Phorbol esters stimulate the phosphorylation of c-Jun but not v-Jun: regulation by the N-terminal delta domain.
c-Jun and its oncogenic counterpart v-Jun are completely conserved within the region from Ser-63 to Ser-73; these serines are sites for phorbol ester-inducible c-Jun phosphorylation. Using a U937 human leukemic cell line stably expressing v-Jun, we have demonstrated that phorbol esters stimulate the in vivo phosphorylation of c-Jun but not v-Jun. We developed an in vitro protein kinase assay to characterize the c-Jun protein kinase and to examine the determinants underlying this differential phosphorylation. Fusion proteins between glutathione S-transferase and the N terminus of c-Jun, v-Jun, or several c-Jun mutants were used as substrates. A c-Jun kinase activity was affinity-purified 5000-fold by using glutathione S-transferase-c-Jun-glutathione-Sepharose beads and was found to phosphorylate the N terminus of c-Jun but not v-Jun or c-Jun containing a 27-amino acid N-terminal deletion found in v-Jun. These effects were also observed in vivo as phorbol 12-myristate 13-acetate did not induce the phosphorylation of v-Jun or the c-Jun deletion mutant in U937 cell lines stably expressing these proteins. These findings indicate that the delta domain of c-Jun (amino acids 34-60), which is deleted in v-Jun, plays a critical role in regulating N-terminal c-Jun phosphorylation.
Adler
V
V
Division of Hematology/Oncology, University of Alabama, Birmingham 35294.
Franklin
C C
CC
Kraft
A S
AS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D016758:Genes, jun; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016759:Oncogene Protein p65(gag-jun) / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D009838:Oligodeoxyribonucleotides; D016759:Oncogene Protein p65(gag-jun); D010710:Phosphates; D016755:Proto-Oncogene Proteins c-jun; D008715:Methionine; D011494:Protein Kinases; D013755:Tetradecanoylphorbol Acetate