(Received for publication, August 25, 1995)
From the
A paradigm has been established whereby mutant tyrosine kinase
receptors such as the v-erbB and v-fms gene products
function as oncoproteins in the absence of ligand. A spontaneously
occurring deletional mutant of the human epidermal growth factor
receptor (EGFRvIII) has been isolated from astrocytic neoplasms and
transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is
constitutively complexed with the majority of cellular GRB2, suggesting
a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D.
Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong,
and A. J. Wong, submitted for publication). In this report, we document
that expression of EGFRvIII in fibroblasts is associated with
downstream activation of mitogen-activated protein (MAP)
kinase/extracellular signal-regulated kinase (MEK) and modest
activation of p42 and p44 MAP kinases. The presence of EGFRvIII
suppresses activation of p42 and p44 MAP kinases by phorbol
12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA
is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase
activity in EGFRvIII-transfected cells is augmented by the tyrosine
phosphatase inhibitor sodium vanadate. EGFRvIII is capable of
transducing downstream signals through MAP kinase as evidenced by
activation of cytoplasmic phospholipase A at levels similar
to that induced by intact EGFR. Our results suggest that EGFRvIII
constitutively activates downstream signal transduction through MAP
kinase, and this chronic stimulation of the MAP kinase pathway may
represent one means by which mutant EGFR transduces an oncogenic
signal.
The epidermal growth factor receptor (EGFR) ()transduces signals for proliferation and differentiation
in neuronal and epithelial tissues. EGFR signal transduction is
initiated by ligand binding, receptor dimerization, and inter- and
intrareceptor activation of kinase activity, with concomitant
phosphorylation of tyrosine residues(1) . Assembly of SH2
containing signal transduction molecules ensues at the receptor with
activation of downstream signaling pathways, several of which appear to
converge at the MAPK/extracellular signal-related kinase kinase (MEK)
with subsequent activation of the p42/p44 MAP kinases, a family of dual
specificity serine/threonine kinases(2) . MAP kinases
phosphorylate a number of cytoplasmic substrates including cytoplasmic
phospholipase A
(cPLA
) as well as the nuclear
ternary complex factor Elk-1 and
Myc(3, 4, 29) .
Overexpression of intact
EGFR in the presence of ligand or truncation of receptor by removal of
the extracellular domain induces transformation in fibroblast
systems(5, 6) . The overexpression of intact EGFR has
been documented in a variety of neuronal and epithelial neoplasms and
carries a poor prognosis for patients with these malignancies,
suggesting that overexpression of receptor may confer a proliferative
advantage in the presence of limiting amounts of growth
factor(7) . The retroviral oncogene v-erbB encodes a
form of the EGFR lacking the majority of the extracellular domain and
containing mutations in the transmembrane region and carboxyl terminus.
Mutational analysis has demonstrated that deletion of the extracellular
domain causes downstream activation of MAP kinase and contributes
significantly to transformation by
v-erbB(6, 8) . In human tumors, various
deletions of the extracellular domain of the EGFR have been identified
in astrocytic
neoplasms(9, 10, 11, 12) . The most
common of these alterations, termed the EGFRvIII, contains a deletion
of nucleotides 275-1075 of the EGFR cDNA(9) . The mutant gene
product is a 145-kDa receptor that undergoes spontaneous
homodimerization in transfected NIH3T3 cells and demonstrates increased
receptor autophosphorylation. ()Expression of this mutant
receptor in NIH3T3 cells is transforming, and a large proportion of
cellular GRB2 associates with the EGFRvIII.
This suggests a
link with the Ras-MAP kinase pathway, which we have explored to
delineate novel aspects of EGFRvIII signaling.
In this study, we
show that in NIH3T3 cells the EGFRvIII stimulates strong downstream
activation of MEK, but the degree of p42 and p44 MAP kinase activation
is more modest, recapitulating chronic EGF exposure in cells containing
full-length human EGFR. Down-regulation of protein kinase C by phorbol
esters does not abrogate constitutive activation by mutant receptor.
The chronic low level activation of MAP kinase suppresses the ability
of other agonists to induce kinase activity. Suppression of MAP kinase
response occurs without suppression of MEK, suggesting that aberrant
regulation occurs at the level of MAP kinase. Vanadate treatment
up-regulates MAP kinase and restores responsiveness to PMA, implicating
a tyrosine phosphatase as the regulatory element stimulated by
EGFRvIII. EGFRvIII is able to stimulate signaling downstream from MAP
kinase, activating constitutive cPLA activity as reflected
by increased arachidonate release. EGFRvIII may utilize this pathway to
transduce a transforming signal in vitro and in vivo.
