(Received for publication, October 19, 1994; and in revised form, November 9, 1994)
From the
The hepatocyte growth factor (HGF) receptor is a tyrosine kinase
receptor that mediates signal transduction upon ligand stimulation.
This receptor is present in mouse tissues as two major isoforms
differing by a 47-amino acid segment in the juxtamembrane domain, an
alternatively spliced cytoplasmic region adjacent to the transmembrane
domain of the receptor. We report here that the juxtamembrane domain of
the receptor is involved in the regulation of downstream signal
transduction. The two receptor isoforms were transiently expressed in
COS-7 cells. Both exogenous receptors underwent autophosphorylation and
subsequently stimulated a set of protein tyrosine phosphorylations that
were not present in control cells. Comparisons of phosphotyrosine
profiles of transfected cell lysates induced by receptor isoforms
demonstrated that at least three phosphorylated proteins of 62,
35, and
30 kDa were differentially induced by the receptor
isoforms, suggesting that the juxtamembrane domain of a kinase receptor
can play a role in selective signal transduction. Furthermore, the p85
subunit of phosphatidylinositol-3-OH kinase (PI
kinase)
co-precipitated with the small isoform of the HGF receptor, and this
association was dramatically inhibited by treatment with
12-O-tetradecanoylphorbol-13-acetate. Since removal of the
juxtamembrane domain facilitates the binding of p85 to the receptor, it
is likely that the juxtamembrane region plays a role in negative
regulation of the binding of PI
kinase to the HGF receptor.
Our study establishes novel molecular sequelae of alternative splicing
of an intracellular domain of the HGF receptor.
The hepatocyte growth factor (HGF) ()receptor/c-met is a transmembrane protein that is
a member of the tyrosine kinase receptor
superfamily(1, 2) . The ligand for this tyrosine
kinase receptor was identified as HGF, also known as scatter
factor(3) . This factor has pleiotropic functions. It is a
mitogen(4) , a morphogenic factor(5) , and an
angiogenic factor(6) . Therefore, the HGF receptor is believed
to be involved in the regulation of cell proliferation, migration, and
differentiation. Upon HGF stimulation, the HGF receptor exhibits
tyrosine kinase activity, including receptor autophosphorylation, and
it activates a cascade of tyrosine phosphorylation involving a series
of signal-transducing proteins. Following autophosphorylation, the HGF
receptor associates with PI
kinase in vivo and in vitro(7, 8) . PI
kinase is a
lipid kinase that phosphorylates phosphatidylinositols at the 3`
position of inositol upon growth factor stimulation. The products
generated by PI
kinase have been implicated in the
regulation of cell growth. The p85 subunit of PI
kinase
binds to tyrosine-phosphorylated sites of the HGF receptor through an
SH2 domain (8) . The regulation of PI
kinase
activity in response to growth factors can also be mediated by an
intermediary cytoplasmic protein. Insulin receptor substrate-1, for
instance, has multiple sequence motifs for PI3 kinase binding and is
highly phosphorylated upon insulin stimulation(9) .
Besides
PI kinase activation, HGF/scatter factor can also stimulate
the Ras-guanine nucleotide exchanger, which promotes the GTP-bound
active state of Ras protein(10) . The Ras pathway is essential
for the scattering effect mediated by HGF receptor(11) .
Recently, Ras protein has been demonstrated to interact directly with
PI
kinase in a GTP-dependent manner(12) . Since
PI
kinase is a key enzyme in the intracellular growth
signaling pathway, inhibitors have been developed as antiproliferative
agents(13) . However, it is not clear whether the activation of
PI
kinase can be differentially regulated by different
forms of a tyrosine kinase receptor.
