Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product.
The regulation of the Erk (extracellular-signal-regulated kinase) gene-encoded protein kinase activity by reversible phosphorylation has been reported to involve either an activator of autophosphorylation or an upstream protein kinase. In this communication we describe assays utilizing the Erk-1 protein fused to glutathione S-transferase that permit the identification of protein kinase(s) that phosphorylate and activate the myelin basic protein kinase activity encoded by the Erk-1 gene. A phorbol ester-stimulated protein kinase activity was identified that phosphorylated a kinase-negative Erk-1 gene product on tyrosine and threonine. The protein kinase phosphorylated and activated wild-type protein expressed in bacteria from 20- to 50-fold. The activation of the Erk-1-encoded myelin basic protein kinase required ATP and correlated directly with the degree of phosphorylation on the same amino acid residues previously shown to be phosphorylated in vivo. Conversion of the tyrosine site of phosphorylation to phenylalanine yielded an Erk-1 gene product that could not be activated. Similar results were obtained when the threonine site was mutated to valine. It is likely that the phorbol ester-stimulated protein-tyrosine/threonine kinase(s) is an up-stream target for multiple extracellular signals.
Alessandrini
A
A
Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138.
Crews
C M
CM
Erikson
R L
RL
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D066298:In Vitro Techniques; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010449:Peptide Mapping; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D010703:Phorbol Esters; D010769:Phosphothreonine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases