Product Pathways - Kinases
Aurora B Kinase #7394
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Description
Purified recombinant full-length human Aurora B (Met1-Ala344) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human Aurora B (Met1-Ala344) (GenBank Accession No. NM_004217) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using GSH-agarose.
Gel Staining
Figure 1. The purity of the Aurora B fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. Aurora B kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: MBP 200 ng/μL, and recombinant Aurora B: variable.
Quality Control
The theoretical molecular weight of the Aurora B fusion protein is 66 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. Aurora B kinase activity was determined using a radiometric assay [Fig.2].
Background
Aurora kinases belong to a highly conserved family of mitotic serine/threonine kinases with three members identified among mammals: Aurora A, Aurora B and Aurora C (1,2). Studies on the temporal expression pattern and subcellular localization of Aurora kinases in mitotic cells suggest an association with mitotic structure. Their functional influences span from G2 to cytokinesis and may be involved in key cell cycle events such as centrosome duplication, chromosome bi-orientation and segregation, cleavage furrow positioning and ingression (3). Aurora A is detected at the centrosomes, along mitotic spindle microtubules and in the cytoplasm of mitotically proliferating cells. Aurora A protein levels are low during G1 and S phases and peak during the G2/M phase of the cell cycle. Phosphorylation of Aurora A at Thr288 in its catalytic domain increases kinase activity. Aurora A is involved in centrosome separation, maturation and spindle assembly and stability. Expression of Aurora B protein also peaks during the G2/M phase of the cell cycle, while kinase activity peaks at the transition from metaphase to the end of mitosis. Aurora B associates with chromosomes during prophase prior to relocalizing to the spindle at anaphase. Aurora B regulates chromosome segregation through the control of microtubule-kinetochore attachment and cytokinesis. Expression of both Aurora A and Aurora B during the G2/M phase transition is tightly coordinated with histone H3 phosphorylation (4,5), while overexpression of both kinases is seen in a variety of human cancers (2,4). Aurora C localizes to the centrosome from anaphase to cytokinesis and both mRNA and protein levels peak during G2/M phase. Although typical Aurora C expression is limited to the testis, overexpression of Aurora C is detected in various cancer cell lines (6).
- Warner, S.L. et al. (2003) Mol. Cancer Ther. 2, 589-595.
- Katayama , H. et al. (2003) Cancer Metastasis Rev. 22, 451-464.
- Andrews, P.D. et al. (2003) Curr. Opin. Cell Biol. 15, 672-683.
- Pascreau, G. et al. (2003) Prog. Cell Cycle Res. 5, 369-374.
- Crosio, C. et al. (2002) Mol. Cell. Biol. 22, 874-885.
- Kimura, M. et al. (1999) J. Biol. Chem. 274, 7334-7340.
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
