Temporal Analysis of PP2A Phosphatase Activity During Insulin Stimulation Using a Direct Activity Probe.
Protein serine/threonine phosphatases (PSPs) are ubiquitously expressed in mammalian cells. In particular, PP2A accounts for up to 1% of the total protein within cells. Despite clear evidence for the role of PP2A in cellular signaling, there is a lack of information concerning the magnitude and temporal dynamics of PP2A catalytic activity during insulin stimulation. Herein, we describe the development of a direct, fluorescent activity probe capable of reporting on global changes in PP2A enzymatic activity in unfractionated cell lysates. Utilizing this new probe, we profiled the magnitude as well as temporal dynamics of PP2A activity during insulin stimulation of liver hepatocytes. These results provide direct evidence for the rapid response of PP2A catalytic activity to extracellular stimulation, as well as insight into the complex regulation of phosphorylation levels by opposing kinase and phosphatase activities within the cell. This study provides a new tool for investigating the chemical biology of PSPs.
Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
Jon R
JR
Beck
Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
Tiffany
T
Truong
Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
Cliff I
CI
Stains
D004789:Enzyme Activation; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D043564:Inorganic Pyrophosphatase / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007328:Insulin; D024101:Mitochondrial Proteins; D043564:Inorganic Pyrophosphatase; C509848:PPA2 protein, human