Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.
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 primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.
Northwood
I C
IC
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605.
Gonzalez
F A
FA
Wartmann
M
M
Raden
D L
DL
Davis
R J
RJ
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D013912:Threonine; D011494:Protein Kinases; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases