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Disease-Related Kinase Activity

Molecular Signaling and Cancer Biology

Description of Disease-related Kinase

Activity: Cells sense, interpret, and respond to their environment through a process known as signal transduction. A major mechanism that propels signal transduction is the phosphorylation of biomolecules by a class of enzymes known as kinases. Under normal circumstances, kinases strictly control important cellular processes such as apoptosis, proliferation, and differentiation. When control over kinase activity is lost, these processes malfunction and often result in disease. Therefore, identifying, or profiling, aberrant kinase activity helps us understand the underlying mechanisms that drive diseases and exposes therapeutic targets for drug development.

There are many biological and technological avenues of research that revolve around characterizing kinase activity. Some labs in the field focus primarily on cancer, as mutations to kinase encoding genes often lead to uncontrolled cell proliferation or reduce the cell’s capacity to undergo apoptosis. Kinases are also implicated in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease by promoting neuronal cell death that results in cognitive degeneration. Students that pursue kinase-related research may focus on discovering new biological roles for these enzymes, develop new technologies to improve activity detection, or synthesize small molecule inhibitors that target specific kinases. A variety of techniques are used to solve these problems, including traditional molecular biology, mass spectrometry, state-of-the-art imaging, and organic synthesis. Graduates from kinase laboratories may go on to work in the pharmaceutical industry, academia, or in non-research oriented fields such as medical writing.

The peptide-biosensor
The peptide-biosensor labeled with a fluorophore (red) is seen here being internalized by HEK293 cells (blue). This provides evidence that phosphorylation detected by the biosensor occurs within live cells.

Research

Placzek EA, Plebanek MP, Lipchik AM, Kidd SR, Parker LL. A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem. 2010 Feb 1;397(1):73-8.

Summary: This article comes from Dr. Laurie Parker’s lab in the Department of Medicinal Chemistry and Molecular Pharmacology. It introduces the lab’s peptide-biosensor concept, and demonstrates a working example with Abl kinase. The biosensor is used in an engineered cell line that expresses an inducible form of Abl kinase, and MALDI-TOF mass spectrometry is used to detect the reporter module of the peptide-biosensor.

Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808441/?tool=pubmed

Parker lab’s peptide-based biosensor for detecting Abl kinase activity in live cells
This image outlines the design of the Parker lab’s peptide-based biosensor for detecting Abl kinase activity in live cells. It contains amino acid sequences that give the sensor functions that include cell penetration (transduction), protein targeting, and kinase activity reporting.

Paris LL, Hu J, Galan J, Ong SS, Martin VA, Ma H, Tao WA, Harrison ML, Geahlen RL. Regulation of Syk by phosphorylation on serine in the linker insert. J Biol Chem. 2010 Dec 17;285(51):39844-54.

Summary: This study was a collaborative project between the labs of Dr. Robert Geahlen, Dr. Marietta Harrison, and Dr. Andy Tao, who all participate in the Cancer Biology and Molecular Signaling training group. In this work they describe the role phosphorylation plays in a linker region of spleen tyrosine kinase (Syk). Using genetically altered B-cells, high-resolution mass spectrometry, and kinase activation assays, they show that phosphorylation of a specific serine residue in the linker region of Syk significantly affects Syk’s kinase activity.

Link: http://www.jbc.org/content/285/51/39844.long

Iliuk A, Martinez JS, Hall MC, Tao WA. Phosphorylation assay based on multifunctionalized soluble nanopolymer. Anal Chem. 2011 Apr 1;83(7):2767-74.

Here, Dr. Andy Tao and Dr. Mark Hall present a new detection method for the phosphorylation of proteins. It is based on a nanopolymer that is functionalized with a titanium trivalent cations, which allows the nanopolymer to complex phosphoproteins, and a biotin affinity tag that allows the efficient capture of the protein-nanopolymer conjugate. Using an ELISA style setup, the researchers are able to quantitatively detect protein phosphorylation from cell lysates.

Link: http://pubs.acs.org/doi/full/10.1021/ac2000708

Abl kinase activity
Abl kinase activity detected by the reporter module of the peptide is attentuated by treatment with the Abl kinase inhibitor, imatinib mesylate (IM), indicating that the biosensor can specifically detect Abl kinase activity.

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