Project Details
Projekt
Molecular characterization of Hunk, a novel regulator of pancreatic β-cell function
Applicant
Dr. Tanja Schallschmidt
Subject Area
Endocrinology, Diabetology, Metabolism
Term
from 2018 to 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 419280124
The present research project deals with the molecular characterization of Hunk, a gene which has recently been identified by quantitative trait locus analysis (QTL) in the Attie lab and was shown to negatively regulate insulin secretion in murine isolated pancreatic islets. Hunk is a Serine / Threonine protein kinase whose direct substrates are not known. However, the identification of these substrates is necessary to understand the underlying mechanism by which the protein kinase regulates insulin secretion. To gain preliminary insight into possible Hunk substrates, a phosphoproteome analysis was already conducted on islets from Hunk wild-type and Hunk knockout mice. This analysis revealed 243 proteins differentially phoyphorylated between the two genotypes, the strongest phosphorylation change occurred on the gene Pard3b. Interestingly, the human PARD3B gene locus has already been associated with type 2 diabetes (T2D) risk in Mexican populations. However, the phosphoproteome analysis was not able to distinguish between direct substrates for Hunk, and proteins that may be differentially phosphorylated as an indirect consequence of the Hunk knockout. As a consequence, the primary research objective of this project consists in the identification of the direct substrates for Hunk. To allow their detection, a chemical-genetic approach is planned that has already been successfully applied for the identification of the physiological substrates of several other protein kinases. This method will firstly require the generation of a so called analogue-sensitive (AS) kinase, which is able to bind ATP analogs. Thus, using molecular cloning methods, the first task will be the creation of the AS-Hunk kinase. Subsequently, this construct will be used in an in vitro kinase assay in which ATP analogs will be applied that can only be utilized by the AS-Hunk kinase and thus allows the determination of phosphorylation specifically by Hunk. Consequently, phosphorylated substrates in pancreatic islets from Hunk knockout mice as well as in human islets are supposed to be purified and proteins will be identified by mass spectrometry, which will be executed in cooperation with Professor Lloyd Smith at the University of Wisconsin. Finally, by expressing specific mutations in the phosphorylation sites of the identified substrates that mimic either the phosphorylated- or the unphosphorylated state, potential impacts of the phosphorylated proteins will be further investigated in functional in vitro assays.
DFG Programme
Research Fellowships
International Connection
USA
