Modular design of peptide-based kinase biosensors for studying cancer cell signaling
Abstract
The Src-family kinases (SFKs) Hck and Lyn are highly expressed and active in chronic myelogenous leukemia patients that exhibit resistance to tyrosine kinase inhibitors. These kinases possess several modular domains that regulate catalytic activity and interaction with substrates. Using a common SFK substrate, we evaluated the effects of exogenous SH3-domain binding (SDB) ligands on substrate selection and activation. We show that the attachment of an SH3 ligand to the peptide substrate increased kinase activation and phosphorylation. Also, the position of the ligand relative to the substrate module affects recognition and kinase activity. The SFK Hck dislikes class II type polyproline ligands while Lyn tolerates both classes of ligands. Furthermore, our results show that Hck is activated through a typical Michaelis-Menten kinetics whereas Lyn undergoes cooperativity with the same biosensor. We discuss the possible utility of these results in designing peptide-based biosensor for tyrosine kinase inhibitor screening. We further utilized thiol-ene Michael addition reactions to ligate different peptide modules together. We demonstrate the chemical ligation of Abl kinase substrate, Abltide, and the Abl SH3 ligand using a maleimide and a phosphine catalyst.
Degree
Ph.D.
Advisors
Parker, Purdue University.
Subject Area
Biochemistry|Pharmacy sciences|Oncology
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