Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669 (original) (raw)

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the pr...