Abstract
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.
Highlights
Because v-Jun cannot be phosphorylated in vivo, we shown to be phosphorylated by H-ras transfection of F9 cells compared the ability of cJAT-PK to bind to glutathione S-transferase (GST)-V- and tomediate c-Jun transcriptional activity in thesecells
Addingphorbol estersto U937 cells induces an increase in JunB protein
Eventsassociated with phorbol esters in theinduction of differentiation of human leukemic cells includes the activation of transcription of the c-jun proto-oncogene [1,2,3], increase inc-Jun protein (I), andstimulation of transcription of genes located downstream from c-Jun DNA-binding sites (AP-1enhancer elements)(1,4)
Summary
AMP-dependent protein kinase stimulates the amino-terminal phosphorylation of c-Jun in these cells [8].To examine the role of this phosphorylation in regulating c-Jun-mediated transcriptional activation, we constructeda fusion protein containing the 84 NH2-terminal amino acids of c-Jun and fused ittothe DNA-binding domain of the yeast GAL4 cally phosphorylatesthese sites in thec-dunamino protein Transfection of this plasmid along with a recorder terminus. Unlike c-Jun, v-Jun is not phosphorylated when a 20-p1reaction containing0.2 pg ofc-Jun/glutathione S-transferasel u937 cells are treatedwith phorbolesters, suggesting that the glutathione-Sepharose beads, 5 p1 of protein extract, and 10 p1 of amino-terminal PKmight need to bind to thedeleted not found in v-Junbefore it can phosphorylate serines region 63 and kinase buffer, 20 mM Hepes, pH 7.6,lmM EGTA, 1mM dithiothreitol,.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
