Detection Of Protein Kinase Activity Based On New Signal Readout Pathways

Protein kinases play critical regulatory roles in a majority of biological processes, including cell proliferation, differentiation, metabolism, and apoptosis. Aberrations in protein kinase activities and abnormal protein phosphorylation states can result in a number of diseases. Therefore, monitoring kinase activities are of great signi?cance for further understanding the molecular mec hanism in clinical diagnosis and development of targeted therapy. So there is an urgent need to develop sensitive, simple, inexpensive new method for detecting the activity of protein kinases. In this dissertation, Zr4+-functionalized magnetic beads(Zr MBs) were synthesized and used as the carrier to identify phosphorylated peptides. Two sensitive protocols have been developed for the detection of protein kinase activities by novel signal readout pathways based on upconversion nanophosphors(UCNPs) and portable glucose meter, respectively.1. In the presence of Protein kinase A(PKA), a proof-of-concept kinase target, the γ-phosphoryl of ATP will be transferred to the hydroxyl group of serine(S) in the biotinylated peptide, which is used as the PKA-specific substrate. Zr4+-functionalized magnetic beads(Zr MBs) are prepared for highly selective capture and enrichment o f phosphopeptides with facile magnetic separation. When the biotin–peptides are phosphorylated by PKA, the phosphopeptides will be specifically bind on the Zr MBs. Biotin–peptides acts as a bridge to further bring Avidin- functionalized UCNPs onto the surface of Zr MBs. The UCNPs enriched on the Zr MBs will be proportional to the PKA activity. The PKA activity could be determined by the elution of the biotin-phosphopeptide/STV-UCNPs from the surface of Zr MBs. PKA activity can be quantitatively detected over a wide concentration range from 0.05 m U/μL to 0.2 U/μL and the detection limit is estimated to be 0.02 m U/μL.2. We have provided a simple, rapid and low-cost method for measuring PKA activity by quantitating the amount of glucose in solution following a kinase reaction, which is monitored by the personal glucometer. By using invertase as the bridge, the glucose level is correlated with the kinase activity, providing a valuable and portable protocol for the proteinkinase-related research. The wide dynamic range was obtained for PKA from 0.1 m U/μL to 0.2 U/μL.