Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.
The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.
Adler
V
V
Division of Hematology/Oncology, University of Alabama, Birmingham 35294.
Polotskaya
A
A
Wagner
F
F
Kraft
A S
AS
D002460:Cell Line; D002846:Chromatography, Affinity; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007938:Leukemia; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D054648:Protein Phosphatase 2; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine
D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D013755:Tetradecanoylphorbol Acetate