(Received for publication, October 19, 1995; and in revised form, January 4, 1996)
From the
Activity of the ubiquitously expressed
Na-H
exchanger subtype NHE1 is
stimulated upon activation of receptor tyrosine kinases and G
protein-coupled receptors. The intracellular signaling pathways
mediating receptor regulation of the exchanger, however, are poorly
understood. Using transient expression of dominant interfering and
constitutively active alleles in CCL39 fibroblasts, we determined that
the GTPases Ha-Ras and G
13 stimulate NHE1 through distinct
signaling cascades. Exchange activity stimulated by constitutively
active RasV12 occurs through a Raf1- and mitogen-activated protein
kinase kinase/extracellular signal-regulated kinase kinase
(MEK)-dependent mechanism. Constitutively active G
13QL, recently
shown to stimulate the Jun kinase cascade, activates NHE1 through a
Cdc42- and MEK kinase (MEKK1)-dependent mechanism that is independent
of Rac1. Constitutively active Rac1V12 does stimulate NHE1 through a
MEKK1-dependent mechanism, but dominant interfering Rac1N17 does not
inhibit G
13QL-mediated or constitutively active Cdc42V12-mediated
stimulation of the exchanger. Conversely, Cdc42N17 does not inhibit
Rac1V12 activation of NHE1, suggesting that Rac1 and Cdc42
independently regulate a MEKK1-dependent activation of the exchanger.
Rapid (<10 min) stimulation of NHE1 with a G
13/G
chimera also was inhibited by a kinase-inactive MEKK. G
13QL,
but not RasV12, also stimulates NHE1 through a RhoA-dependent pathway
that is independent of MEKK, and microinjection of mutationally active
G
13 results in a Rho phenotype of increased stress fiber
formation. These findings indicate a new target for Rho-like proteins:
the regulation of H
exchange and intracellular pH. Our
findings also suggest that a MEKK cascade diverges to regulate
effectors other than transcription factors.
The ubiquitously expressed Na-H
exchanger subtype NHE1 (
)plays a major role in
intracellular pH (pH
) homeostasis and in cell
volume regulation(1) . NHE1 activity is stimulated by hormones,
cytokines, and growth factors, resulting in an increase in
pH
. Hyperosmotic shock (2) and cell
adhesion (3) also activate NHE1. Increases in NHE1 activity are
associated with increased cell proliferation(4, 5) ,
differentiation(6, 7) , and neoplastic
transformation(8, 9, 10) . Receptor (11, 12) , but not osmotic(2) , activation of
NHE1 is associated with increased phosphorylation of the exchanger on
serine residues, suggesting kinase-dependent regulatory mechanisms.
Although activation of protein kinase C stimulates NHE1, growth factors
and vasoactive agents can stimulate the exchanger independently of this
kinase. Mutational activation of three GTPases,
Ha-Ras(8, 9) ,
G
(13, 14, 15) , and
G
13(13, 14, 15) , stimulates NHE1
activity. Of these GTPases, only G
activates the
exchanger through a protein kinase C-dependent mechanism (14) . (
)The downstream signaling events mediating Ha-Ras and
G
13 stimulation of NHE1 have not been identified. Ha-Ras and
G
13 regulate two parallel MAP kinase signaling cascades, and these
cascades include serine/threonine protein kinases that could
potentially modulate exchange activity.
Stimulation of one of these
cascades, the extracellular signal-regulated kinase (ERK) cascade, by
activated growth factor receptor tyrosine kinases is mediated by the
GTPase Ras. GTP-bound Ras recruits an immediate downstream effector,
Raf1, to the plasma membrane, where it is activated by an unidentified
mechanism(16, 17) . The serine/threonine kinase Raf1
activates the MAP kinase kinases MEK1 and MEK2(18) . These
dual-specificity kinases phosphorylate and activate the MAP kinases
ERK1 and ERK2(19) . Activated ERKs regulate a wide range of
cytosolic and nuclear proteins involved in cell proliferation and
neoplastic transformation, including phospholipase A,
p90
, c-Myc, and c-Fos(19, 20) .
Heterotrimeric G proteins, both
(21, 22, 23) and
(23, 24, 25) subunits, also
regulate the ERK cascade, although their action on ERK is more cell
type-specific than that of Ras. For example, mutational activation of
the ubiquitously expressed G
13 subunit stimulates growth and
neoplastic transformation in Rat1 (22) and NIH3T3(26, 27) fibroblasts; however, it enhances epidermal growth
factor-stimulated ERK activity in the former cell type(22) ,
but does not affect this kinase in the
latter(26, 27) . In already transformed COS-7 cells,
mutationally activated G
13 has been found both to inhibit (
)and to have no effect (24, 28) on ERK
activity. In contrast to its cell-specific effects on the ERK cascade,
G
13 consistently stimulates NHE1 activity in a wide range of cell
types, suggesting a divergence in its actions on ERK and the exchanger.
