Diacylglycerol kinase alpha mediates HGF-induced Rac activation and membrane ruffling by regulating atypical PKC and RhoGDI

Abstract

Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into phosphatidic acid (PA), acting as molecular switches between DAG- and PA-mediated signaling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKalpha are required for growth-factor-induced cell migration and ruffling, through the control of Rac small-GTPase activation and plasma membrane localization. Herein we unveil a signaling pathway through which DGKalpha coordinates the localization of Rac. We show that upon hepatocyte growth-factor stimulation, DGKalpha, by producing PA, provides a key signal to recruit atypical PKCzeta/iota (aPKCzeta/iota) in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. Then, DGKalpha-dependent activation of aPKCzeta/iota mediates the release of Rac from the inhibitory complex with RhoGDI, allowing its activation and leading to formation of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKalpha as the central element of a lipid signaling pathway linking tyrosine kinase growth-factor receptors to regulation of aPKCs and RhoGDI, and providing a positional signal regulating Rac association to the plasma membrane.

Fig. 1.

DGKα provides the signal directing Rac to the nascent ruffle. MDCK cells, transfected with either DGKαWT or myr-DGKα, were cultured overnight in the absence of serum, fixed, and stained for Rac (green), myc tag (red), and actin (blue). Arrows indicate DGKαWT- or myr-DGKα-transfected cells. (Scale bar, 10 μm.) C, 30 transfected cells, scored for the presence of Rac at ruffling sites. n = 6, with SEM; ***P = 0.0002.

Fig. 2.

Rac plasma membrane targeting requires Rac/RhoGDI interaction. MDCK cells were transiently transfected with mCherry-RacR66E, treated with 10 ng/mL HGF for 15 min, fixed, and stained for actin (green). (Scale bar, 24 μm.) C, 30 transfected cells, scored for the presence of ruffles or RacR66E plasma membrane localization. n = 3, with SEM; *P = 0.0057.

Fig. 3.

DGKα regulates RhoGDI targeting to the plasma membrane. (A) MDCK cells were stimulated with 10 ng/mL HGF for 5 min in the presence or absence of 1 μM R59949, fixed, and stained for RhoGDI (green) and actin (blue). Arrows indicate RhoGDI at membrane ruffles. (Scale bar, 24 μm.) C, 70 cells, scored for RhoGDI localization at ruffling sites. n = 3, with SEM; *P < 0.05. (B) MDCK cells, transfected with either DGKαWT or myr-DGKα, were cultured overnight in the absence of serum, fixed, and stained for RhoGDI (green), myc tag (red), and actin (blue). Arrowheads indicate transfected cells. (Scale bar, 10 μm.)

Fig. 4.

aPKCζ/ι mediates both HGF- and myr-DGKα-induced extension of membrane protrusions. (A) MDCK cells were transfected with three combinations of PKCζ (79, 80) and PKCι (1–3) specific siRNAs. Whole-cell lysates were analyzed for levels of aPKCζ/ι expression by western blot. (B) MDCK cells were transfected as in A, treated with 10 ng/mL HGF for 15 min, fixed, and stained for Rac or RhoGDI and actin. C, 110 cells, analyzed for the presence of ruffles and Rac or RhoGDI at the plasma membrane. n = 4 (Rac and RhoGDI), n = 8 (ruffles), with SEM; **P < 0.005, ***P < 0.0005. (C) MDCK cells were transiently transfected either with myr-DGKα alone or cotransfected with myr-DGKα and PKCζKW, cultured overnight in the absence of serum, fixed, and stained for myc and flag tags, actin, and Rac or RhoGDI. C, 20 transfected cells, scored for the presence of ruffles and Rac or RhoGDI at protrusion sites. n = 4, with SEM; **P < 0.001, ***P < 0.0001.

Fig. 5.

DGKα regulates aPKCζ/ι function. (A) MDCK cells were transfected either with control siRNA or DGKα siRNA c1 and c2, treated with 10 ng/mL HGF for 15 min, fixed, and stained for aPKCζ/ι (green) and actin (red). (Scale bar, 10 μm.) (B) MDCK cells, transfected either with DGKαWT or myr-DGKα, were cultured overnight in the absence of serum, fixed, and stained for aPKCζ/ι (green), myc tag (red), and actin (blue). Arrowheads indicate transfected cells. (Scale bar, 10 μm.) (C) MDCK cells were stimulated with either 250 μM C8-PA, C8-DAG, or C6-LPA or left untreated, and fixed and stained for actin (red) and aPKCζ/ι (green). Arrowheads indicate cortical actin rearrangements, while the arrow indicates aPKCζ/ι membrane localization. (Scale bar, 19 μm.) (D) MDCK cells were stimulated with either 250 μM C8-PA, 10% FCS medium (as positive control), 250 μM C8-DAG, or left untreated. Whole-cell lysates were analyzed by western blot and the intensity of phospho-aPKCζ/ι bands was quantified by densitometry. For each condition, nine replicate points were performed in four independent experiments. The densitometry of each band was normalized as the percentage of the densitometry mean of control points in the same experiment, and is shown in the histogram, with SEM; **P < 0.005. A representative picture is shown.

Fig. 6.

DGKα regulates Rac/RhoGDI complex dissociation. MDCK/empty vector or MDCK/DGKα-DN cells were stimulated with 50 ng/mL HGF for 15 min. Cell lysates were immunoprecipitated for RhoGDI and analyzed by western blot.

Fig. 7.

DGKα does not affect myr-PKCζ-induced events. MDCK cells were transiently transfected with myr-PKCζ, grown overnight in the absence of serum, and treated with 1 μM R59949 for 1 h. Cells were fixed and stained for flag tag (red), RhoGDI (green), and actin (blue). Arrows indicate RhoGDI staining at protrusion sites. (Scale bar, 24 μm.) C, 30 transfected cells, scored for the presence of protrusions and RhoGDI at protrusion sites. n = 3, with SEM.

Fig. 8.

Model proposed for Rac-localized activation at the leading edge upon growth-factor stimulation. Upon growth-factor stimulation, DGKα is activated in a Src-dependent manner and recruited to the plasma membrane. The production of PA is the crucial signal to direct the recruitment of aPKCζ/ι, in complex with RhoGDI and Rac. PKCζ/ι, in turn, mediates the dissociation of Rac from the inhibitory complex with RhoGDI, which may become prone to activation by a RacGEF.