(Received for publication, May 10, 1995; and in revised form, June 12, 1995)
From the
We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.
Vascular endothelial growth factor (VEGF) ()promotes
microvascular permeability and is a specific mitogen for endothelial
cells(1, 2, 3) . VEGF plays a significant
role during angiogenesis, a process required during development, wound
healing, and pathological conditions such as solid tumor growth and
metastasis, rheumatoid arthritis, diabetic retinopathy, and
atherosclerosis(1, 2, 3) . Two transmembrane
receptors that bind this mitogen have recently been cloned, namely flt-1 (4) and KDR (flk-1)(5, 6) . Both proteins contain an
extracellular domain consisting of seven IgG-like loops, a single
transmembrane-spanning region, and a kinase insert within the tyrosine
kinase domain.
Activated growth factor receptors interact with a
variety of intracellular proteins containing Src homology 2 (SH2)
domains(7) . These modules recognize phosphotyrosine motifs in
the receptor tyrosine kinases and specificity is afforded by the
particular context of the phosphotyrosine moiety(8) . It has
recently been shown that Flt-1 couples with Fyn and Yes(9) ,
PLC, and RasGAP (10) following VEGF stimulation. Many
receptor tyrosine kinases couple to PI 3-kinase, a heterodimer
consisting of an 85-kDa regulatory subunit and a 110-kDa catalytic
subunit. In intact cells PI 3-kinase phosphorylates the
phosphatidylinositol (PtdIns) ring of PtdIns(4,5)P
at the
3-position to produce the potential second messenger
PtdIns(3,4,5)P
(11) . One role for
PtdIns(3,4,5)P
is the activation of the
isozyme of
protein kinase C(12) . The 110-kDa subunit was recently
characterized as a dual specificity enzyme since it also possesses
serine/threonine kinase activity(13) .
Binding of the SH2
domains in the 85-kDa subunit to specific phosphotyrosine motifs of
activated growth factor receptors results in a conformational change of
the dimer and activation of PI 3-kinase (14) . This enzyme was
discovered as an activity associated with the middle
T-antigen-c-Src complex in polyomavirus-transformed cells and
has been continually implicated in cell growth regulation(11) .
Mutant PDGF receptors that couple solely to PI 3-kinase and Nck remain
capable of initiating DNA synthesis following receptor
activation(15) . The recent discovery that PI 3-kinase can
directly activate Ras (16) provides further proof for its role
in cell proliferation. PI 3-kinase activity is also necessary for
trafficking of receptor tyrosine kinases(17) , PDGF-stimulated
actin rearrangements(18) , insulin-induced membrane
ruffling(19) , and cell survival(20) . Most
interestingly, it has recently been discovered that p85 can act as an
adapter molecule linking the insulin receptor with a p62
GTPase-activating protein(21) . Surprisingly, this complex does
not contain the p110 subunit and thus would not lead to elevated
PtdIns(3,4,5)P levels.
In this paper we take a novel approach to study receptor tyrosine kinase/SH2 domain interactions by using the yeast two-hybrid system (22) . We have examined the interactions of Flt-1 with the p85 subunit of PI 3-kinase in order to define additional second messenger pathways that may be activated following VEGF stimulation of Flt-1.
PI 3-kinase is recruited following growth factor receptor
activation by some but not all growth factors(25) . In order to
determine a potential role for this second messenger system in
VEGF-stimulated angiogenesis, we separately synthesized both p85 SH2
domains to examine their interactions with the intracellular domain of
Flt-1 in the yeast two-hybrid system. In the absence of extracellular
domains, recombinant intracellular domains of receptor tyrosine kinases
are constitutively active (26, 27) (Fig. 2).
The SFY526 strain used to analyze interactions possesses a lacZ reporter gene under the control of a GAL1 upstream activating
sequence (Clontech). The GAL4 binding domain-Flt-1 fusions associate
with the upstream activating sequence. Interaction of a GAL4
activation/SH2 domain fusion protein with the Flt-1 receptor would
align the activation domain with the promotor resulting in
-galactosidase gene transcription.
Figure 2:
Elution profiles of phosphorylated (P) peptides from trypsin digests of recombinant
glutathione S-transferase-Flt-1 proteins. Either wt Flt-1 (A) or mutant Flt-1 Y1213F (B) autophosphorylated
protein digests were separated on a reverse phase HPLC C18 column.
Autophosphorylation of Flt-1 in the presence or absence of vanadate
resulted in identical peaks. The arrowhead in A indicates the unique phosphopeptide absent in the Y1213F profile.
The total time in each panel is 120 min.
Table 1displays the
-galactosidase activities of yeast expressing either Flt-1 alone
or in combination with the p85 SH2 domains. It is clear from this assay
that p85 is capable of interacting with Flt-1 through either the amino
or carboxyl SH2 domains. In order to prove that p85 interactions were
occurring through specific phosphotyrosine residues on Flt-1, we
mutated lysine in the ATP binding site (K861A) to produce an
autophosphorylation-deficient mutant. Mutation of this conserved lysine
has previously been shown to inhibit the intrinsic tyrosine kinase
activity of the epidermal growth factor receptor with minimal
structural alterations(28) . As predicted, Flt-1 K861A is
unable to interact with p85 resulting in zero
-galactosidase
activity (Table 1). To verify that this kinase-deficient receptor
was being synthesized in yeast, we performed Western blot analysis of
the GAL4 binding domain fusion expressed in the SFY526 strain. Western
blot analysis of yeast lysates using an anti-GAL4 binding domain
antibody (Upstate Biotechnology, Inc.) are shown in Fig. 2. The
estimated molecular mass of the GAL4 binding domain is 17 kDa, and the
Flt-1 intracellular domain is 63 kDa. It is clear that an 80-kDa band
is apparent in yeast expressing both wt Flt-1 (lane2) and Flt-1 K861A (lane3).
The results raised the question as to whether different motifs were recognized by each SH2 domain or whether a common site on the activated Flt-1 was capable of interacting with both domains. It is now well established that the SH2 domains of p85 bind to phosphotyrosine moieties in the consensus YMXM/YXXM (8, 11) , although there are exceptions to this rule. Most notable is the use of a YVXV motif by the hepatocyte growth factor receptor (HGFR)(29) . An examination of the Flt-1 sequence revealed only one consensus motif (YQIM), which was located just within the boundaries of the kinase domain itself. This sequence is conserved within most tyrosine kinases (30) and is not responsible for binding of p85 in those receptors that interact with PI 3-kinase. Mutation of this site does decrease partially the levels of p85 binding to Flt-1 (Table 1, Y1130F). However, this conserved residue is probably important for other structural or catalytic functions of the kinase. From comparison with other receptor tyrosine kinases, it is unlikely to represent a direct p85 binding site. In addition, all SH2 domain interactions thus far reported have been mapped outside of the catalytic kinase domain.
Since the
autophosphorylation sites of the Flt-1 receptor have not yet been
sequenced, the following rationale was used to identify tyrosine
residues involved in this interaction. The Y1213 and Y1327 sites were
targeted since they resided in a motif similar to the HGFR p85 binding
sites. The Y1309F mutant was included as a potential control since its
context did not conform to either the YXXM or
YVXV motifs. The triple YSTP mutant was investigated since the
repetition of this motif suggested a conserved sequence that may be
involved in protein-protein interactions. Table 1summarizes the
results of these tyrosine to phenylalanine mutations. It is apparent
that p85 interactions are only ablated with Flt-1 Y1213F. This site
does not affect the binding of the amino SH2 domain of PLC, (
)which suggests specificity, and Fig. 1(lane4) shows that the Flt-1 Y1213F protein is intact. If p85
was capable of directly interacting with other phosphotyrosines in
Flt-1, the Y1213F mutant would result in only a partial inhibition of
-galactosidase activity. Thus, these results demonstrate that a
single phosphotyrosine motif is responsible for the independent binding
of both amino and carboxyl SH2 domains. A similar mechanism has been
described for the binding of both SH2 regions of PLC
to the
-PDGF receptor(31) . Several kinases have been described, e.g. PDGF receptor (32) and HGFR (29) that
contain two consecutive phosphotyrosine motifs that participate in p85
binding. It is believed that two juxtaposed sites strengthen the
interactions with proteins containing two SH2 domains. Alternatively,
since activated growth factor receptors exist as dimers, Flt-1 may
present YVNA motifs in close apposition enabling each SH2 domain of a
single p85 molecule to bind simultaneously to a phosphotyrosine moiety.
