(Received for publication, January 4, 1996)
From the
We report that expressing interfering mutants of the small
Ras-related GTPase Rac, using either recombinant vaccinia virus or
stable DNA transfection, eliminates epidermal growth factor-induced
Ca signaling, without affecting Ca
mobilization or influx from G protein-coupled receptors.
Platelet-derived growth factor-dependent Ca
influx,
however, is only partly sensitive to dominant negative Rac proteins.
Thus, whereas epidermal growth factor-induced Ca
influx is completely mediated by Rac proteins, platelet-derived
growth factor-induced Ca
influx involves
Rac-dependent and -independent signaling pathways.
Epidermal growth factor (EGF) ()and its receptor are
important regulators of cell growth and differentiation. Binding of EGF
to its receptor results in receptor dimerization and subsequent
activation of the EGF receptor tyrosine kinase, which mediates further
signal transduction(1, 2, 3, 4) .
Among the biochemical changes initiated by the EGF receptor is a
transient increase in
[Ca
]
(5) ,
which is essential for EGF-induced
proliferation(6, 7) . This Ca
transient has been linked to EGF-stimulated hydrolysis of
polyphosphoinositides by phospholipase C, as the resulting production
of inositol 1,4,5-trisphosphate causes release of Ca
from intracellular stores(8, 9) . Ca
release, however, constitutes only a minor part of the overall
EGF-induced elevation in
[Ca
]
, since the major
part originates from the activation of voltage-independent,
Ca
-specific ion channels, resulting in a
Ca
influx through the plasma
membrane(5, 10, 11, 12, 13) .
The molecular mechanism, responsible for the induction of this
Ca influx, is unclear. Several types of
Ca
-permeable channels are activated in response to
EGF, including a 10-pS Ca
-selective
conductance(12, 14) . Stimulation of the latter
channel requires the liberation of AA from cellular membranes and its
subsequent conversion to leukotrienes(15, 16) . The
importance of activation of the 10-pS Ca
channel,
with respect to the EGF-induced Ca
transient, is at
present unclear. Apart from EGF, other growth factors open plasma
membrane-localized calcium channels as well. PDGF, for instance, is a
potent inducer of Ca
influx in Rat-1
fibroblasts(17) . The underlying Ca
channels,
as well as the signaling mechanisms leading to their activation, have
remained unexplored.
A candidate molecule to function in the
signaling upstream of growth factor-induced Ca influx
is the Rac protein. Rac is a member of the Rho family of Ras-related
GTPases (for a review, see (18) ). Recently, it has been shown
that Rac has an essential function in the cellular transformation
induced by oncogenic Ras(19) , although the downstream elements
of the Rac signaling cascade leading to transformation are not clear.
Rac proteins are implicated in growth factor-induced cytoskeletal
remodeling. For instance, Rac mediates polymerization of actin at the
plasma membrane (so-called ruffling (20) ). Rac also activates
the Rho protein, an event necessary for growth factor-dependent stress
fiber formation(21) . Interestingly, apart from Rac, AA release
and subsequent leukotriene production are implicated in growth
factor-induced actin remodeling as well(22) , suggesting a
relationship between Rac and AA metabolism. Indeed, we recently
established that growth factor-induced AA release requires functional
Rac protein and that Rac-dependent Rho activation is dependent on the
metabolism of AA to leukotrienes(23) . This connection between
Rac and leukotriene production and the requirement for leukotriene
synthesis in the EGF-dependent activation of the 10-pS Ca
channel therefore raise the possibility that Ca
influx is one of the effectors of the Rac signaling cascade.
In the present study, we have investigated the possible relationship
between the Rac signaling and the growth factor-dependent
Ca transient. The results show that EGF-induced
Ca
signaling is mediated by Rac. PDGF-induced
Ca
influx, however, is only partly dependent on
functional Rac protein. These results identify Ca
influx as a Rac-specific effector and define this Rac-mediated
Ca
influx as the major pathway by which EGF activates
Ca
influx.
Figure 1:
Inhibition of the EGF-induced
Ca transient by Rac N17. Effects are shown of EGF and
histamine (HA) on the intracellular Ca
concentration in indo-2-AM-loaded HeLa cells (Hela), in
wild type vaccinia virus-infected HeLa cells (Hela vv-WT), and
in HeLa cells infected with a vaccinia virus expressing Rac N17 (Hela vv-Rac N17). Also the effects of EGF on ERK2
phosphorylation (as assayed by altered gel mobility; for experimental
procedures, see (17) ) in vv-wt-infected cells and vv-Rac
N17-infected HeLa cells are shown.
