(Received for publication, November 7, 1995)
From the
Interaction of hepatocyte growth factor with its high affinity
receptor c-met initiates a cascade of intracellular events
leading to epithelial motility. An 11-amino acid sequence from the
c-met receptor has been found to cause cell transformation in
transfected fibroblasts (Ponzetto, C., Bardelli, A., Zhen, Z., Maina,
F., Dalla, Z. P., Giordano, S., Graziani, A., Panayotou, G., and
Comoglio, P. M.(1994) Cell 77, 261-271). We inserted
this sequence into a mutant platelet-derived growth factor receptor
(F5) to determine if this region of c-met can initiate cell
motility and which signaling pathways it activates. The
platelet-derived growth factor (PDGF) receptor/c-met hybrid
(F5 met) initiated PDGF-dependent chemotaxis in renal epithelial cells
(8.0 ± 2.3 versus 70.5 ± 4.8
cells/mm), while the parental construct, F5, did not.
Addition of PDGF to cells expressing F5 met caused activation of the
phosphatidylinositol (PI) 3-kinase (control 2.0 ± 0.8,
+PDGF 17.1 ± 5.1, n = 3, p <
0.05) and phospholipase C (control 478.5 ± 67 dpm/well,
+PDGF 1049.3 ± 93, n = 4, p = 0.003), while neither pathway was activated in cells
expressing F5. The chemotactic response of F5 met was inhibited by both
the PI 3-kinase inhibitor wortmannin and the phospholipase C inhibitor
U-71322. Selective activation of the PI 3-kinase utilizing a PDGF
receptor mutant (F3) containing the native high affinity PI 3-kinase
binding site also resulted in PDGF stimulated chemotaxis, although less
than that generated by the c-met sequence.
These findings demonstrate that the 11-amino acid sequence from c-met initiates epithelial motility via coincident activation of the PI 3-kinase and phospholipase C and that selective activation of the PI 3-kinase can initiate a partial chemotactic response.
Hepatocyte growth factor (HGF) ()has been
independently characterized by its ability both to induce mitogenesis (1) and to cause ``scattering'' of epithelial cells
in culture(2) . Further study has implicated HGF and its high
affinity receptor, c-met, in diverse biologic events that
involve the motile response: chemotaxis and branching tubulogenesis in
renal epithelial cells (3, 4) and wound healing and
angiogenesis in endothelial cells(3, 5) . The motile
response induced by c-met is a complex one, which involves
dissociation of cell contacts, ruffling of the leading edge, extension
of lamellipodia, and finally migration(6) . The role of
c-met in this series of events has not yet been well
characterized, but it is likely to involve the integration of multiple
signaling pathways.
Examination of the COOH-terminal region of
c-met reveals a single 11-amino acid sequence,
YVHVNATY
VNV, which is predicted to be a
low affinity site for the two SH2 domains of phospholipase C
(PLC
) and the phosphatidylinositol 3-kinase (PI 3-K), and a high
affinity site for the ras activating complex
GRB2-hSos1(7) . No other domain outside of the tyrosine kinase
region of c-met is predicted to bind any known signaling
proteins, making it possible that this 11-amino acid domain is a site
for competitive interaction of many, if not all, of the
receptor's signaling proteins. Indeed, Ponzetto et al.(8) were able to demonstrate co-immunoprecipitation of
overexpressed c-met with PLC
, GRB2-hSos1, and PI 3-K as
well as pp60
(8) . Altering both
tyrosines in this 11-amino acid sequence eliminated association with
all four proteins, leading these researchers to term this region a
``multifunctional docking site.'' Thus, this 11-amino acid
sequence might be sufficient to activate all of the signaling activity
induced by HGF and mediate the motility response.
Work in our laboratory has demonstrated the importance of the PI 3-K in HGF-mediated motility(4) . Using the PI 3-K inhibitors wortmannin and LY-294002, we found striking inhibition of both motogenesis and morphogenesis. These experiments demonstrate that c-met-mediated activation of the PI 3-K is required for the full chemotactic response, but they do not address whether the PI 3-K alone is sufficient to initiate chemotaxis.
In this report, we used
an altered platelet-derived growth factor receptor (PDGFR), which is
unable to bind to PI 3-K, RasGAP, Syp/PTP, or PLC (9) as a
vehicle to introduce and study the role of the 11-amino acid c-met sequence in the motility response of epithelial cells. Use of a
PDGFR mutant in our inner medullary collecting duct cells (which do not
express endogenous PDGFR) makes it possible to study selective tyrosine
phosphorylation of this met sequence in the setting of normal
epithelial signaling machinery. In addition, a second PDGFR mutant with
the native high affinity PI 3-K binding site (Y
MDM . . .
Y
VPM) intact but with Tyr
Phe substitutions of the
tyrosines critical for association with RasGAP, Syp/PTP and PLC
(F3) were utilized to examine more selective activation of the PI 3-K
in epithelial chemotaxis.
