To determine whether H+
efflux by NHE1 is necessary for activation of Cdc42, we used NHE1-deficient fibroblasts expressing wild-type (WT) NHE1 or a mutant NHE1 that contains an E266I substitution and lacks H+
efflux (Denker et al., 2000
). To biochemically determine Cdc42 activity in migrating cells, multiple wounds were created in a confluent monolayer with a multichannel pipette. The abundance of active Cdc42-GTP at the indicated times before and after wounding was determined by precipitation with GST–p21-activated kinase (PAK)–Cdc42/Rac-interactive binding domain (CRIB) and immunoblotting for Cdc42 (). After wounding, the abundance of Cdc42-GTP in WT cells increased twofold at 5 min and remained elevated at 5 h. In E266I cells, the abundance of Cdc42-GTP before wounding was 60% of that in WT cells, and after wounding there was no increase in Cdc42-GTP. The abundance of total Cdc42 was similar in WT and E266I cells before and after wounding.
Figure 1. H+ efflux by NHE1 is necessary for activation of Cdc42 by different extracellular cues. (A) Time course of Cdc42 activity in WT and E266I cells after multiple wounding of a confluent monolayer. At the indicated times, Cdc42-GTP was determined (more ...)
Monolayer wounding triggers multiple stimuli, including activation of integrins and release of growth factors. Integrin engagement with extracellular matrix proteins activates Cdc42 (Price et al., 1998
) and stimulates NHE1 activity (Schwartz et al., 1991
; Tominaga and Barber, 1998
). We found that H+
efflux by NHE1 is necessary for haptokinetic migration toward fibronectin (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200704169/DC1
) and for integrin-induced activation of Cdc42 (). Plating on fibronectin for 1 h increased Cdc42-GTP fourfold in WT cells compared with cells in suspension. In E226I cells, the abundance of Cdc42-GTP in cell suspension was less than in WT cells and there was no increase after plating on fibronectin. Integrin affinity for fibronectin, determined by binding FITC-labeled fibronectin, and expression of β1 integrin, determined by immunoprecipitating lysates of biotinylated cells with β1 antibodies, were similar in WT and E266I cells (Fig. S1).
PDGF also increases Cdc42-GTP (Jimenez et al., 2000
) and NHE1 activity (Yan et al., 2001
), and we found that H+
efflux by NHE1 is necessary for activation of Cdc42 by PDGF (). 50 ng/ml PDGF increased the abundance of Cdc42-GTP in WT cells, with a maximum of 2.2-fold increase at 2 min. In contrast, PDGF did not activate Cdc42 in E266I cells, although the abundance of total Cdc42 was similar in subconfluent WT and E266I cells. PDGF-induced activation of Cdc42 also was inhibited in NHE1-deficient PS120 cells, which were used to make WT and E266I cells, but not in parental CCL39 cells that express NHE1 (). Hence, H+
efflux by NHE1 is necessary for maintaining Cdc42 activity in quiescent cells and for increased Cdc42 activity with monolayer wounding, integrin engagement, and PDGF.
In migrating cells, Cdc42-GTP is predominantly localized at cell protrusions (Nalbant et al., 2004
). NHE1 is localized at the leading-edge membrane of migrating cells (Denker and Barber, 2002
; Patel and Barber, 2005
), which suggests that H+
efflux might be necessary for Cdc42 activity at cell protrusions. This was confirmed by using a merocyanine–Cdc42-binding domain (MeroCBD) biosensor for Cdc42-GTP (; Nalbant et al., 2004
). MeroCBD, an environmentally sensitive fluorescent dye covalently coupled to the Cdc42/Rac binding domain of the neural Wiskott-Aldrich syndrome protein, increases fluorescence intensity upon binding activated Cdc42, which enables detection of spatially localized endogenous Cdc42-GTP in living cells. EGFP attached to the Cdc42/Rac binding domain allows ratiometric image analysis, thereby normalizing for cell thickness and concentration artifacts. The MeroCBD probe, which is insensitive to pH 4.5–8.0, was injected into cells at the edge of a wounded monolayer 15 h after wounding, and images were acquired after 30 min. In WT cells, Cdc42-GTP was elevated in cell protrusions (n
= 22 cells), however, in E266I cells, Cdc42-GTP was more uniform and notably reduced even where cells protruded (n
= 52 cells; ). Acquired images were also used to quantify active Cdc42 in microinjected cells, and, like biochemical assays with GST-PAK-CRIB, they indicated attenuated Cdc42-GTP in E266I cells (). The ratio intensity was 344.4 ± 28.1 U in WT cells and 170.9 ± 7.0 U in E266I cells. The Mero/EGFP fluorescence ratio of cells injected with an insensitive control probe (MeroCBD mutated to greatly reduce Cdc42 binding; Nalbant et al., 2004
) was 196.9 ± 24.8 U (n
= 5 cells; unpublished data), indicating that activation in E2661 cells was near the minimum level detectable by the biosensor. Hence, biochemical and imaging analyses indicate that H+
efflux by NHE1 is necessary to maintain the abundance of Cdc42-GTP in quiescent and stimulated cells and to maintain the localization of Cdc42-GTP in migrating cells.
Figure 2. H+ efflux by NHE1 is necessary for active Cdc42-GTP at the front of migrating cells. (A) Probe used as a biosensor for endogenous Cdc42-GTP. MeroCBD is composed of the CRIB domain of neural Wiskott-Aldrich syndrome protein (blue), covalently labeled (more ...)
We previously reported that H+
efflux by NHE1 increases in fibroblasts expressing an active GTPase-deficient Cdc42-V12 and that H+
efflux by NHE1 stimulated by a constitutively active Gα13-QL is suppressed by coexpression of mutationally inactive Cdc42-N17 (Hooley et al., 1996
). In wound-edge WT cells, expression of Cdc42-N17 inhibited H+
efflux by NHE1, resulting in decreased intracellular pH (pHi
) of 7.10 ± 0.09 (n
= 43 cells), compared with pHi
of 7.30 ± 0.10 (n
= 30 cells)
in cells not expressing Cdc42-N17. In subconfluent WT cells, PDGF stimulated NHE1 activity and increased pHi
from 7.15 ± 0.03 to 7.47 ± 0.05, which was attenuated in cells expressing Cdc42-N17 to 6.99 ± 0.02 and 7.10 ± 0.03 (n
= 3 cell preparations).
Activation of Cdc42 requires release of inactive Cdc42-GDP from Rho GDP dissociation inhibitor (RhoGDI) in the cytosol, recruitment to the plasma membrane, and activation at the plasma membrane by a guanine nucleotide exchange factor (GEF) that catalyzes the exchange of GDP for GTP. Immunoprecipitating RhoGDI and immunoblotting for total Cdc42 indicated that PDGF induced a decrease in the abundance of coprecipitating Cdc42 that was similar in WT and E266I cells (), which suggests that NHE1 activity is not necessary for the regulated dissociation of Cdc42 from RhoGDI. Immunoblotting particulate fractions (P100) for Cdc42 showed that abundance in E266I cells was comparable to that in WT cells after PDGF stimulation (), which indicates that H+ efflux by NHE1 is not necessary for membrane recruitment of Cdc42.
Figure 3. H+ efflux by NHE1 is necessary for GEF activity but not for Cdc42 dissociation from RhoGDI or translocation to particulate fraction. (A) Lysates prepared from quiescent cells (t = 0) and cells treated with PDGF for the indicated times (more ...)
These findings suggest that H+
efflux by NHE1 might be necessary for GEF-induced guanine nucleotide exchange, which we confirmed by determining GEF activity in cell lysates (Fukuda et al., 2002
). Exchange of [32
P]GTP by Cdc42 for cold GTP was used as an index of GEF activity. The amount of Cdc42-[32
P]GTP decreased to 49.2% in lysates from WT cells treated with PDGF (). Lysates from PDGF-stimulated E266I cells did not show a marked exchange of [32
P]GTP for cold GTP compared with unstimulated cells (). These data suggest that GEF activity is impaired in E266I cells.
We asked whether GEF activity might be pH sensitive because in WT cells the quiescent pHi
of 7.15 increases to 7.45 with growth factors, but in E226I cells, the quiescent pHi
of 7.00 does not change (Denker et al., 2000
; Yan et al., 2001
). At least 20 members of the Dbl family of GEFs stimulate guanine nucleotide exchange by Cdc42, and we tested whether the activity of two GEFs for Cdc42, Dbs (Dbl's big sister) and intersectin, is pH dependent. Because Dbs contains a His residue (H814) in the α6 helix critical for interacting with switch two of Ccd42 and because it contacts a His residue in Cdc42 (H103 in the α3b region; Rossman et al., 2002
), we reasoned that pH-dependent titration of these histidines might regulate GEF activity or contact with Cdc42. Using the minimal Dbl homology (DH)–pleckstrin homology (PH) segment of Dbs necessary for activity and a GST-fusion of Cdc42 loaded with the fluorescent analogue methylanthraniloyl (mant)-GDP, which has reduced fluorescence when not bound to Cdc42 (Nomanbhoy and Cerione, 1996
), we found no change in activity from pH 6.5 to 8.0 for Dbs () or intersectin (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200704169/DC1
). Additionally, in the absence of Dbs, the release of mant-GDP from Cdc42 was similar from pH 6.5 to 8.0 (), indicating that guanine nucleotide exchange by Cdc42 was pH insensitive.
