Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.
Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.
Kisfalvi
Krisztina
K
Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine, Molecular Biology Institute, University of California, Los Angeles, California 90095-1786, USA.
Hurd
Cliff
C
Guha
Sushovan
S
Rozengurt
Enrique
E
D002277:Carcinoma / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D002452:Cell Count; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000187:drug effects / Q000378:metabolism; D009496:Neurotensin / Q000037:antagonists & inhibitors / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D006133:Growth Substances; D009496:Neurotensin; D004441:Ecdysterone; C008999:ponasterone A; D004247:DNA; C089212:protein kinase D; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human