Implication of Ca²⁺‐Dependent Protein Tyrosine Phosphorylation in Carbachol‐Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract

Abstract:The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled pheochromocytoma P012 cells with respect to the involvement of protein tyrosine phosphorylationand Ca2~. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111,91, 84, 74,(65)(66)(67)(68)(69)(70) 44, and 42 kDa) in P012 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65-70-kDa proteins peaked within 1 mm, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK,and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 mm. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84,(65)(66)(67)(68)(69)(70) in a preincubation time-and concentration-dependent fashion. In Ca 2~-freebuffer, CCh-induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2~ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65-70 kDa only in the presence of extracellular Ca2~. Extracellular Ca2~depen-dency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111 -kDa protein.These results suggest that Ca2-dependent protein tyrosine phosphorylation is closely implicated in OCh-induced PLO activation in P012 cells.

Possible Involvement of Mitogen‐Activated Protein Kinase in Phospholipase D Activation Induced by H2O2, but Not by Carbachol, in Rat Pheochromocytoma PC12 Cells

“…Monolayer cultures of PCI2 cells were maintained in 100-mm-diameter tissue culture dishes in growth medium composed of Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum and 5% (vol/ vol) horse serum (Kanoh et al, 1992;Ito et a!., 1997a).…”

Phenylarsine Oxide and H2O2 Plus Vanadate Induce Reverse Translocation of Phorbol-Ester-Activated PKC.BETA.II

“…This is indicated by numerous observations that vanadate in combination with H 2 O 2 , or PAO, stimulates phospholipase D activity in vivo (Bourgoin and Grinstein, 1992;Kumada et al, 1994;Kumada et al, 1996;Min et al, 1998;Taher et al, 1998). Further support comes from the observations that tyrosine kinase inhibitors such as genistein, herbimycin or tyrphostin, inhibit phospholipase D activation in several cell types (Uings et al, 1992;Schmidt et al, 1994;Ward et al, 1995;Jinsi et al, 1996;Natarajan et al, 1996;Ito et al, 1997;Exton, 1999). These reports prompted us to investigate whether phospholipase D activity is involved in PTPase-inhibitor-induced PKCβII reverse translocations.…”

Phospholipase D—Structure, regulation and function

“…Thus, the inhibitors decrease the activation of PLD by ATP in U937 promonocytes Kusner et al 1993), thrombin in platelets (Martinson et al 1994), FMLP in neutrophils or Iti.60 cells (Uings el al. 1992;Itoule et al 1999), endothelin 1, norepinephrine and angiotensin II in vascular smooth muscle cells (Wilkes et al 1993;Jinsi et al 1996;Suzuki et al 1996b), bombesin in fibroblasts (Briscoe et al 1995), carbachol in PC12 pheochromocytoma cells and ItEK cells expressing the M3 muscarinic receptor (Ito et al 1997a;Schmidt et al 1994) and endothelin 1 in myometrium (Le Stunff et al 2000b).…”

“…19, Gomez-Cambronero 1995;Ito et al 1997b;Marcll et al 1997;Min et al 1998a). Although the tyrosine phosphorylation of cellular proteins was correlated with the activation of PLD, the tyrosine phosphorylation of PLD itself was not (Bourgoin & Grinstein 1992;Ito et al 1997aIto et al , 1997bMin et al 1998a). Furthermore, the effects ofPKC inhibitors and PKC down-regulation indicated that PKC mediated most of the PLD activity change seen with HzO2 in Swiss 3T3 cells (Min et al 1998a) although not in endothelial cells (Natarajan et al 1993).…”

“…The nature of the tyrosine kinases activated by G protein-coupled receptors and HzOz is not well defined, but there is evidence that they are Ca 2+-dependent (Ito et al 1997a(Ito et al , 1997b. All of the G protein-coupled receptors that mediate PLD activation through a tyrosine kinase mechanism are known to activate Gq and phospholipase C, and thus increase cytosolic Ca 2 § (Exton 1996).…”