Figure 4. PI(4,5)P2 binding, but not guanine nucleotide exchange activity, for Cdc42 by the DH-PH domain of Dbs is pH sensitive. (A) Recombinant Cdc42-GST was loaded with mant-GDP and mixed with GTP alone (dashed lines) or with the recombinant DH-PH domain of Dbs (more ...)
Although in most Rho family GEFs the DH domain is sufficient to catalyze nucleotide exchange, a tandem PH domain that binds phosphoinositides is invariant. In cells, phosphoinositide binding by the PH domain can regulate activity of the DH domain of some GEFs (Rossman et al., 2005
). We asked whether phosphoinositide binding to Dbs or intersectin might be pH sensitive because phosphoinositides bind to positively charged residues in PH domains, which might titrate with changes in pH, and phosphates on phosphoinositides have pKa
s near neutral (van Paridon et al., 1986
). Additionally, FYVE domains, which share structural similarity with PH domains for binding phosphoinositides at loops between β strands, have pH-dependent affinity for phosphoinositides (Kutateladze, 2006
). By using liposome sedimentation, we found that the DH-PH domain of Dbs bound phosphotidylinositol 4,5–bisphosphate (PI(4,5)P2), as previously reported (Russo et al., 2001
; Snyder et al., 2001
), and that binding was pH dependent (). Maximal specific binding seen at pH 6.5 (52 ± 13%) was significantly reduced at pH 7.5 and 8.0 (P < 0.05; n
= 4), suggesting a lower affinity at higher pH. Although PI(4,5)P2 binding by the DH-PH domain of intersectin was maximal at pH 6.5 (42 ± 8%), binding was relatively insensitive to pH (). We speculate that pH-sensitive binding of PI(4,5)P2 to Dbs is caused by the presence of a His (H843) in the same position as H355 in the Arf GEF Grp1 that is critical for binding phosphoinositides (Lietzke et al., 2000
; Barriero, G., and M. Jacobson, personal communication). Computational modeling (unpublished data) indicates that a spatially conserved His in close proximity to predicted PI(4,5)P2–binding sites is present in other GEFs activating Cdc42, including Fgd1 (H985), αPix (H38), ASEF (H513; H505), and Dbl (H701; H756), but is absent in intersectin, Fgd3, Tiam1, and PDZRhoGEF (unpublished data; Barriero, G., and M. Jacobson, personal communication). Hence, whether Dbs or another predicted pH-sensitive GEF mediates NHE1-dependent activation of Cdc42 remains to be determined.
It also remains to be determined whether pH-dependent PI(4,5)P2 binding by GEFs contributes to NHE1-dependent activation of Cdc42. PI(4,5)P2 binding to Sos2, a Ras GEF, inhibits nucleotide exchange activity, possibly by retaining a cis inhibition of the DH domain by the adjacent PH domain (Jefferson et al., 1998
; Das et al., 2000
). The functional significance of PI(4,5)P2 binding to Rho family GEFs, however, is less clear. PI(4,5)P2 binding to recombinant DH-PH domains in vitro is reported to stimulate (Crompton et al., 2000
), inhibit (Han et al., 1998
; Russo et al., 2001
), or not affect (Fleming et al., 2000
; Snyder et al., 2001
) activity. Additionally, we cannot rule out other pH-dependent mechanisms, such as scaffolding or conformation changes independent of phosphoinositide binding, because attenuated GEF activity is retained in lysates of E266I cells () and for some proteins conformational changes, ligand-binding affinities, and macromolecular assemblies are sensitive to small changes in physiological pH (Srivastava et al., 2007
). A suggested pH-dependent scaffolding by NHE1 (Baumgartner et al., 2004
) is also a putative mechanism because NHE1 binds PI(4,5)P2 (Aharonovitz et al., 2000
) and the ezrin-radixin-moesin protein ezrin (Denker et al., 2000
), and ezrin is suggested to sequester Dbl to plasma membrane microdomains (Prag et al., 2007
) and to regulate Dbl activation of Cdc42 (Batchelor et al., 2007
). Moreover, H+
efflux by NHE1 could regulate an upstream activator of Cdc42-GEFs, although activity of Rap1B, which is necessary and sufficient to initiate polarity in neurons via activation of Cdc42 (Schwamborn and Puschel, 2004
), was not impaired in E266I cells compared with WT cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200704169/DC1
Our data indicate positive feedback signaling between Cdc42 and NHE1 activity that is likely critical for polarity in migrating cells by asymmetrically amplifying both signals at the leading edge. Our findings also suggest that RhoGDI dissociation and membrane recruitment of Cdc42 are distinct signaling events that can be regulated independently of guanine nucleotide exchange. Beyond our current focus on regulated Cdc42 activity our data raise the possibility that activity of other GTPases and GEFs, and the affinities of protein modules for binding phosphoinositides, might be pH sensitive and regulated by NHE1 activity.