Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D.
Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic (32)P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.
Waldron
Richard T
RT
Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, USA. rwaldron@mednet.ucla.edu
Whitelegge
Julian P
JP
Faull
Kym F
KF
Rozengurt
Enrique
E
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005982:Glutathione Transferase / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D012694:Serine; D005982:Glutathione Transferase; C089212:protein kinase D; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases