To determine the effect of Gβ1
(Gβγ) on basal, unstimulated PLD1 activity, Gβγ was incubated with soluble, purified hPLD1 in complex lipid vesicles containing PIP2
, and PLD activity was measured in this reconstituted system. [3
H]PC was cleaved over 30 min by PLD, and soluble [3
H]choline released as a result of PLD activation was recovered and measured by scintillation counting. As seen in , Gβγ inhibited basal activity of purified full-length PLD1. To determine the role of the amino-terminal domain of PLD in this interaction, which contains the PX/PH domains of PLD, we used a truncated rPLD1 isoform lacking residues 1 to 311 of full-length PLD (PLD1.d311) (). PLD1.d311, like fulllength PLD1, requires PIP2
for activity and is regulated by Arf and Rho family proteins Cdc42, RhoA, and Rac1 (Henage et al., 2006
). This truncated rPLD1 isoform, expressed as a MBP fusion protein, demonstrates high expression (>100-fold compared with wild-type hPLD) and robust enzymatic activity in vitro and in vivo. PLD1.d311 retains sensitivity to monomeric G proteins, with activation kinetics similar to that of wild type, but it demonstrates a reduced sensitivity to PKCα, because of elimination of one of two putative PKC interaction sites located within the amino terminus. We tested the ability of Gβγ to inhibit both full-length and the N-terminally truncated PLD isoform PLD1.d311, which are both PIP2
-dependent (Henage et al., 2006
). Although fulllength PLD1 was inhibited by a relatively modest Gβ1
concentration (1 µM), PLD1.d311 was not inhibited by even higher levels of Gβ1
, compared with their respective basal activities in the absence of Gβγ (), suggesting Gβ1
inhibits full-length PLD1.
PLD1 activity is stimulated by PKCα, Arf, and RhoA family proteins, and PKCα acts synergistically to activate PLD in the presence of monomeric G proteins. To determine the effect of Gβγ on stimulated PLD activity, PLD1 was incubated with indicated activators () in the presence and absence of Gβ1γ1, and PC hydrolysis was measured as indicated above. We found that Gβγ inhibited PLD-mediated PC hydrolysis in the presence of each activator tested (), whereas using boiled Gβγ, Gβγ storage buffer, or bacterial PLD had no effect on PC hydrolysis, nor did Gβγ or activators alone in the absence of PLD (data not shown). In addition, synergistic PLD1 activation was also strongly inhibited by Gβ1γ1 (, far right).
Fig 2 Gβγ inhibits stimulated PLD1 activities. Comparison of effect of 5/µM Gβ1γ1 on 1.6 nM PLD1-mediated PC hydrolysis (mean basal PLD activity, 3.4 pmol of PC hydrolyzed/30 min) in the presence and absence of PLD activators (more ...)
was found to inhibit both basal and stimulated PLD1 activity, we next examined the isoform specificity of the interaction. Because PLD1 is more sensitive to regulation by monomeric G proteins and PKC activators, PLD2 stimulation by selected activators has also been reported, albeit to a lesser extent than PLD1 (Lopez et al., 1998
; Chen and Exton, 2004
). Membranes from Sf21 cells expressing PLD1 or PLD2 (mPLD1and mPLD2) were incubated with a combination of Gβγ and activators PKCα and Arf. PLD-mediated PC hydrolysis was measured, relative to basal PLD activity, for each isoform in the absence of Gβγ and activators. We found Gβ1
inhibited both PLD1 and PLD2 activity () stimulated by Arf and PKCα. Despite higher basal activity of PLD2 (results normalized to basal), only modest increases were seen upon activation of PLD2 by PKC and Arf, consistent with the observation that this isoform is less sensitive to activators (Lopez et al., 1998
). Although Gβ1
inhibited both stimulated PLD1 and PLD2 activity, Gβ1
reduced stimulated PLD2 activity to a level below that of basal, an effect not seen with mPLD1, nor with soluble PLD1 stimulated by activators (), suggesting a strong inhibition of PLD2 by Gβγ subunits. The Gβγ-mediated inhibition of PLD1 in membrane preparations is less complete than that seen using purified PLD1 (compare with ), which may reflect the accessibility of Gβγ to the PLD enzyme in a membrane environment.
Fig 3 Gβγ inhibits PLD1 and PLD2 activities in membranes. PC hydrolysis was measured in the presence and absence of purified Gβ1γ1 added to membranes expressing either PLD1 or PLD2 (26 and 10 ng of total membrane protein, respectively), (more ...)
