title: Phospholipase D1 is threonine-phosphorylated in human-airway epithelial cells stimulated by sphingosine-1-phosphate by a mechanism involving Src tyrosine kinase and protein kinase Cdelta.
abstract: The regulatory role of protein kinase C (PKC) delta isoform in the stimulation of phospholipase D (PLD) by sphingosine-1-phosphate (SPP) in a human-airway epithelial cell line (CFNPE9o(-)) was revealed by using antisense oligodeoxynucleotide to PKCdelta, in combination with the specific inhibitor rottlerin. Cell treatment with antisense oligodeoxynucleotide, but not with sense oligodeoxynucleotide, completely eliminated PKCdelta expression and resulted in the strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKCalpha, beta, delta, epsilon and zeta isoforms expressed in these cells, only PKCdelta was activated on cell stimulation with SPP, as indicated by translocation into the membrane fraction. Furthermore, pertussis toxin and genistein eliminated both PKCdelta translocation and PLD activation. In particular, a significant reduction in phosphatidylbutanol formation by SPP was observed in the presence of 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1), an inhibitor of Src tyrosine kinase. Furthermore, the activity of Src kinase was slightly increased by SPP and inhibited by PP1. However, the level of PKCdelta tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCdelta. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas the PLD1 isoform alone was threonine-phosphorylated in SPP-treated cells. PLD1 threonine phosphorylation was strongly inhibited by rottlerin, by anti-PKCdelta oligodeoxynucleotide and by PP1. In conclusion, in CFNPE9o(-) cells, SPP interacts with a membrane receptor linked to a G(i) type of G-protein, leading to activation of PLD, probably the PLD1 isoform, by a signalling pathway involving Src and PKCdelta.
authors.affiliation: Dipart. di Biologia Ev. Sp., Via Irnerio 42, Università di Bologna, Bologna, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Ghelli
authors.forename: Anna M
authors.initials: AM
authors.lastname: Porcelli
authors.forename: Annalisa
authors.initials: A
authors.lastname: Facchini
authors.forename: Silvana
authors.initials: S
authors.lastname: Hrelia
authors.forename: Flavio
authors.initials: F
authors.lastname: Flamigni
authors.forename: Michela
authors.initials: M
authors.lastname: Rugolo
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D019833:Genistein / Q000494:pharmacology; D020404:Glycerophospholipids / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D008246:Lysophospholipids; D009296:Nasal Cavity / Q000166:cytology; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D020545:Respiratory Mucosa / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013997:Time Factors
chemical_list: D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D020404:Glycerophospholipids; D007527:Isoenzymes; D008246:Lysophospholipids; D016376:Oligonucleotides, Antisense; D010712:Phosphatidic Acids; D020033:Protein Isoforms; C068564:phosphatidylbutanol; C060506:sphingosine 1-phosphate; D013912:Threonine; D012694:Serine; D019833:Genistein; C085746:rottlerin; D016392:Proto-Oncogene Proteins pp60(c-src); C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D010739:Phospholipase D; C106801:phospholipase D1; D013110:Sphingosine