHaspin-KD was not autophosphorylated even in the presence of ATP (Figure 2A, second lane). However, GST-AtHaspin was autophosphorylated in the presence or absence of ATP (Figure 2A, first and third lanes). This result indicated that autophosphorylation of AtHaspin was not dependent on addition of ATP, and that this lysine residue is also required for autophosphorylation of AtHaspin. This result also suggested that GST-AtHaspin was autophosphorylated during production in Escherichia coli.

Bottom Line: Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana.Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT

Background: Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.

Results: Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.

Conclusions: Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

Show MeSH