We have previously reported that a novel type of structural variant of the tyrosine kinase HGF receptor exists in mouse tissues(14) . This new isoform transcript, which is shorter by 141 base pairs, occurs through mRNA alternative splicing and results in a deletion of 47 amino acids in the juxtamembrane domain of the cytoplasmic domain of the receptor. The juxtamembrane domain appears to be involved in the negative regulation of kinase activity(15) , mitogenesis, and transforming activity(16) . These studies seem to suggest that this juxtamembrane domain may contain crucial determinants that could direct specificity of the signaling pathway of this kinase receptor.
The
purpose of this study was to investigate the role of the juxtamembrane
domain of the HGF receptor in the regulation of downstream signaling
pathways by using HGF receptor isoform cDNA constructs. We report that
the juxtamembrane domain is involved in selective tyrosine
phosphorylation of three unidentified proteins. It is also a negative
controlling element for the association of PI kinase with
the HGF receptor, since removal of the juxtamembrane region greatly
facilitates binding of the p85 subunit of PI
kinase to the
HGF receptor. Our data suggest a novel mechanism by which the HGF
receptor may greatly reinforce its physical association with PI
kinase, promoting a specific signal transduction pathway among
c-met-associated signaling cascades by using receptor isoforms
that differ in a cytoplasmic juxtamembrane domain.
Figure 1:
Detection of exogenous expressed HGF
receptor isoforms by immunoblotting. Cell lysates were
immunoprecipitated with c-met antibody followed by
immunoblot analysis with the same antibody (A) or with
anti-phosphotyrosine (PY) antibody (B). Cells
transfected with plasmid DNA of large and small HGF receptor isoforms
are shown in lanes1 and 2, respectively,
while mock transfection with control plasmid is shown in lane3 (A and B). Bands corresponding to HGF
receptor isoforms are indicated by brackets in the center
(HGFR). Protein size markers are indicated in kilodaltons (kd). IP,
immunoprecipitate.
Figure 2:
Comparison of phosphotyrosine profile
induced by receptor isoforms in transfected (lanes 1 and 2) and mock transfected (lane 3) COS-7 cells. The
tyrosine-phosphorylated products that were differentially regulated by
HGF receptor isoforms are indicated by arrows on the left. Protein size markers are shown on the right. IP, immunoprecipitate; PY,
anti-phosphotyrosine.
We next
examined whether any of the tyrosine-phosphorylated proteins are
receptor-associated proteins. The immunoprecipitates of c-met antibody were Western-blotted with anti-phosphotyrosine to detect
tyrosine phosphorylation of c-met-precipitated proteins. As
shown in Fig. 1B, both receptor isoforms underwent
autophosphorylation, including the 190-kDa immature form; however,
no additional tyrosine-phosphorylated protein was observed in this
assay. This was confirmed by overexposing the x-ray film (data not
shown). This result indicates that little or none of the tyrosine
phosphorylated protein is associated with the HGF receptor.
Figure 3:
Association of p85 and HGF receptor
isoforms in COS-7 transfected cells. Lysates of cells transfected with
the HGF receptor isoforms were immunoprecipitated with
c-met antibody followed by immunoblot analysis. The
same blot was probed with anti-p85 antibody (UBI) (A) and then
reprobed with anti-phosphotyrosine antibody (B).
12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment
at 10 min (lanes3 and 4) and controls (lanes1 and 2) are shown as indicated at
the top. Bands corresponding to HGF receptor isoforms are
indicated by brackets in the center (HGFR). Protein
size markers are indicated in kilodaltons (kd). IP,
immunoprecipitate; PY,
anti-phosphotyrosine.
We have previously identified a HGF receptor isoform that has
a deletion of 47 amino acids in the juxtamembrane region. The removal
of the 47-amino acid segment appears to occur by mRNA alternative
splicing. The differential splicing occurs in a variety of mouse
tissues, suggesting biological significance of the two isoforms.
Removal of the alternatively spliced domain produces a tyrosine kinase
receptor that lacks the protein kinase C target phosphorylation site
and that would lack this mechanism for down-regulation of receptor
kinase activity. To investigate additional roles of the juxtamembrane
domain of the HGF receptor in cellular regulation, we used a transient
expression system to express exogenous HGF receptor isoforms in COS-7
cells. The signaling proteins that were differentially regulated by
receptor isoforms were analyzed by immunoblotting. We report here that
at least three tyrosine-phosphorylated proteins are differentially
induced by the receptor isoforms differing in the juxtamembrane region.
In addition, studies using antisera to p85 of PI kinase
suggested that the 47-amino acid segment of the juxtamembrane domain is
a negative controlling element for the binding of the p85 subunit of
the PI
kinase, since deletion of this domain greatly
enhanced the binding of PI
kinase to the receptor.
Our
findings that the presence of the juxtamembrane domain has a
significant effect on differential affinity of signaling proteins
provides evidence that the juxtamembrane domain plays a potentially
pivotal role in regulating signaling transduction pathways. Two newly
phosphorylated proteins were specifically produced by the large
isoform, i.e. in the presence of the juxtamembrane domain. The
induction of this protein phosphorylation is presumably a further
downstream signaling event, although the molecular mechanism is not
clear. On the other hand, the juxtamembrane domain is also a negative
controlling region for the binding of the PI kinase to the
HGF receptor. The autophosphorylated EGF receptor and HGF receptor bind
weakly to PI
kinase in
vivo(7, 19) . Compared with platelet-derived
growth factor receptors, for instance, EGF receptors containing the
juxtamembrane domain appear to show much weaker binding to PI
kinase even though the EGF receptor was overexpressed at 10 times
or more higher levels than the platelet-derived growth factor
receptor(19) . The evidence for complex formation of the
PI
kinase with EGF receptor or HGFR involves detecting
PI
kinase activity in vitro in anti-receptor
immunoprecipitates or overexpressing either p85 subunit or the
receptors. The binding of an intermediary protein(s) to the
juxtamembrane domain may be involved in interrupting complex formation
between PI
kinase and the receptor tyrosine kinase domain,
since deletion of the region facilitates PI
kinase binding
to the receptor. Alternatively, removal of the juxtamembrane domain may
cause a conformational change in the receptor that facilitates its
binding to PI
kinase.
Ras protein was recently shown to
play an integral part in mediating the mobility signal of HGF/scatter
factor(11) , suggesting involvement of Ras, PI kinase, and activated HGF receptor in functional complex
formation. Our finding that deletion of the juxtamembrane domain
facilitates the binding of PI
kinase to the receptor
clearly suggests that signals transmitted by each of the two HGF
receptor isoforms may couple diverse signaling pathways and result in
distinct cellular responses. Our results, therefore, provide the first
direct evidence that a cytoplasmic juxtamembrane domain of the HGF
receptor interacts with different signal transduction pathways. These
findings may also partly explain the diverse biological functions of
the HGF receptor.
Functional importance of the juxtamembrane region has also been reported for the EGF receptor(20) . Phosphorylation of a threonine residue by protein kinase C in the juxtamembrane region significantly decreases receptor kinase activity. A point mutation at the threonine residue in the sequence motif for protein kinase C phosphorylation prevents the receptor kinase from being down-regulated. The same regulatory mechanism involving protein kinase C was recently observed for the HGF receptor(15) . Within the juxtamembrane domain, however, there are also regions other than the protein kinase C target site that are important for functions of the receptor. A receptor mutant with a deletion (residues 660-667) six amino acids downstream from the protein kinase C regulatory site alters the mitogenic activity of the EGF receptor(21) . A point mutation within this region strongly affects the specificity of signal transduction, indicating that the juxtamembrane domain may impart specificity for certain cytoplasmic proteins(22) . In addition, the juxtamembrane region of the HGF receptor is also involved in transforming activity in focus formation and in nude mice(16) . These studies further strongly suggest that the juxtamembrane domain of kinase receptors has crucial and distinct biological and biochemical properties, underscoring the functional significance of its modification by alternative splicing.