Recently, a parallel MAP kinase cascade, the Jun kinase (JNK) or
stress-activated protein kinase cascade, has been
described(18, 29, 30) . Identified substrates
of JNK/stress-activated protein kinase are the transcription factors
c-Jun and AFT2(31) . The JNK cascade is activated by epidermal
growth factor (32) and UV irradiation (33) through a
Ras-dependent mechanism and by cytokines in a Ras-independent
manner(30, 34) . Recently, G13 was found to
activate the JNK cascade through a mechanism that is interrupted by the
dominant interfering allele RasN17(28) . Extracellular signals
activating JNK are mediated through activation of MEK kinase (MEKK1), a
mammalian homolog of the STE11 kinase involved in the yeast pheromone
mating pathway(35) . MEKK1 directly activates the
stress-activated protein-kinase kinase SEK1 (JNK kinase), a direct
upstream regulator of JNK(36, 37) . Upstream, although
probably not direct regulators of MEKK1, are two members of the Rho
subfamily of GTPases, Rac1 and Cdc42 (38, 39) . These
GTPases are also involved in the organization of the actin
cytoskeleton. Rac1 regulates membrane ruffling and lamellipodia (40, 41) , and Cdc42 controls the formation of
filopodia(41, 42) . Although morphological studies
suggest that Cdc42 acts upstream of Rac1 and
Rac2(41, 42) , their sequential regulation of JNK
remains unclear. Coexpression of a dominant negative Rac1N17 with
constitutively activated Cdc42V12 does not inhibit(38) , or
only partially inhibits (39) , JNK activation, indicating that
these GTPases may independently regulate the JNK cascade.
Ha-Ras and
G13 are highly oncogenic, and their regulation of the ERK and JNK
cascades may be critical for their transforming actions. The ability of
these GTPases to constitutively stimulate NHE1 activity and the
correlation of increased exchange activity with transformed phenotypes
indicate the importance of determining the mechanisms mediating GTPase
regulation of NHE1. In this study, we used dominant interfering alleles
of kinases and GTPases to determine the mechanisms by which Ha-Ras and
G
13 activate NHE1. Although Ha-Ras and G
13 regulate both the
ERK and JNK cascades, our results indicate that Ha-Ras stimulates the
exchanger through a Raf1- and MEK1-dependent mechanism that is
independent of MEKK1. In contrast, G
13 activates NHE1 through a
MEKK1-dependent mechanism that requires Cdc42, but not Rac1. G
13,
but not Ras, also stimulates exchange activity through a Rho-dependent
mechanism that is independent of MEKK1.
Figure 1:
Distinct kinases mediate RasV12 and
13QL stimulation of NHE1. The rate of pH
recovery (dpH
/dt
10
pH/s) from an acute acid load was determined in
CCL39 cells transiently expressing empty vector (pcDNA) or the
indicated kinases and GTPases. Data are expressed as the recovery rate
at pH
6.75. A, Raf1 regulation of NHE1
activity was determined by measuring recovery rates in cells expressing
pcDNA or constitutively active v-Raf, RasV12, and
13QL alone (control) or coexpressed with a dominant interfering Raf1 (+ Naf). Recovery rates also were determined in cells
coexpressing RasV12, Naf, and wild-type Raf (+ Naf + wtRaf). B, MEK-regulated NHE1 activity was determined in
cells expressing pcDNA, RasV12, and
13QL in the absence (control) or presence (+ MEK-Km) of a dominant
interfering MEK. C, the role of MEKK1 in stimulating NHE1 was
determined in cells expressing pcDNA or constitutively active
MEKK1
, RasV12, and
13QL alone (control) or
coexpressed with a dominant interfering MEKK1 (+ MEKK-Km). Data represent the mean ± S.E. of recovery rates
in four to five separate transfections.
Although Raf1 may act on several substrates, the MAP kinase kinase
proteins MEK1 and MEK2 are its preferred targets(18) . We used
a catalytically inactive MEK-Km allele (35) to investigate
MEK-dependent activation of NHE1. Coexpression of MEK-Km completely
inhibited RasV12 activation of NHE1 (Fig. 1B) and
lowered RasV12-induced resting pH from 7.13 ± 0.02
to 6.97 ± 0.03 (n = 4). Ras therefore activates
NHE1 by the same pathway as it activates ERK. MEK-Km, however, had no
effect on
13QL-induced NHE1 activity (Fig. 1B) or
on resting pH
(data not shown).
Ras (29, 30) and G13 (28) also activate the
JNK cascade, which suggests that another signaling pathway may mediate
the effect of these GTPases on NHE1. The lack of suitable dominant
interfering or constitutively active alleles of JNK and its upstream
regulator JNK kinase limited our study to the role of MEKK1, an
upstream regulator of JNK(36, 37) , in stimulating the
exchanger. Expression of a constitutively active carboxyl-terminal
truncated MEKK1 (MEKK1
) increased NHE1 activity (Fig. 1C) and increased the resting pH
from
7.00 ± 0.02 in vector controls to 7.16 ± 0.04 (n = 3). Coexpression of a kinase-inactive MEKK1-Km with
RasV12 had no effect on Ras-stimulated exchange activity (Fig. 1C) or on Ras-induced increases in pH
(7.12 ± 0.02 in the absence and 7.11 ± 0.01 in the
presence of MEKK1-Km; n = 4). Coexpression of MEKK1-Km
with
13QL, however, completely inhibited G13-stimulated exchange
activity (Fig. 1C) and reduced the G13-induced resting
pH
from 7.12 ± 0.02 to 7.00 ± 0.03 (n = 5). G
13, but not Ras, therefore uses a
MEKK1-dependent pathway to stimulate NHE1.
Figure 2:
Rho family proteins stimulate NHE1
activity. Rates of pH recovery from an acid load
were determined at pH
6.75 in CCL39 cells
transiently expressing pcDNA or constitutively active Rac1V12,
Cdc42V12, and RhoAV14 alone (control) or coexpressed with
either dominant interfering MEK1 (+ MEK-Km) or dominant
interfering MEKK1 (+ MEKK-Km). Data represent the mean
± S.E. of recovery rates in three separate
transfections.
Figure 3:
13QL stimulation of NHE1 activity is
inhibited by Cdc42N17 and RhoAN19, but not Rac1N17. The rates of
pH
recovery at the indicated pH
values were determined in CCL39 cells transiently expressing
empty vector (pcDNA) or
13QL or coexpressing
13QL and the
indicated dominant interfering Rho family proteins. Data represent the
mean ± S.E. of recovery rates in four to six separate
transfections. A, coexpression of Rac1N17 had no effect on
13QL stimulation of NHE1 activity. B, coexpression of
Cdc42N17 blocked
13QL-stimulated exchange. C,
coexpression of RhoAN19 also blocked
13QL-stimulated exchange. D, expression of Myc-tagged Rac1N17 and Cdc42N17 was
determined by immunoblotting with an anti-Myc
antibody.
Figure 4:
Rho family proteins have independent
effects on NHE1 activity. Rates of pH recovery at
pH
6.75 were determined in CCL39 cells transiently
expressing pcDNA or the indicated constitutively active Rho family
proteins in the absence or presence of the indicated dominant
interfering Rho family proteins. Data represent the mean ± S.E.
of recovery rates in three separate
transfections.
Members of the Rho family have distinct effects on
the actin cytoskeleton. Rac1 regulates membrane ruffling and
lamellipodia(40, 41) ; Cdc42 controls the formation of
filopodia(41, 42) ; and RhoA induces stress fiber
formation(40) . We next examined the effect of 13QL on the
actin cytoskeleton by plasmid microinjection followed by
immunofluorescence microscopy. CCL39 cells expressing
13QL were
contracted and showed a marked increase in stress fiber formation (Fig. 5, A and B), which was very similar to
the phenotype induced by expression of RhoAV14 (Fig. 5, C and D). In contrast, expression of Rac1V12 caused cell
spreading and stimulated lamellipodia formation (Fig. 5, E and F), whereas Cdc42V12-expressing cells showed enhanced
formation of filopodia and, to a much lesser extent, of lamellipodia as
well (Fig. 5, G and H). These results suggest
that of the Rho family members, RhoA may be the primary target of
G
13 activation.
Figure 5:
Expression of 13QL causes enhanced
stress fiber formation. Shown are fluorescence micrographs of CCL39
cells expressing
13QL (A and B), RhoAV14 (C and D), Rac1V12 (E and F), and Cdc42V12 (G and H). A, C, E, and G, fluorescein isothiocyanate-labeled phalloidin staining; B, protein A staining visualized by indirect
immunofluorescence using anti-Myc antibody to mark injected cells; D, F, and H, Myc staining visualizing
expression of the respective epitope-tagged GTPases. Rac1-induced
lamellipodia are indicated by thick arrows in F.
Cdc42-induced filopodia are indicated by thin arrows in H. Bar = 10
µm.
Figure 6:
Acute activation of NHE1 by an
13/
chimera is inhibited by dominant interfering
MEKK-Km. A, rates of pH
recovery from an
acid load were determined at pH
6.75 in CCL39
cells transiently expressing pcDNA or the D
R or
coexpressing the D
R and
13/
chimeric
protein. Recovery rates were determined in the absence (
) or
presence (
) of the D
R agonist quinpirole (100
nM). B, shown is the time course of pH
recovery from an NH
Cl prepulse in CCL39 cells
expressing the D
R and
13/
. Recoveries
were determined in HEPES buffer in the absence (left trace)
and presence (right trace) of quinpirole. C, acute
activation of NHE1 by
13/
was inhibited by
coexpression of MEKK-Km, but not MEK-Km.
The ubiquitously expressed Na-H
exchanger NHE1 is one of several ion exchangers involved in
cytoplasmic pH homeostasis. Activation of NHE1 increases the rate of
H
efflux from the cell, resulting in a rise in
pH
. Although the kinetics of NHE1 activation by growth
factors, hormones, and cytokines has been extensively
studied(1) , post-receptor signaling mechanisms regulating the
exchanger remain largely unknown. This study focuses on the signaling
pathways mediating activation of NHE1 by Ha-Ras and G
13. Although
these GTPases regulate similar kinase cascades, our findings indicate
they use distinct mechanisms to stimulate NHE1 activity (Fig. 7). It is not surprising that Raf1 and MEK, which act
downstream of Ras, mediate RasV12 activation of the exchanger. ERK, a
selective substrate for MEK(19) , is also a likely component in
Ras activation of NHE1, although there is currently no direct evidence
for an ERK-dependent regulation of the exchanger. G
13 also
regulates ERK(22) ; however, it stimulated NHE1 activity
independently of the Ras/Raf pathway. It is unknown how G
13
regulates ERK; if the G
13 signal acts directly on ERK, then the
dominant interfering alleles of Raf1 and MEK used in this study would
not block an ERK-mediated activation of NHE1.
Figure 7:
Distinct signaling pathways mediate Ha-Ras
and G13 stimulation of NHE1 activity. Ha-Ras stimulation of NHE1
occurs through activation of Raf1 and MEK, but independently of the Rho
family of GTPases and MEKK. Although the Rho family of GTPases (Rac1,
Cdc42, and RhoA) stimulate NHE1, their upstream regulation and coupling
to the exchanger are distinct. Rac1 and Cdc42 activate NHE1 through a
MEKK1-dependent mechanism, whereas RhoA couples to the exchanger
independently of MEKK1. G
13 activation of NHE1 is mediated by
Cdc42 and RhoA, but not by Rac1. The downstream signaling pathways used
by MEK, MEKK1, and Rho to stimulate NHE1 are
undetermined.
Our findings identify
NHE1 as a previously undescribed effector of a MEKK1 signaling pathway.
A role for MEKK1 in activating the exchanger was determined by several
experiments. First, a constitutively activated amino-terminal truncated
MEKK1 increased NHE1 activity and pH. Second, a
kinase-inactive MEKK1-Km inhibited NHE1 activation by constitutively
activated Rac1V12, Cdc42V12, and
13QL. This inhibition was
specific, as MEKK1-Km had no effect on NHE1 activity stimulated by
RasV12 or RhoAV14. Third, acute stimulation of the exchanger by
D
R activation of an
13/
chimeric
protein was also inhibited by MEKK1-Km. This latter finding argues
against a role for transcriptional regulation in a G
13-MEKK1-NHE1
pathway, suggesting a divergence in the actions of MEKK1 on nuclear
factors and NHE1, an integral plasma membrane protein. JNK, a
downstream effector of MEKK(37) , is stimulated by Ras through
a Rac-dependent mechanism(38, 39) . Ras activation of
NHE1, however, occurs independently of Rac and MEKK1. A likely
explanation for this is that perhaps a critical threshold of MEKK1
activation must be attained to signal downstream to the exchanger. Ras
stimulates only a modest increase in JNK activity (29, 38, 39) that is much less potent than
its activation of ERK. Additionally, we consistently find that JNK
activation by
13QL is 3-4 times greater than that by RasV12. (
)The distinct mechanisms whereby Ras and G
13 stimulate
NHE1 are not consistent with recent findings that G
13 stimulates
JNK through a Ras-dependent mechanism(28) . A Ras-mediated
activation of JNK by G
13, however, could function in regulating
effectors other than NHE1.
Our findings also identify NHE1 as a
previously undescribed downstream effector of the Rho family of
GTPases. Although three members of this family, Rac1, Cdc42, and RhoA,
stimulated NHE1 activity, their upstream regulation and their coupling
to the exchanger were distinct. Rac1 and Cdc42, recently shown to
stimulate JNK activity(38, 39, 52) ,
activated NHE1 through a MEKK1-dependent mechanism, whereas RhoA
coupled to the exchanger independently of MEKK1. G13 activation of
NHE1 was mediated by Cdc42 and Rho, but not by Rac, indicating that
G
13 does not regulate Rac. Dbl, a tissue-specific proto-oncogene,
functions as a guanine nucleotide exchange factor for Cdc42 and RhoA,
but not Rac1(53) . Although speculative, our findings suggest
that perhaps a Dbl homolog expressed in CCL39 cells may act downstream
of G
13. If Cdc42- and Rho-mediated pathways are divergently
regulated by G
13, it is uncertain why a dominant interfering
allele of either monomeric GTPase completely blocked
13QL
activation of NHE1. One possibility is that G
13 activation of the
exchanger requires a cooperative action of Cdc42 and RhoA. Another
explanation is that Cdc42N17 or RhoAN19 could competitively bind to a
guanine nucleotide exchange factor common to Cdc42 and RhoA and thus
indirectly inhibit activation of endogenous proteins.
Our
morphological studies suggest that RhoA is the primary target of
G13, as expression of constitutively activated
13QL increased
stress fiber formation, similar to a RhoAV14-induced phenotype. How
acute activation of G
13 regulates the actin cytoskeleton is
currently unknown, and we cannot exclude the possibility that transient
activation of G
13 might also induce a Cdc42 phenotype. Concertina, a Drosophila homolog of G
13, also
functions in cytoskeletal reorganization(58) . Concertina is critical for early gastrulation, as it coordinates cell shape
changes during ventral furrow formation. Hence, regulation of the actin
cytoskeleton may be a phylogenetically conserved function of G
13.
It is currently unknown whether the Rho-like GTPases divergently
regulate NHE1 and the actin cytoskeleton or whether these effectors lie
within a single signaling pathway. If the latter is true, then a
critical question is whether activation of NHE1 occurs upstream or
downstream of cytoskeletal reorganization. Cytoplasmic pH may be a
potent modulator of cytoarchitecture, as the bundling and cross-linking
of actin filaments are pH-dependent processes(54) . In Dictyostelium, changes in pH over the
physiological range seen in our study (6.9-7.2) produce dramatic
reorganization of the actin cytoskeleton(54) . Additionally,
pH
-influenced cytoskeletal remodeling has been suggested to
modulate cell motility (55) , differentiation(56) , and
protein synthesis(57) . NHE1 regulation could also occur
downstream of cytoskeletal reorganization by the Rho-like GTPases, as
osmotically induced changes in cell shape regulate
Na
-H
exchange(2) .
At this
point, it is not clear how MEK-, MEKK-, and Rho-mediated pathways
couple to NHE1. Ultimately, these pathways must converge at the
exchanger or at an upstream regulator of the exchanger. Using
interaction cloning, we recently identified a novel protein, NIP1, that
coprecipitates with NHE1 in vivo and regulates its activity. ()Overexpression of NIP1 interferes with Ras and G
13
activation of the exchanger, suggesting that the signaling pathways
identified in this study may converge at NIP1 to regulate NHE1. The
functional consequence of increased NHE1 activity remains unresolved.
Constitutive activation of the GTPases and kinase cascades we studied
induces neoplastic transformation, and transformed cells have a higher
exchange activity and resting pH
than nontransformed
cells(8, 9, 10) . The Rho and JNK pathways
may also participate in cell volume regulation, and NHE1 is activated
by osmotic challenge(2) . Whether activation of NHE1 plays
either an obligatory or a permissive role in the cellular actions
regulated by these three distinct signaling pathways remains to be
determined.
Note Added in Proof-Buhl et al. recently reported that 13QL stimulates a Rho-dependent
increase in stress fiber formation (Buhl, A. M., Johnson, N. L.,
Dhanasekaran, N., and Johnson, G.(1995) J. Biol. Chem.270, 24631-24634).