The specificity of the
inhibitory effect of Rac N17 on EGF-induced Ca signaling was demonstrated in experiments in which the effect of
vv-Rac N17 infection on the EGF-dependent phosphorylation of ERK2 was
analyzed. This phosphorylation of ERK2 is mediated by a complex signal
transduction cascade (reviewed in e.g.(28) ). vv-Rac
N17 infection did not inhibit EGF-dependent ERK2 phosphorylation in
comparison with uninfected or vv-wt-infected cells (Fig. 1). We
concluded that Rac proteins act specifically in the signal transduction
pathway leading to EGF-induced Ca
signaling.
Figure 2:
Effects of EGF and ATP on
[Ca]
in Rat-1
fibroblasts (Rat1), Rat-1 fibroblasts expressing an empty
transfection vector (Rat1 Vector Control), Rat-1 fibroblasts
expressing Rac N17 (Rat1 Rac N17), and Rat-1 fibroblasts
expressing Rac V12 (Rat1 Rac Val12). Also the effects of EGF
on ERK2 gel mobility in vector control, Rac N17-expressing, and Rac
V12-expressing Rat-1 fibroblasts are shown.
Figure 3:
PDGF-induced Ca transients in Rat-1 fibroblasts and Rat-1 fibroblasts expressing
Rac N17.
Figure 4: Effects of EGF and ATP in Rho V14-expressing Rat-1 fibroblasts.
In the present study, we have investigated a possible
relationship between the growth factor-induced Ca transient and the small GTPase Rac. We showed that expressing
interfering mutants of the Rac protein, using either a vaccinia virus
expression system or stable DNA transfection, abolished Ca
signaling in response to EGF but did not affect the
Ca
transients induced by histamine, endothelin, and
ATP. PDGF-induced Ca
influx, however, was only partly
inhibited by Rac N17 expression, showing that the PDGF receptor can
activate Ca
channels via Rac-dependent and
-independent signaling pathways. We concluded that Ca
influx is a Rac-specific effector mechanism and that this
Rac-dependent Ca
influx is the major signaling
pathway mediating the EGF-induced increase in
[Ca
]
.
Our results are the
first example for a role of a small GTPase in the control of
intracellular Ca levels. Ca
is an
important second messenger in the cell, and our finding that Rac
mediates growth factor-induced calcium influx stresses the importance
of this protein in cellular processes. In addition to calcium influx,
Rac mediates growth factor-induced release of AA (23) and is
implicated in the control of the JNK/SAPK pathway (31, 32) as well as in the stimulation of the
mammalian STE20 homologue PAK65(33) . In particular the finding
that Rac mediates AA metabolism is interesting, since in patch clamp
analysis we have shown that the opening of a 10-pS calcium channel by
EGF can be blocked by inhibitors of AA metabolism and can be partially
restored by leukotrienes(16) . Therefore, Rac-mediated AA
release and leukotriene production may account for the effects of Rac
N17 expression on Ca
signaling. Alternatively,
Rac-dependent Ca
influx may act upstream of AA
release, as activation of the cytosolic form of phospholipase A
is Ca
-dependent(23) .
Expression of
Rac N17 caused a 50-70% decrease in the PDGF-induced
Ca transient in Rat-1 fibroblasts. Therefore, a
function of Rac in the production of Ca
influx is not
limited to the EGF receptor. The PDGF receptor, however, can apparently
partly bypass the requirement for Rac for elevating
[Ca
]
, since expression of Rac
N17 was not able to completely inhibit this response. In agreement, in
cells expressing PDGF receptors lacking the kinase insert domain, PDGF
does not activate Rac (34) but still induces Ca
signaling. (
)Therefore, whereas the EGF-induced
increase in [Ca
]
is mostly
mediated by Rac, the PDGF receptor employs Rac N17-sensitive and
-insensitive signal transduction pathways to produce Ca
signaling. Interestingly, in cell-attached patch clamp
experiments, PDGF, but not EGF, activates a 6-pS
Ca
-permeable ion channel, (
)which may
mediate this Rac N17-insensitive Ca
influx.
The
functioning of Rac in signal transduction is most clear with respect to
the growth factor-induced cytoskeletal reorganization. Rac mediates the
formation of lamellipodia and membrane ruffles(21) .
Furthermore, as an upstream regulator of Rho, it also regulates stress
fiber formation(20) . The biochemical pathways, leading to
cytoskeletal reorganization, are not clear. Importantly, the activity
of many actin-controlling proteins is highly dependent on
[Ca]
. Increases in
[Ca
]
directly activate actin
filament-severing proteins(35) . Preventing Ca
signaling, by intracellular Ca
chelation with
quin-2, inhibits lamellipodia formation upon activation of human blood
platelets(36) . This suggests that at least some of the
cytoskeletal effects of Rac require an increase in
[Ca
]
. Therefore, Rac-mediated
Ca
influx may serve as one of the biochemical signals
by which Rac proteins effect cytoskeletal organization.