Figure 1: Western blot analysis of GAL4 binding domain/Flt-1 fusions expressed in yeast. Total cell lysate from SFY526 cells expressing GAL4 binding domain alone (lane1), GAL4 wt Flt-1 (lane2), GAL4 Flt-1 K861A (lane3), and GAL4 Flt-1 Y1213F (lane4) were probed with an anti-GAL4 binding domain polyclonal antibody. Cross-reactive bands were detected using the ECL method. The arrow indicates the GAL4-Flt-1 fusion proteins.
Songyang et al.(8) , using a phosphopeptide library
to study the binding specificities of various SH2 domains in
vitro, reported that valine at position +1 is favorable for
binding of the p85 NH-terminal SH2, although it did not
enhance interactions with the COOH-terminal SH2. Both of these domains
had a strong preference for methionine at position +3, whereas
neither valine or alanine conferred enhanced binding at this position.
Nevertheless, as shown for the HGFR, and now here for the Flt-1
receptor, these amino acids appear to be favorable for in vivo interactions. It should be noted that the general
Tyr(P)-hydrophobic-hydrophilic-hydrophobic pattern is maintained. KDR
does not possess an optimal YXXM motif, and an
alignment with Flt-1 shows that a YLQN sequence is substituted for
YVNA. This site has not been reported to autophosphorylate in
KDR(26) . Thus it is possible that only Flt-1 has the potential
to couple with PI 3-kinase following VEGF stimulation of endothelial
cells.
In order to confirm that Y1213 is autophosphorylated, we
employed an insect cell expression system to prepare fusion proteins of
glutathione S-transferase with intracellular domains of wt
Flt-1 and Flt-1 Y1213F. As predicted, the purified proteins
demonstrated constitutive kinase activity and Fig. 2A shows HPLC analysis of seven radiolabeled peaks derived from a
trypsin digest of autophosphorylated wt Flt-1. Strikingly, only six
peaks are apparent in the digest from the Flt-1 Y1213F kinase domain (Fig. 2B). The disappearance of the peak at 20 min
strongly argues that indeed this site is autophosphorylated by the
Flt-1 tyrosine kinase. In addition, we can conclude that mutation of
this tyrosine does not prevent autophosphorylation of other sites in
the receptor. Thus we have identified this Y1213 as a key
autophosphorylation site that is important for functional coupling of
p85 with Flt-1 in the yeast two-hybrid system. In preliminary
experiments we have also analyzed the binding of PLC amino SH2 to
Y766 of fibroblast growth factor receptor-1, a previously characterized in vivo interaction(33, 34) . Fibroblast
growth factor receptor-1/PLC
interactions (2.5 ± 0.5 units
of
-galactosidase/min) were quantitatively similar to those for
Flt-1/p85 and were abolished by mutation of Tyr-766 to phenylalanine
(0.04 ± 0.017 units of
-galactosidase/min). Thus, it is
likely this system reflects true in vivo interactions.
Furthermore, while this paper was in preparation, another group
reported that VEGF stimulation of bovine aortic endothelial cells,
which express both KDR and Flt-1, results in phosphorylation of p85 and
its recruitment into a signaling complex (35) .
The results
presented here demonstrate the feasibility of analyzing SH2
domain-receptor tyrosine kinase interactions in the yeast two-hybrid
system. We have made the first determination of an autophosphorylated
site on the Flt-1 receptor and identified it as a binding site for the
regulatory subunit of PI 3-kinase. This data implicates that p85 may
play a role in coupling Flt-1 to intracellular signal transduction
systems and thus elevated PtdIns(3,4,5)P levels may be
important during angiogenesis.