Expression and regulation of phospholipase D in the human keratinocyte cell line HaCaT

• Published in: FEBS letters 1998-03, Vol.425 (2), p.199-203
• Main Authors: Müller-Wieprecht, Verena, Riebeling, Christian, Alexander, Christian, Scholz, Frank-Rüdiger, Höer, Ariane, Wieder, Thomas, Orfanos, Constantin E, Geilen, Christoph C
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - immunology; ARF1, ADP-ribosylation factor 1; BCA, bicinchoninic acid; BSA, bovine serum albumin; DPPC, dipalmitoylphosphatidylcholine; EGTA, ethylene glycol-bis[β-aminoethylether]-N,N,N′,N′-tetraacetic acid; Gene Expression Regulation - drug effects; GTPγS, guanosine 5′-O-(3-thiotriphosphate); Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; HaCaT cell; Humans; Keratinocytes - metabolism; MgATP, adenosine triphosphate magnesium salt; Na-HEPES, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] sodium salt; PC, phosphatidylcholine; PE, phosphatidylethanolamine; Peptide-specific antibody; PEt, phosphatidylethanol; Phospholipase D; Phospholipase D - genetics; Phospholipase D - immunology; PIP2, 1-phosphatidyinositol-4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; PMSF, phenylmethylsulfonyl fluoride; Rabbits; RNA, Messenger - metabolism; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; Signal transduction; TPA, 12-O-tetradecanoyl phorbol-13-acetate
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)00227-0

The generation of lipid second messengers via phosphatidylcholine (PC)-specific phospholipase D (PLD) has emerged as an important step leading to transduction of extracellular signals. In the present investigation the expression of human cytosolic PLD isoenzymes in the immortalized human keratinocyte cell line HaCat was determined. At the mRNA level we found the expression of hPLD1b and for the first time in human cells also the expression of hPLD2. For further analysis of enzyme expression at the protein level, hPLD1 peptide fragments were synthesized and specific antibodies were generated (rabbit) to be used for detection of hPLD1 in Western blot experiments. Furthermore, small G-proteins were found to be involved in the regulation of PLD activity in HaCaT cells using the guanine nucleotide analogue GTPγS.