Kinases are a class of highly regulated enzymes that play a central role in the regulation of numerous cellular processes. Due to the prevalence of aberrant kinase expression in many types of cancer, kinases have become a popular drug target in the war against cancer. But kinases also happen to be extremely difficult to target. Specifically targeting a single kinase in a pool of up to 500 different kinases in a human cell is no small feat, especially since kinases share a significant amount of structural similarity with one another.
To help assist in the development of anticancer therapies that target specific kinases, researchers have developed kinase assays for a variety of applications, ranging from fundamental biological studies to kinase inhibitor discovery and characterization.
At the recent American Association for Cancer Research conference in Chicago, a number of scientists discussed how they utilize various kinase assays in their investigations on cancer.
Potential drug targets are often identified by looking for proteins that are overexpressed in cancer cells. However, if researchers do not understand how a particular protein works in concert with others, it can be difficult to develop inhibitors that effectively downregulate the overexpressed protein in diseased cells.
Aurora B kinase is overexpressed in many types of cancer. To lay the groundwork to develop more effective therapeutics that target Aurora B, researchers need to understand how the kinase is regulated by other proteins in the cell. With this goal in mind, Ming-Ying Tsai, Ph.D., assistant professor at the University of Nebraska Medical Center, and graduate student Jyoti Iyer performed a research study that helps shed light on the cellular mechanism of Aurora B kinase.
The team studied the role of mitotic protein TPX2 (targeting protein for Xenopus kinesin-like protein 2) in Aurora B regulation. In the cell, Aurora B is part of an enzyme complex known as the chromosomal passenger complex (CPC), along with three other proteins: INCENP, Survivin, and Borealin. Iyer first performed in vitro binding assays to determine which residues of TPX2 are sufficient to enhance the interactions between Aurora B and Survivin, a known activator. Using this fragment of TPX2, composed of residues 138 through 328, she performed an in vitro kinase assay with Aurora B and found that the TPX2 peptide fragment was able to increase Aurora B kinase activity.
In a subsequent in vitro kinase assay, Iyer incubated recombinant, bacterially expressed CPC proteins and histone H3, a known substrate of Aurora B, in the presence or absence of the TPX2 fragment. She performed a Western blot with an antibody specific to phosphorylated histone H3 and found an increase in histone H3 phosophorylation in the presence of the TPX2 fragment compared to the control. The results suggest that TPX2 functions as a coactivator of Aurora B and serves as a scaffold protein for the assembly of the CPC.
“I anticipate these findings will open lots of doors for future research” on the role of TPX2 in regulating Aurora B and the CPC, Iyer says.