Interaction of PLD1b with actin in antigen-stimulated mast cells.
Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.
Farquhar
M J
MJ
Molecular Physiology Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Powner
D J
DJ
Levine
B A
BA
Wright
M H
MH
Ladds
G
G
Hodgkin
M N
MN
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000941:Antigens / Q000502:physiology; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D008407:Mast Cells / Q000378:metabolism*; D009154:Mutation; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D012694:Serine / Q000737:chemistry; D014162:Transfection
D000199:Actins; D000941:Antigens; D012694:Serine; D010739:Phospholipase D; C106801:phospholipase D1