Human Phospholipase D1 Can Be Tyrosine-phosphorylated in HL-60 Granulocytes

Abstract

The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase C␣, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V 4؉ -OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V 4؉ -OOH, PLD1 was tyrosinephosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.

The uncovering of a novel regulatory mechanism for PLD2: Formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

“…In HL-60 granulocytes, the isoform PLD1 is tyrosyl phosphorylated in response to peroxides of vanadate [19]. In Swiss 3T3 fibroblasts, hydrogen peroxide induces PLD1 activation via protein tyrosine kinases [20].…”

Involvement of Tyrosine Phosphorylation and Protein Kinase C in the Activation of Phospholipase D by H2O2 in Swiss 3T3 Fibroblasts

“…H 2 O 2 has been reported to enhance tyrosine phosphorylation of the platelet-derived growth factor (PDGF) receptor, the epidermal growth factor receptor, Src kinase, and mitogen-activated protein kinase, leading to activation of gene expression including c-fos, c-myc, c-jun, NF-B, and AP-1 (12)(13)(14)(15)(16). Tyrosine phosphorylation has also been implicated in PLD activation mediated by epidermal growth factor (17), PDGF (18), pervanadate (19,20), fMet-Leu-Phe (21), and immunoglobulin E (22). Furthermore, inhibitors of protein-tyrosine phosphatases such as peroxides of vanadate stimulate PLD activity by G proteinindependent (19) and -dependent (23) mechanisms.…”

“…Furthermore, inhibitors of protein-tyrosine phosphatases such as peroxides of vanadate stimulate PLD activity by G proteinindependent (19) and -dependent (23) mechanisms. Recently, it was reported that, in response to stimulation by peroxides of vanadate, human brain PLD1 was tyrosine-phosphorylated and associated with several undefined, tyrosine-phosphorylated proteins in HL-60 cells (20).…”

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

“…Indeed, although the tyrosine kinase inhibitors ST-638, MDHC and genistein inhibit fMLP-induced PLD activation, we were unable to detect tyrosine phosphorylation of hPLD1 in fMLPstimulated granulocytes (data not shown). Furthermore, a small fraction of total PLD1 was found tyrosine phosphorylated after a 10-min stimulation with V 4ϩ -OOH [30]. Finally, the time course of PLD activation and tyrosine phosphorylation of PLD1 elicited by V 4ϩ -OOH were not similar, with PLD activation preceding tyrosine phosphorylation of hPLD1 by several minutes [14,30].…”

“…Furthermore, a small fraction of total PLD1 was found tyrosine phosphorylated after a 10-min stimulation with V 4ϩ -OOH [30]. Finally, the time course of PLD activation and tyrosine phosphorylation of PLD1 elicited by V 4ϩ -OOH were not similar, with PLD activation preceding tyrosine phosphorylation of hPLD1 by several minutes [14,30].…”