PLDl exists in two splice variants, one of which has a 38 amino acid deletion between conserved
Numerous studies have shown that phorbol esters activate PLD in a variety of cell types, and, as described in the previous sections, many agonists stimulate PIP2 hydrolysis and thereby activate PKC. These findings indicate that PKC is a major regulator of PLD. Certain PKC isozymes are able to directly activate PLD1 isozymes in vitro. Surprisingly the activation does not involve ATP and phosphorylation, and is restricted to the conventional isozymes, as no other PKC isozymes produce activation under these conditions. The activation involves the regulatory domain of the PKC isozymes and is unaffected by inhibitors of PKC kinase activity. It therefore occurs by a nonphosphorylating protein-protein interaction. The PKC interaction site on PLD1 has been localized to two sequences in the N-terminus. Deletion of these sequences abolishes PKC activation of the enzyme. Deletion of the most N-terminal sequence also causes a large increase in activity. It has been proposed that this sequence contains an inhibitory domain and that PKC acts to reverse the effect of this domain. When PLD1 is simultaneously activated by PKC and Rho and/or ARF, marked synergism is observed. The mechanistic basis for this is presently unknown.
Despite the evidence that conventional PKC iso-zymes activate PLD by a nonphosphorylating mechanism in vitro, many studies have shown that inhibitors of PKC kinase activity decrease the activation of PLD by phorbol esters and agonists in intact cells. The magnitude of the inhibition is variable, but in many cases it is substantial. PLD can be phosphorylated by PKCa in vitro, but this does not cause activation. Therefore, direct phosphorylation of PLD by PKC cannot be a mechanism by which it is activated in vivo. Other possible mechanisms include the phos-phorylation of regulatory proteins that act on the enzyme and the phosphorylation of another protein kinase(s) that phosphorylates and activates PLD. An alternative mechanism, for which there is some support, is that PKC phosphorylates a scaffolding protein and that the phosphorylation promotes the binding and productive interaction of PKC and PLD.
Members of the Rho subfamily of Ras GTPases activate PLD1 in vitro. RhoA, RhoB, and RhoC are the most effective Rho proteins, but Rac1, Rac2, and Cdc42Hs have partial effects. Their potency is greatly increased by geranylgeranylation. When the Rho proteins are expressed in cells, RhoA, RhoB, and RhoC produce greater stimulation of PLD than Rac1, but Cdc42Hs is ineffective. The Rho proteins interact with PLD1 through specific residues in their Switch I (activation loop) region, which is the common effector domain for Ras GTPases. However, differences in Rho protein efficacy are determined by addi tional residues in or adjacent to the Switch II region. Concerning the site on PLD1 at which the Rho proteins interact, this is in the C-terminal third of the enzyme. PLD1 displays synergistic activation when concurrently stimulated by Rho proteins and PKCa. The molecular basis for the synergism is unknown.
The in vivo role of Rho proteins in agonist regulation of PLD has been explored using the C3 exoen-zyme of Clostridium botulinum, which ADP-ribosy-lates and inactivates Rho proteins. The exoenzyme interacts preferentially with Rho rather than Rac and Cdc42Hs in vitro, and in vivo is selective for Rho also. Treatment of many cell types with C3 exoenzyme markedly inhibits agonist activation of PLD, although other cells may show little or no effect. These results indicate that Rho plays a major role in mediating agonist activation in PLD in some, but not all, cells. Dominant negative and dominant active forms of Rho proteins have also been utilized in a limited number of studies. These have shown that Rho and Rac play a role in the activation of PLD by growth factors and agonists linked to heterotrimeric G proteins (Gq and G13) in several cell lines. In summary, there is evidence that Rho proteins are important mediators of agonist activation of PLD in many, but not all, cells.
The mechanism(s) by which Rho proteins regulate PLD in intact cells is unclear. As the proteins can directly activate the enzyme in vitro, it is logical to conclude that this is the mechanism in vivo. However, Jakobs and associates have obtained evidence suggesting that Rho acts by controlling the intracellular concentration of PIP2. Rho activates PI 4-P 5-kinase, which is the enzyme that synthesizes PIP2, and treatment of cells with C3 exoenzyme or tyrosine kinase inhibitors causes a decrease in PIP2. However, it has not been shown that agonist activation of PLD is associated with an increase in PIP2 in any cell line.
Members of the ADP-ribosylation factor (ARF) subfamily of Ras GTPases activate PLD1 isozymes. There is little difference in the potency of the different ARF members, but myristoylation increases their potency markedly. The in vitro efficacy of the myris-toylated ARF proteins is greater than that of the gera-nylgeranylated Rho proteins, but they exhibit similar concentration dependencies. The PLD1 interaction site has been localized to the N-terminus of the ARFs, but the region of PLD1 that interacts with the ARFs is unknown. When combined with Rho proteins and/
or PKCa, ARF1 produces a synergistic activation of PLD1.
As in the case of Rho, some cells exhibit a dependency on ARF for agonist activation of PLD, whereas others show no dependency. Thus brefeldin A, an inhibitor of guanine nucleotide exchange on ARF, inhibits the PLD-stimulatory effects of some agonists, but not others. There have also been reports that addition of ARF restores the activation of PLD by growth factors in permeabilized cells, and that expression of dominant negative mutants of ARF blocks the stimulation of PLD in intact cells.
Whereas there is much evidence that certain agonists activate PKC and Rho proteins in cells, there are fewer data showing that agonists activate ARF. Evidence that agonists activate ARF comes from studies showing that the agonists increase the membrane association of ARF. As this association is also increased by GTPyS in vitro, it is reasoned that the agonists promote GTP binding to ARF.
PLD1 is localized to the perinuclear region of the cell, which includes the ER, Golgi apparatus, and late endosomes. The Golgi-associated enzyme is stimulated by ARF in the presence of a nonhydrozable analog of GTP and the activation is blocked by brefeldin A. ARF-sensitive PLD is also found in the secretory vesicles of neutrophils and has been reported to be translocated to the plasma membrane by the vesicles upon stimulation by f-Met-Leu-Phe. Translocation of PLD1 from secretory granules/lyso-somes to plasma membranes has also been observed in RBL-2H3 basophilic cells upon crosslinking of IgE receptors. There are also reports that ARF-stimulated PLD is present in plasma membranes as well as Golgi, endomembranes, and nuclei in HL60 promyelocytic cells and liver. The functional significance of the PLD1 activity in these intracellular organelles, its regulation by ARF, and its translocation to the plasma membrane is at present unclear.
Was this article helpful?