Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

• Published in: Molecular & cellular proteomics Vol. 6; no. 2; pp. 356 - 366
• Main Authors: Kinoshita-Kikuta, Emiko, Aoki, Yuri, Kinoshita, Eiji, Koike, Tohru
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01-02-2007
• Subjects: Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel - methods; Humans; Manganese - chemistry; Phosphates - metabolism; Phosphates - pharmacokinetics; Phosphoproteins; Phosphorylation; Phosphotransferases - metabolism; Proto-Oncogene Proteins c-abl - metabolism; Signal Transduction; tau Proteins - metabolism
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.T600044-MCP200

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-3β, 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 up-shifted 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 μ m in the presence of 0.10 m m ATP.