A nonradioactive fluorescent gel-shift assay for the analysis of protein phosphatase and kinase activities toward protein-specific peptide substrates.
Synthetic peptides are important tools with which to study the activities of protein kinases and phosphatases toward specific substrate sequences which are present within selected regions of a protein. Most existing assays for the phosphorylation or dephosphorylation of such peptides utilize 32P and either affinity chromatography or HPLC separation and require extensive characterization and validation. Here, we describe a method for monitoring the phosphorylation or dephosphorylation of almost any peptide of interest which does not require the use of radioactivity, making its reagents stable for a prolonged period, and which can be performed in any standard laboratory. For this, after performance of kinase or phosphatase reactions with the peptide of interest, products are derivatized with fluorescamine and are separated according to charge by agarose gel electrophoresis. Phosphorylated and nonphosphorylated peptides are readily separated and can be both identified and quantified by uv detection. The lower limit for detection of peptide in the agarose gel was 0.02 nmol using the gel-shift kinase assay with cAMP-dependent kinase and Kemptide as substrate. This had sensitivity and reproducibility similar to those of a standard assay using [gamma-32P]ATP with this substrate. Dephosphorylation of a synthetic phosphopeptide corresponding to a segment of the cholecystokinin receptor was tested in an analogous assay with known amounts of protein phosphatase 2A. Phosphopeptide and dephosphopeptide were easily detected and quantified with as little as 0.03 mU/ml protein phosphatase 2A activity. Therefore, with this assay, most synthetic peptides and phosphopeptides can be used as substrates without further modification. This will be of particular interest for monitoring the purification of highly specific protein kinase and phosphatase activities.
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
M P
MP
Lutz
D I
DI
Pinon
L J
LJ
Miller
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005450:Fluorescamine; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D054648:Protein Phosphatase 2; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009842:Oligopeptides; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D011949:Receptors, Cholecystokinin; D005450:Fluorescamine; C037642:kemptide; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2