Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis

• Published in: Biochimica et biophysica acta 2000-09, Vol.1487 (2), p.163-176
• Main Authors: Thorsen, Vidar A.T, Bjørndal, Bodil, Nolan, Gary, Fukami, Miriam H, Bruland, Ove, Lillehaug, Johan R, Holmsen, Holm
• Format: Article
• Language: English
• Subjects: 1-Butanol; Amino Acid Sequence; Animals; Carbon Radioisotopes; Choline - metabolism; Enzyme Activation - drug effects; Fibroblast C3H/10T1/2; Mice; Molecular Sequence Data; Peptides - chemistry; Peptides - metabolism; Phorbol myristoyl acetate; Phosphatidylcholine synthesis; Phosphatidylcholines - biosynthesis; Phosphatidylcholines - metabolism; Phospholipase D; Phospholipase D - antagonists & inhibitors; Phospholipase D - metabolism; PKC; Protein Kinase C - metabolism; RACK1; Receptors for Activated C Kinase; Tetradecanoylphorbol Acetate
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/S1388-1981(00)00092-5

The C3H/10T1/2 Cl8 HAβC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAβC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of α/βPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAβC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAβC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [ 14C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between α/βPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.