Abstract
In the cell, reversible phosphorylation, controlled by protein phosphatases and protein kinases, initiates and regulates various signaling-dependent processes such as enzyme-substrate interactions, the cell cycle, differentiation, and immune responses. In addition to these processes, in unicellular parasites like Trypanosoma brucei, the causative agent of African sleeping sickness, additional signaling pathways have evolved to enable the survival of parasites in the changing environment of the vector and mammalian host. In this chapter, we describe two in vitro kinase assays and the use of the phosphoprotein chelator Phos-tag and show that these three polyacrylamide gel-based assays can be used for rapid target validation and detection of changes in phosphorylation.
Highlights
Investigations of signal transduction pathways can be challenging in the trypanosome field due to the early divergence of these organisms from other eukaryotic lineages that may have resulted in the evolution of unique mechanisms and components
Two families of antagonistic enzymes are responsible for such regulation, that is, protein kinases that transfer the γ-phosphate of ATP molecules on the substrate acceptor residues and protein phosphatases that are responsible for the dephosphorylation of these proteins
There are cases when low cost, rapid, semiquantitative methods can be useful as the first step to identify a phosphosubstrate or the phosphorylation state of a target protein or to manually validate hits obtained by high-throughput technologies
Summary
Investigations of signal transduction pathways can be challenging in the trypanosome field due to the early divergence of these organisms from other eukaryotic lineages that may have resulted in the evolution of unique mechanisms and components. Two families of antagonistic enzymes are responsible for such regulation, that is, protein kinases that transfer the γ-phosphate of ATP molecules on the substrate acceptor residues and protein phosphatases that are responsible for the dephosphorylation of these proteins Based on their analysis of the human phosphoproteome, in which 50,000 distinct phosphopeptides were detected, Sharma et al [1] have revealed that at least 75% of the proteome is phosphorylated. If a stronger signal is required, we describe an alternative approach that uses classical radioactive ATP, labeled on the γ-phosphate by the 32P isotope This method is based on the migration of the phosphorylated substrates on SDS-PAGE and their transfer onto PVDF membrane. The activity for this “Hot” kinase assay is detected on
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