Protein kinase D complexes with C-Jun N-terminal kinase via activation loop phosphorylation and phosphorylates the C-Jun N-terminus.
Protein kinase D (PKD), a downstream effector of protein kinase C (PKC), is implicated in suppression of the c-Jun N-terminal kinase (JNK) signaling pathway, however, its mechanism of action is unclear. Transphosphorylation of the PKD activation loop at serines 744/748 by a PKC mediated signal transduction pathway enhances its catalytic activity. Here we show that PKD activation loop phosphorylation at serines 744/748 via PKC, or mutation of these serines to glutamic acid (PKD-S744/748E) also results in complex formation with JNK, indicating that suppression of JNK signaling by PKD involves a direct interaction with JNK. Because catalytically active PKD associates with JNK we determined whether it could phosphorylate the c-Jun N-terminus as a potential mechanism by which it suppresses c-Jun Ser 63 phosphorylation when it complexes with JNK. Purified human PKD and either wild-type PKD from phorbol 12, 13-dibutyrate (PDB)-stimulated cells or unstimulated constitutively active PKD (PKD-S744/748E), phosphorylated the c-Jun N-terminus between amino acids 1-89 at sites distinct from those phosphorylated by JNK. These results demonstrate, for the first time, phosphorylation dependent association of PKD with another signaling molecule and reveal a potential mechanism by which PKD could modulate the ability of JNK to phosphorylate c-Jun by phosphorylating alternative sites in the c-Jun N-terminus when it is complexed with JNK.
Hurd
Cliff
C
Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California, CA 90095-1786, USA.
Waldron
Richard T
RT
Rozengurt
Enrique
E
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002855:Chromatography, Thin Layer; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D046911:Macromolecular Substances; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D007527:Isoenzymes; D046911:Macromolecular Substances; D010748:Phosphopeptides; D016755:Proto-Oncogene Proteins c-jun; C089212:protein kinase D; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases