If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
∗ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
). 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(
) 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)P2 at the 3-position to produce the potential second messenger PtdIns(3,4,5)P3(
). 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.
Yeast Two-hybrid Interactions
The intracellular domain (amino acids 781-1338) of the Flt-1 receptor tyrosine kinase was obtained by the polymerase chain reaction (PCR) and subcloned into the EcoRI site of the yeast pGBT9 GAL4 DNA binding domain expression vector (Clontech). The amino-terminal SH2 domain of p85 was obtained by PCR between glutamine 329 and 435 and the carboxyl-terminal SH2 between aspartic acid 613 and arginine 724 and subcloned into the pGAD424 GAL4 transcriptional activation domain vector. All products were obtained by PCR from reverse transcribed human umbilical vein endothelial cell (HUVEC) poly(A) mRNA and sequenced. SH2 domains were cotransformed with Flt-1 in SFY526 cells and co-expressing colonies selected on agar deficient in tryptophan and leucine. β-Galactosidase activities were assayed from overnight cultures of individual colonies in Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0) using o-nitrophenylgalactosidase. The reactions were stopped with Na2CO3 and the absorbance measured at 420 nm. Activities were calculated according to Miller(
). Assay reproducibility was assessed using p53/SV40 large T antigen interactions by employing the pVA3/pTD1 vectors (Clontech, data not shown).
Western Blotting of GAL4 Fusion Proteins
Log phase yeast cultures (50 ml) expressing the various flt-1 constructs were pelleted and resuspended in 1 ml of 2 × SDS electrophoresis sample buffer, 10 mM dithiothreitol and denatured at 100°C for 5 min. Total protein (15 μl) was electrophoresed through 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed using a polyclonal antibody raised against the GAL4 binding domain (Upstate Biotechnology, Inc.). Protein bands were visualized using enhanced chemiluminescence (ECL, Amersham Corp.).
Fragments of the flt-1 intracellular domain were subcloned into M13 vectors for site-directed mutagenesis according to the method of Kunkel et al.(
). The receptor was made kinase-deficient by mutating lysine in the ATP binding site (VAVK) to an alanine residue (K861A). Five of the seven potential autophosphorylation sites in the COOH-terminal tail were mutated to phenylalanine. The remaining two Y/F mutations were performed in the kinase domain.
Sf21 cells were maintained in suspension culture and grown in Grace's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin. The intracellular domains of flt-1 and flt-1 Y1213F were subcloned into the pBakPak 8 vector (Clontech) with glutathione S-transferase (amino acids 1-219) fused to the amino terminus. Recombinant virus and protein were prepared according to standard protocols. Sf21 cells were lysed in ice-cold lysis buffer (1% Triton X-100, 10 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride HCl (AEBSF), 10 μg/ml aprotinin, 10 μg/ml leupeptin) and the glutathione S-transferase fusion protein captured using glutathione-agarose (Pharmacia Biotech Inc.).
Kinase Assay and Peptide Mapping
Purified protein (approximately 5 μg) was washed in kinase buffer (25 mM HEPES, pH 7.2, 10 mM MgCl2, 0.5 mM EGTA) and autophosphorylated in the presence of 25 μCi of [γ-32P]ATP and 0.1 mM ATP for 5 or 10 min at 30°C. The reaction was terminated by the addition of 4 × SDS sample buffer with heating. The samples were loaded onto 10% acrylamide gels in SDS, electrophoresed, and electrophoretically transferred to nitrocellulose. The nitrocellulose was briefly stained with Ponceau S in 100 mM acetic acid and destained with water. Individual bands were excised, washed, and blocked with 0.5% polyvinylpyrrolidone in 100 mM acetic acid at 37°C for 30 min. After extensive washing with water, the proteins were digested and eluted from the slices by overnight incubation at 37°C in 100 mM NaHCO3, 5% acetonitrile containing 40 μg of acetylated trypsin (Promega). Supernatants were collected from the slices and acidified to a 1% final concentration of trifluoroacetic acid. Efficiency of 32P-peptide elution was monitored by Cerenkov counting and ranged from 87% to 93%. Peptide mapping was accomplished by reverse phase HPLC on a μBondpack C18 column (Waters) using gradient elution of 0.05% trifluoroacetic acid to 80% acetonitrile in 0.05% trifluoroacetic acid. The elution profile of each gradient was monitored at A210 and the radioactive peptides containing 32P were detected on-line using a Flo-one/beta detector (model CR/DS; Radiomatic Instruments).
RESULTS AND DISCUSSION
PI 3-kinase is recruited following growth factor receptor activation by some but not all growth factors(
). 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 (
) (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 β-galactosidase gene transcription.
Table 1displays the β-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(
). As predicted, Flt-1 K861A is unable to interact with p85 resulting in zero β-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 (
). 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 (
) 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 PLCγ,
S. A. Cunningham, P. Arrate, T. A. Brock, and M. Neal Waxham, manuscript in preparation.
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 β-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 PLCγ to the β-PDGF receptor(
) 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.
), 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 NH2-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(
). 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 PLCγ amino SH2 to Y766 of fibroblast growth factor receptor-1, a previously characterized in vivo interaction(
). Fibroblast growth factor receptor-1/PLCγinteractions (2.5 ± 0.5 units of β-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 β-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 (
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)P3 levels may be important during angiogenesis.
We thank Kay Sughrue for maintenance of the insect cell cultures.