Figure 2: Directional chemotaxis of mIMCD-3 cell clones toward a PDGF gradient. Independent clones for each mutant PDGFR were screened for the ability to migrate in response to PDGF. F5 PDGFR clones did not exhibit chemotaxis, while F5 met clones did chemotax toward PDGF.
Figure 1:
Schematic depiction
of the PDGF receptor mutants. The wild-type PDGFR is shown highlighting
the 5 tyrosines critical for association with the PI 3-kinase
(740/751), RasGAP (771), SHPTP2(1009), and PLC(1021). The F5 clone
has phenylalanine substitutions for all 5 residues. F5 met has the
sequence from 750-760 replaced with the 11-amino acid c-met sequence YVHVNATYVNV. F3 clone retains the 740/751 tyrosines for
PI 3-kinase binding.
Each of the constructs was then cloned into pCMV expression vectors and transfected into mIMCD-3 cells using the lipofectin reagent (Life Technologies, Inc.), and independent clones were selected on G418 (400 mcg/ml).
In contrast, we identified eight F5 met clones that demonstrated
chemotaxis in response to PDGF and found that all eight clones
expressed the PDGFR. Additionally, no clone exhibited PDGF-dependent
chemotaxis, which did not express the receptor. Among these clones, the
amount of chemotaxis correlated well with the level of expression of
the PDGFR (Fig. 3). Interestingly, clones F5 met and F5 met
subsequently failed to chemotax to PDGF
and were found to no longer express the PDGFR by Western analysis. One
clone, F5 met
, which stably expressed the mutant PDGFR,
was used for further experiments and displayed consistent ability to
chemotax toward PDGF (control, 4.6 ± 3.5 cells/mm
, n = 9; PDGF, 78.6 ± 12.7 cells/mm
, n = 11; p < 0.001).
Figure 3: Top panel, expression of F5 met in cells that chemotax to PDGF. Anti-PDGFR blot of anti-PDGFR immunoprecipitates from clones transfected with F5 met mutant PDGFR. Bottom panel, directional chemotaxis of mIMCD-3 cells and F5 met PDGFR clones toward a PDGF gradient.
Figure 4: Expression and phosphorylation of mutant PDGFR in mIMCD-3 cell clones. a, whole cell lysates from confluent plates of cells were analyzed with an anti-PDGFR Western blot. The 190-kDa PDGF receptor is evident in all but parent IMCD cells. b, mutant PDGFRs are tyrosine-phosphorylated in response to PDGF. Lysates of PDGF-stimulated or unstimulated mIMCD-3 cells and PDGFR clones were immunoprecipitated with anti-PDGFR. Complexes were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine.
Figure 5:
PDGF stimulated PI 3-K activity in IMCD
cells expressing the PDGFR mutants. Cells were stimulated with PDGF or
vehicle control, immunoprecipitated with an antibody to
phosphotyrosine, and then incubated with phosphatidylinositol (PI) substrate and [-
P]ATP.
Labeled lipids were separated by thin-layer chromatography (top
panel), and the PI3P was cut out and counted in a
scintillation counter and expressed in disintegrations/min/g of protein
immunoprecipitated (bottom panel). Results are the mean of
three separate experiments. *, p < 0.05 in comparison with
unstimulated control;**, p < 0.025 in comparison with
unstimulated control.
The importance of the association of the mutant PDGFR with the PI 3-K in the initiation of chemotaxis was confirmed by incubation of the cells with the PI 3-K inhibitor wortmannin. In these experiments, 10 nM wortmannin was found to markedly inhibit PDGF-dependent chemotaxis in F5 met cells (+PDGF, 179.1 ± 42.1 versus +PDGF + 10 nM wortmannin, 70 ± 10.3, n = 9, p = 0.023).
Figure 6:
HPLC analysis of inositol phosphate from
F5 met cells. F5 met cells were labeled with
[H]inositol as described and stimulated for 2 min
with either vehicle control or 10 ng/ml PDGF. Cytosolic lipids were
then separated via HPLC. Inositol 1,4,5-phosphate eluted at 28.3
min.
A PLC inhibitor, U-73122 was then tested
for its effect on F5 met chemotaxis(14) . U-73122 had no effect
on the activity of the PI 3-K (control 126.9 ± 37 dpm
PI3P/µg; 1 µM U-73122, 144.8 ± 46 dpm/µg, n = 3; p = not significant) and did not
inhibit chemotaxis initiated by the protein kinase C activator
diacylglycerol (diacylglycerol 142.5 ± 42.4
cells/mm, n = 12; diacylglycerol +
U-73122, 131.7 ± 9.6, n = 9; p =
not significant). However, at concentrations as low as 10 nM,
there was a 70% inhibition of PDGF dependent chemotaxis, supporting a
major role for PLC in c-met mediated chemotaxis (Fig. 7).