Unless otherwise indicated, all experiments were performed at least three times, and representative data are presented.
Figure 1:
Effect of EGF on MAP kinase activity in
CO12 cells. Serum-starved CO12 fibroblasts were treated with 25 ng/ml
EGF for indicated time periods. Cells were then lysed as described
under ``Experimental Procedures,'' and in-gel kinase assays
were performed using MBP incorporated into the gel. Activation (-fold)
relative to basal activation in unstimulated CO12 cells is indicated.
The autoradiogram reflects P-labeled MBP immediately
adjacent to kinases and was obtained after exposure of the gel to film
for 3 days.
Figure 2: MAP kinase activity in HC2 cells. In-gel kinase activity in CO12 and HC2 cells was analyzed as per Fig. 1. Autoradiogram was exposed for 3 days.
Figure 3: MAP kinase response to EGF, PMA, and FCS. Serum-starved HC2 and CO12 fibroblasts were treated with 25 ng/ml EGF for 15 min, and PMA (100 nM) or FCS (20%) was exposed for 15 min. Cells were then lysed, and in-gel kinase assay was performed as described in Fig. 1.
The inability to stimulate MAP kinase activity in HC2 cells with agonists could be due to diminished MAP kinase expression in HC2 cells. However, immunoblots of whole cell lysates from both cell lines demonstrated essentially equivalent amounts of p44 in both cell lines (Fig. 4). Recently, specific negative regulators of p42 and p44 in the form of dual specificity tyrosine/threonine phosphatases have been described, including CL100 in human fibroblasts and its murine homologue MKP-1, as well as PAC-1 in lymphocytes(18, 19, 20) . These phosphatases are immediate early gene products whose synthesis is induced by activators of MAP kinases and are believed to mediate the subsequent attenuation of kinase activity. Each appears to be vanadate sensitive and okadaic acid insensitive.
Figure 4: p44 MAP kinase content of CO12 and HC2 cells. Whole cell lysates of cells were prepared, analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using anti-p44 MAP kinase antibody.
We evaluated the possibility that vanadate exposure may restore the ability to activate MAP kinases in cells transformed by mutant receptor. Both cell lines were exposed to 4 mM vanadate for 1 h followed by stimulation with serum and PMA. As shown in Fig. 5, A and B, vanadate increased basal activity in cells transfected with mutant receptor but not in CO12 cells. Subsequent exposure to PMA further activated MAP kinases, demonstrating that vanadate restored the responsiveness of p42 and p44 in cells containing constitutively activated MAP kinase. Okadaic acid alone or in combination with PMA had no effect on MAP kinase activity (data not shown).
Figure 5:
A, vanadate (VAN) restores MAP
kinase response to PMA. serum-starved HC2 cells were treated with
vanadate (4 mM 1 h) alone or followed by PMA (100
nM
15 min). In gel kinase assays were performed as
described in Fig. 1. B, vanadate effect on MAP kinase
in CO12 cells. Serum-starved CO12 cells were treated with vanadate (4
mM
1 h) alone or followed by PMA (100 nM
15 min). In-gel kinase assays were performed as described
in Fig. 1.
We analyzed MEK response to PMA and vanadate using a kinase-inactive MAP kinase substrate as described under ``Experimental Procedures.'' In a representative experiment, exposure of CO12 cells to 25 ng/ml EGF and 100 nM PMA for 15 min induced an 11-fold and 8-fold increase in MEK activity, respectively (Fig. 6). Basal MEK activity in HC2 cells was 4-fold higher than that of basal CO12 activity. PMA exposure doubled MEK activity in HC2, and the presence of vanadate and okadaic acid did not amplify the response to PMA. Therefore, the presence of mutant EGFR increases basal MEK activity approximately 4-fold but does not suppress PMA stimulation of MEK. Vanadate did not modulate the level of MEK activation. These data suggest that EGFRvIII transduces a chronic low level signal through MEK and MAP kinase and also induces a tyrosine phosphatase that acts at the level of MAP kinase.
Figure 6:
MEK activation in CO12 and HC2 cells. Cell
lysates from CO12 and HC2 cells were prepared as described under
``Experimental Procedures'' after exposure to 25 ng/ml EGF,
vanadate (VAN, 4 mM 1 h), okadaic acid (OA, 200 nM
1 h), or both, followed by 100
nM PMA for 15 min. Enzymatic activity of MEK was assayed as
described under ``Experimental Procedures.'' Positions of
molecular weight markers and MEK substrate are
indicated.
