Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Abstract

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn 2؉ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3␤, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly upshifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC 50 value of 1.6 M in the presence of 0.10 mM ATP. Molecular & Cellular Proteomics 6:356 -366, 2007.Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells (1, 2). It occurs on several amino acid residues, including histidine, aspartic acid, glutamic acid, lysine, arginine, and cysteine on which it is very labile and difficult to detect, whereas more stable and well studied phosphorylation takes place on the three specific residues serine, threonine, and tyrosine (3). The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Many of the genetic changes that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases (4). T...

Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes

“…The fact that the phosphorylated forms of claudin-2 are differentially retarded in the Phos-tag gels does not imply that more slowly migrating species are more heavily phosphorylated; the degree of retardation using the Phos-tag reagent has been reported to be a function of specific sequences around the phosphorylated sites (Kinoshita-Kikuta et al, 2007). This means that migration rates of individual bands have been shown to be characteristic of specific phosphorylated isoforms (Kinoshita-Kikuta et al, 2007).…”

“…To determine if the endogenous non-tagged claudin-2 were phosphorylated in MDCK cells, we performed phosphate-affinity SDS-PAGE with the acrylamide-pendant Phos-tag reagent; covalent incorporation of this ligand in the acrylamide polymer produces a gel matrix which specifically retards phosphorylated proteins during SDS-PAGE (Kinoshita-Kikuta et al, 2007;Kinoshita and Kinoshita-Kikuta, 2011). Immunoblots of MDCK lysates analysed by Phos-tag SDS-PAGE reveals that a fraction of the endogenous claudin-2 migrates at its characteristic (C, left panel) Claudin-2 immunoblot of Phos-tag SDS PAGE of mouse kidney cell lysate reveals a similar major phosphorylated band as that seen in MDCK II cells; treatment of lysate with l phosphatase results in decreased signal from this higher MW species; unphosphorylated claudin-2 migrates below the major phosphorylated species.…”

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress

“…The CAT3 phosphorylation level was detected using Phos-Biotin technology as described previously (Kinoshita-Kikuta et al, 2007). Under normal conditions, CAT3 phosphorylation was very low in Col/35S pro :CAT3-GFP and cpk8/35S pro :CAT3-GFP plants ( Figure 3F).…”

“…CAT3-GFP proteins were immunoprecipitated using Anti-GFP mAb agarose (MBL). CAT3-GFP proteins were then separated by 10% SDS-PAGE, and phosphorylated proteins were detected by immunoblotting using Biotinylated Phos-tag as described previously (Kinoshita-Kikuta et al, 2007). Calf intestinal alkaline phosphatase-treated (Takara) proteins were used as control.…”

A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis

“…We have previously reported a novel phosphate-affinity SDS-PAGE technique for the separation and detection of phosphorylated forms of proteins (Kinoshita, Kinoshita-Kikuta, & Koike, 2009;Kinoshita, Kinoshita-Kikuta, Takiyama, & Koike, 2006;Kinoshita-Kikuta, Aoki, Kinoshita, & Koike, 2007). The affinity electrophoresis technique, Mn 2+ -Phos-tag SDS-PAGE, which uses a polyacrylamide-bound dimanganese(ΙΙ) complex of the phosphate-binding ligand Phos-tag in conjunction with Laemmli's buffer system (an alkaline-pH gel system), has been widely used in determining the phosphorylation status of a wide variety of proteins.…”