Figure 7: Inhibition of PLC blocks F5 met-mediated chemotaxis. Directional chemotaxis of IMCD cells toward a gradient of either 10 ng/ml PDGF or 40 ng/ml HGF with and without 10 nM U-71322, a PLC inhibitor.
The potent chemotactic effect of HGF on epithelial cells makes the study of its receptor, c-met, a particularly relevant system for determining the signaling cascade involved in the epithelial motility response. The use of a hybrid F5-c-met receptor (F5 met) allowed us to selectively examine the role of an 11-amino acid region of c-met in epithelial cell motility and signal transduction.
We initially determined that all three of the
transfected receptor constructs were successfully expressed in mIMCD-3
cells and underwent PDGF-dependent tyrosine phosphorylation. The F5
mutant PDGFR, which does not bind PI 3-K, PLC, PTP, or RasGAP but
is still capable of associating with src (via
Y
IYV) and possibly GRB-2 (via Y
SNA) (15) did not initiate epithelial chemotaxis in response to
PDGF. However, expression of the F5 met hybrid receptor did initiate
chemotaxis in a ligand-dependent manner, demonstrating that
phosphorylation of the inserted c-met sequence
Y
VHVNATYVNV activated those signaling pathways necessary
for epithelial cell motility.
Based on our observation that the PI 3-K inhibitors wortmannin and LY294002 could partially block HGF/c-met-mediated epithelial chemotaxis(4) , as well as data from Kundra and co-workers (16) that demonstrated that PDGF constructs excluding PLC activation exhibited diminished chemotactic responses(16) , we examined the ability of the 11-amino acid c-met sequence to activate these two candidate signaling pathways in epithelial cells. Indeed, both the PI 3-K and PLC were activated in a ligand-dependent manner by the F5 met hybrid receptor, but not by the parental F5 construct. The importance of these two pathways in chemotaxis was then examined using inhibitors of PI 3-K and PLC as well as a receptor construct, which selectively activates PI 3-K. Both wortmannin, an inhibitor of PI 3-K, and U-73122, an inhibitor of PLC, caused marked reductions of F5 met-stimulated chemotaxis. Thus, the PI 3-K and PLC are activated in vivo by the 11-amino acid sequence from c-met and contribute to the chemotactic response initiated by this receptor.
In addition, the F3 PDGFR in which the
native high affinity PI 3-K binding site (Y MDM . . .
Y
VPM) has been selectively restored (but which still
lacks PLC, RasGAP, and PTP binding sites) also caused epithelial
chemotaxis in response to PDGF. It should be noted that one study has
described the small adaptor molecule Nck as competing for binding to
the Y
VPM sequence of F3 as well(17) . The above
data demonstrate a critical role for both the PI 3-K and PLC in
epithelial cell migration and are the first to show that selective
activation of PI 3-K is sufficient to initiate chemotaxis.
Our
findings are complemented by data from several laboratories. Wennstrom et al.(18) examined the ruffling response in porcine
aortic endothelial cells transfected with a mutant PDGFR, which
selectively excluded binding of the PI 3-K (Tyr Phe
substitutions at 740 and 751)(18) . This construct, the
opposite of our F3 PDGFR, failed to mediate ruffling in response to
PDGF, implicating a requirement for PI 3-K in membrane ruffling.
Likewise, Kundra et al.(16) examined the same
Y740F/Y751F mutation in chemotactic assays of a canine kidney
epithelial cell line(16) . Again, the chemotactic response was
obliterated by an inability to activate the PI 3-K, as well as a mutant
excluding activation of PLC. Here, we demonstrate that selective
activation of PI 3-K initiates a chemotactic response, while
coactivation of PI 3-K and PLC produces a marked increase in this
response.
Other signaling pathways important in cell movement
involve the small GTP binding proteins Ras, Rac and Rho. In a recent
study by Ridley et al.(19) , microinjection of
Madin-Darby canine kidney cells with constituitively active Ras
reproduced HGF-mediated ruffling but did not initiate cell
scattering(19) . Thus, it may be that isolated activation of
ras is sufficient to generate actin filament rearrangements but not
actual cell movement. In agreement with this, our F5 PDGFR clones did
not mediate PDGF-dependent chemotaxis, even though ras activation may
occur via GRB2/hSos1 nucleotide exchange factor association at
YSNA in the F5 receptor(20) . However, since both
the F5 met and F3 receptors are capable of Grb2/hSos1/Ras activation,
we cannot rule out a contributory or co-stimulatory effect of Ras
activation on chemotaxis in these experiments.
The c-met receptor mediates diverse phenotypic changes in several cell types including cell motility and tubule formation. An 11-amino acid sequence from this receptor, when phosphorylated in a ligand dependent manner, activates both PLC and the PI 3-K, triggering the intracellular events necessary for epithelial chemotaxis.