A quantitative method for measuring protein phosphorylation.
We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5).
Mckenzie
J Andres
JA
Department of Biology, Northeastern University, Boston, MA 02115, USA.
Strauss
Phyllis R
PR
D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism
D004676:Myelin Basic Protein; D011506:Proteins; D010770:Phosphotransferases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase