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

Abstract

We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-LeuPhe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V 4؉ -OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase C␣ (PKC␣), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V 4؉ -OOH only elicited RhoA translocation and PMA induced ARF and PKC␣ translocation. Three tyrosine kinases inhibitors, ST-638, methyl 2,5-dihydroxycinnamate, and genistein reduced fMLP-stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP-induced increase of GTP␥S-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKC␣ to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKC␣ in the signaling pathway leading to PLD activation by fMLP. RO 31-8220, a specific inhibitor of PKC, reduced PMA-induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP-stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31-8220 the levels of membrane-bound RhoA were increased. The levels of membrane-bound ARF and PKC␣ were unaffected by RO 31-8220 during PMA stimulation. In contrast, fMLP-induced recruitment of ARF and RhoA was insensitive to RO 31-8220 but PKC␣ translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATPindependent manner. Furthermore, increased fMLP-induced PKC␣ translocation in the presence of RO 31-8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31-8220 are used together in intact HL60 cells.

Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTPγS-stimulated polymorphonuclear leukocytes

“…1 Phosphorylation processes appear to be involved with PLD activation; for example, tyrosine kinase inhibitors reduce PLD activation and membrane recruitment of ARF, RhoA, and PKC␣. 27 However, ARF1 and RhoA translocation were not affected by using apyrase to hydrolyze the ATP in a cell-free system from HL-60 leukemic cells. 19 We incubated the fractions in our cell-free system with 5 U/mL apyrase for 10 minutes at 37°C before GTP␥S activation.…”

Involvement of Phosphatidic Acid in both Degranulation and Oxidative Activity in fMet-Leu-Phe Stimulated Polymorphonuclear Leukocytes

“…This inhibitor is known to diminish the fMet-Leu-Phe induced and tyrosine kinase dependent translocation of ADPribosylation factor (ARF), RhoA and protein kinase Cα (PKCα). All these proteins are implicated to be involved as cofactors in the PLD activation [19,20]. In comparison to butanol, ST-638 was less efficient in the inhibition of degranulation.…”

“…In fMet-Leu-Phe stimulated PMNs, tyrosine kinases are known to be responsible for translocation of small GTPases such as ADP-ribosylation factor (ARF) and RhoA as well as the protein kinase Cα (PKCα). All these proteins are cofactors for PLD activation [19,20].…”

Prostaglandin E2Inhibits the Phospholipase D Pathway Stimulated by Formyl-methionyl-leucyl-phenylalanine in Human Neutrophils. Involvement of EP2Receptors and Phosphatidylinositol 3-kinase γ

“…There is no evidence for a direct tyrosine phosphorylation of PLD after fMLP stimulation in neutrophils (Marcil et al, 1999), but the translocation of PLD cofactors (PKC␣, Arf, and RhoA) as well as Rac2 and Cdc42 activation has been shown to occur downstream of tyrosine phosphorylation events in fMLP-stimulated HL-60 cells, providing a functional link between tyrosine kinases and PLD activation (Benard et al, 1999;Houle et al, 1999). PGE 2 (or CAY 10399) reduced the fMLP-stimulated tyrosine phosphorylation of a set of substrates around 120 kDa by as-yet-unidentified kinases.…”