To determine the potency of inhibition of PLD1 by Gβγ, increasing concentrations of Gβγ were used to inhibit PKC-stimulated PLD1 activity. Because Gβγ inhibited both basal and stimulated PLD activity, Gβγ-mediated inhibition of PLD was measured after stimulation with PKCα (to increase signal amplitude in these assays). We found that increasing amounts of Gβ1γ1 inhibited stimulated PLD1 activity in a dose-dependent manner, compared with the maximal activation in the absence of Gβγ (). For comparison, Gβ1γ2 was also examined for its ability to inhibit PLD. Gβ1γ2 was a more potent inhibitor of PLD activation than Gβ1γ2 (); Gβ1γ2 (1.2 µM) was sufficient to inhibit PLD1 activation by 50%, compared with 2.4 µM Gβ1γ1 required to mediate the same level of PLD inhibition.
Fig 4 Gβ1γ2 inhibits PLD1 with greater potency than Gβ1γ1. PKCα (654 nM) and increasing concentrations of indicated Gβγ subtypes were incubated with purified PLD1 (1.6 nM) in PC vesicles containing PIP (more ...)
The higher potency of Gβ1
is not surprising; in other signaling systems, Gβ1
has been shown to interact with higher affinity to effectors than Gβ1
For example, Gβ1
more potently stimulated PLCβ (Ueda et al., 1994
) and inhibited SNARE fusion machinery (Blackmer et al., 2005
) than Gβ1
. This may be due largely to post-translation modifications of these Gβγ isoforms. Gβ1
is farnesylated, whereas Gβ1
is geranylgeranylated, and these modifications influence membrane association. Geranylgeranylation of Gβ1
may allow this isoform to more effectively bind phospholipids, thus conferring greater potency toward membrane associated effectors such as PLD.
The ability of Gβγ ability to inhibit full-length PLD is in contrast to its lack of effect on PLD1.d311, in which the first 331 residues of full-length PLD1 have been ablated (). To further investigate this result, residues 3 to 311 encompassing the PX/PH of PLD1 domain were expressed and purified as MBP-fusion proteins (N-PLD), as was PLD1.d311. Because the PX/PH domain itself is relatively insoluble, creation of a MBP fusion protein allows for a significant improvement in solubility and moderate levels of protein expression. Binding of purified N-PLD to Gβγ was measured using fluorescently labeled Gβγ. Gβ1γ1 was labeled with M8, a thiol-reactive, environmentally sensitive fluorescent probe that increases its emission upon a binding event as a result of an increase in the hydrophobicity of the environment of the probe. Binding is detected as an increased emission from the labeled protein compared with emission in the absence of the interacting protein. M8-Gβ1γ1 bound N-PLD in a dose-dependent manner (), in contrast to quenched label alone (data not shown). Both the amino-terminally truncated PLD1.d311 and the N-PLD proteins contain an MBP tag; however, the PLD1.d311 protein did not interact with labeled Gβγ (). Only the N-PLD protein demonstrated increases in fluorescence upon incubation with M8-Gβγ. These results indicate the amino-terminal region of PLD is necessary and sufficient for interaction with Gβ1γ1 subunits. PLD1 constructs lacking this domain do not interact with Gβ1γ1, consistent with the inability of Gβγ to regulate PLD1.d311 activity ().
Fig 5 Gβγ binds to the amino-terminal domain of PLD1. Fluorescence of MIANS-labeled Gβ1γ1 (100 nM), excitation/emission 320/420 nm, upon interaction with increasing concentrations of N-PLD, encompassing the PX/PH domain of PLD (more ...)
The ability of Gβγ to inhibit full-length PLD activity in vitro, both basal and stimulated (in contrast to the N-terminally truncated PLD1.d311), suggests Gβγ directly inhibits PLD. This interaction is likely to be mediated through the amino-terminal 311-amino acid stretch of PLD1, which binds to Gβγ with a dose-response relationship consistent with its effect on PC hydrolysis as measured in reconstitution assays.
To determine the effect of Gβγ on PLD activity in cells, we overexpressed Gβ1
in MDA-MB-231 cells (), which express both PLD1 and PLD2 (Meier et al., 1999
). Gβγ reduced basal PLD activity in these cells significantly compared with control (). Furthermore, Gβγ modestly reduced PLD activation in PMA and LPA stimulated MDA-MB-231 cells (), indicating Gβγ may play a modulatory role in PLD activation in vivo.
Fig 6 Inhibition of PLD activity in vivo. MDA-MB-231 cells were transiently transfected with plasmid DNA encoding either empty vector (control) or cotransfected with plasmid DNA encoding Gβ1 and Gγ2. A, transfected MDA-MB-231 cells were analyzed (more ...)