Since the discovery that the the oncogenic viral gene
v-erbB encoded a truncated and transforming homologue of the
EGFR, investigators have sought to define a role for the EGFR in human
malignancies. Several mutant forms of the EGFR have been discovered in
astrocytic neoplasms that contain either amino- and/or
carboxyl-terminal deletions. The EGFRvIII analyzed in this study
encodes a receptor that does not detectably bind EGF, and the deletion
of the EGF binding domain results in spontaneous homodimerization and
receptor autophosphorylation. Cells expressing normal EGFR
or EGFRvIII have similar MEK activity, but the EGFRvIII results in p42
and p44 MAP kinase activation similar to that seen with chronic EGF
exposure. The inability to further stimulate the p42/p44 MAP kinases in
cells expressing EGFRvIII is related to induction of a tyrosine
phosphatase activity as evidenced by the increase in MAP kinase
activity in the presence of vanadate and the restoration of
responsiveness to phorbol esters. Because MEK activation by EGF and PMA
in cells containing intact receptor and MEK activation by PMA in cells
containing mutant receptor appears to be relatively equivalent, the
majority of signal suppression occurs at the level of MAP kinase.
Other investigators have demonstrated that suppression of MEK and MAP kinase responsiveness, apparently by phosphatases, is dependent on the cell line being studied and the agonist used. Samuels et al.(14) utilized an estradiol-inducible Raf mutant to show that in rat 1a cells activation of MEK and MAP kinase by the mutant raf was restored by treatment with the phosphatase inhibitors okadaic acid and vanadate. However, in C7 3T3 cells, there was no evidence for constitutive suppressors of MEK and MAP kinase. In another study utilizing the same estradiol-dependent Raf protein, these authors showed that constitutive activation of Raf inhibited platelet-derived growth factor activation of MAP kinase, which could be restored by pretreatment with vanadate. No evidence for suppression of MEK was found, suggesting inhibition at the level of MAP kinase(24) . However, they found no inhibition of MAP kinase activation by serum or PMA. In this study, we also found no constitutive suppression of MEK and MAP kinases in 3T3 cells transfected with intact EGFR, but the presence of EGFRvIII induced suppression primarily at the level of p42 and p44 MAP kinase. In our hands, serum and PMA signaling through MAP kinase was also abrogated, consistent with a desensitization of MAP kinase to all agonists. We are currently exploring the identity of the MAP kinase phosphatase in these cells.
Because the degree of MAP kinase activation by EGFRvIII is comparatively low, the biological relevance of this finding is of some concern. Both amplitude and duration of activity have been postulated to be important in determining the biological phenotype, which results from MAP kinase activation(25) . Transient MAP kinase activation mediates a proliferative response while sustained, low level activation transduces a differentiative response in neuronal PC12 cells(26, 27) . It is possible that prolonged, relatively low amplitude MAP kinase activation, such as that mediated by Ras, may be an important determinant of transforming potential(28) .
The subcellular localization of activated
MAP kinase and activation of nuclear or cytoplasmic substrates may also
modulate the phenotypic response. A significant proportion of MAP
kinase undergoes nuclear translocation upon activation, and all of the
MAP kinase-specific phosphatases defined to date are
nuclear(18, 19) . EGFRvIII activates cPLA to a level similar to that induced by activated full-length EGFR.
The mechanistic basis for this is not readily apparent; however the
MKP-1/CL100 MAP kinase phosphatases are primarily nuclear and would not
have a significant effect on activated cytoplasmic MAP kinase. If
MKP-1/CL100 is the primary suppressor of MAP kinase activity stimulated
by mEGFR, then activation of cytoplasmic substrates would be expected
to be equivalent. This would suggest that MAP kinase, which is
activated but remains cytosolic, is fully capable of activating
cytoplasmic substrates. It also suggests that activation of cPLA
may play a role in the transforming signal initiated by EGFRvIII.
In summary, our data demonstrate that a transforming mutant of human EGFR activates signaling through the MAP kinase pathway, recapitulating the changes seen with chronic stimulation of intact EGFR. Partial suppression of MAP kinase activity by a vanadate-sensitive phosphatase does not inhibit the signal for transformation, although it is not clear that this signal is necessarily mediated through the MAP kinase pathway. Continued investigation of the mechanisms behind signal suppression at each level of the Ras-MAP kinase cascade should provide new insights into how oncogenes that utilize the same signaling pathways transduce different biological phenotypes. Further study of the signaling pathways activated by mutant EGFR may reveal novel means of inhibiting the oncogenic signal either through direct interference with receptor function or by suppressing components of activated signaling pathways.