©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Tyrosine Mutations on the Kinase Activity and Transforming Potential of an Oncogenic Human Insulin-like Growth Factor I Receptor (*)

(Received for publication, August 22, 1995; and in revised form, October 5, 1995)

Yixing Jiang Joseph L.-K. Chan Cong S. Zong Lu-Hai Wang (§)

From the Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The tyrosines in the cytoplasmic domain of an oncogenic human insulin-like growth factor I receptor (gag-IGFR) were systematically mutated to phenylalanines to investigate the role of those tyrosines in the enzymatic and biological function of the gag-IGFR. Our results indicate that tyrosines 1131, 1135, 1136, and 1221 are important for the receptor protein-tyrosine kinase (PTK) activity. However, mutation of Tyr-1136 only slightly affects the kinase activity but dramatically reduces the transforming ability and overall substrate phosphorylation, in particular, annexin II, which is strongly phosphorylated by the gag-IGFR but not by the Phe-1136 mutant. Single mutation of either Tyr-943 or Tyr-950 resulted in significantly reduced phosphorylation of the receptor but not on its PTK activity or transforming ability. Tyr-950 together with its surrounding sequence is involved in mediating the interaction between the gag-IGFR and insulin receptor substrate 1. Our data also suggest that Tyr-1316 is involved in phosphorylation of phospholipase C-, which is, however, not important for cell transforming activity. Overall, our study has identified several tyrosine residues of IGFR important for its PTK activity and substrate interaction. The transforming potential of the gag-IGFR correlates well with its ability to phosphorylate overall cellular substrates and to activate phosphatidylinositol 3-kinase via insulin receptor substrate 1.


INTRODUCTION

Receptor protein-tyrosine kinases (RPTKs) (^1)are transmembrane glycoproteins with intrinsic protein kinase activity(1, 2) . Ligand binding to an RPTK results in its oligomerization, PTK activation, autophosphorylation, and phosphorylation of cellular substrates leading to gene activation, DNA synthesis, and eventual cell proliferation or differentiation. Interactions between RPTKs and cellular substrates are mediated by receptor tyrosine autophosphorylation(3) . Aside from activating the kinase activity, phosphorylation of tyrosine residues also provides binding sites for Src homology 2-containing signaling proteins such as Grb2, Shc, Nck, PI 3-kinase p85 regulatory subunit, GAP, PLC, and Src family kinases(4, 5) . Mutation of tyrosine residues in those binding sites often leads to a loss of interaction with their corresponding substrates. Alternatively, receptor-substrate interactions can be mediated through a recently characterized phosphotyrosine binding domain interacting with a NPXY motif (6) . IRS1, the major phosphorylation substrate of insulin receptor, lacks a Src homology 2 domain but interacts with insulin receptor at tyrosine 972 via such a sequence(7, 8, 9) . It has also been shown that Shc is phosphorylated in response to insulin stimulation and can also interact with insulin receptor at tyrosine 972 through its phoshpotyrosine binding domain(7, 10) .

The human insulin-like growth factor I receptor (IGFR) is an RPTK closely related to insulin receptor (IR). IGFR and IR share an 84% amino acid sequence identity in their kinase domains; however, their biological functions differ somewhat(11) . Cells treated with insulin rapidly increase glucose uptake, and lipid and glycogen synthesis but only increase DNA synthesis after a prolonged stimulation. IGF-I, however, appears to be a more potent stimulator of DNA synthesis and cell growth(12) . IGFR has been reported to be overexpressed in human breast cancers(13) . Dominant negative mutants and antisense mRNAs of IGFR can inhibit the growth of tumor cells and tumor formation in nude mice(14, 15) . Transfection of EGFR into IGFR knockout mouse cell lines cannot induce EGF-dependent cell transformation, while transfection of an exogenous IGFR can rescue EGF-induced cell transformation, suggesting that IGFR is required for EGFR-mediated transformation(16) . Collectively, they suggest that overexpression or constitutive activation of IGFR may play some role in cell transformation and tumorigenicity.

Our previous studies on IGFR have shown that the cell-transforming activity of native IGFR was significantly enhanced in an amino terminus truncated receptor, coding for 36 amino acids of the extracellular domain, the entire transmembrane, and cytoplasmic domains of IGFR beta subunit, fused to the avian sarcoma virus UR2 gag sequence(17) . The gag-IGFR encoding virus called UIGFR has an enhanced transforming potential over that of native IGFR in cultured CEF but is not tumorigenic in vivo. The gag-IGFR is a dimerized transmembrane protein(17) . Further examination of this extracellular 36-amino acid sequences revealed that it has a negative modulating effect on IGFR transforming and tumorigenic potential(18) . Deletion of the entire 36 amino acids resulted in a strong transforming and tumorigenic mutant called NM1(18) . Similar studies were done on the carboxyl terminus of IGFR by deleting the carboxyl terminus of the UIGFR fusion receptor(19) . A deletion of 27 amino acids from the carboxyl terminus of the receptor, including a potential PI 3-kinase binding motif YXXM (amino acids 1316-1319), resulted in a gag-IGFR that still retains its kinase activity and transformation ability. Further deletion of 20 amino acids abolished the kinase and transforming activities of the receptor. Surprisingly, deletion of 67 amino acids restored both kinase activity and transforming ability, and deletion of 88 amino acids abolished all the activities. Overall, previous studies on the gag-IGFR indicate a strong correlation between the receptor kinase activity and its transforming ability. In this study, we further explored the mechanism of cell transformation induced by this oncogenic gag-IGFR. Since receptor-substrate interactions play important roles in RPTK signal transduction and phosphorylated tyrosines on the receptor provide binding sites for the substrates, tyrosine residues within the cytoplasmic domain of NM1 gag-IGFR were systematically replaced by phenylalanines. In addition, since IRS1 is a substrate of IGFR, a more extensive study of tyrosine 950 and its neighboring sequence, the presumed docking site for IRS1, was undertaken. Although substantial studies on mutation of tyrosine residues in insulin receptor have been carried out (see ``Discussion''), the role of tyrosine residues in IGFR has largely been inferred from insulin receptor thus far. Our study provides a direct systematic examination of the role of tyrosine residues in an oncogenic form of IGFR.


EXPERIMENTAL PROCEDURES

Cells and Viruses

The preparation of CEFs and colony formation assay of virus-infected CEFs were done according to published procedures(17) . Cultures were maintained in F10 medium supplemented with 5% calf serum and 1% chicken serum (Life Technologies, Inc.). UR2 and its associated helper virus have been described previously(17) . Mutant virus stocks were obtained by collecting medium 10 days after transfection.

Construction of Recombinant Plasmids

With the exception of the Tyr-950 mutations, mutant IGFRs were constructed by using site-directed mutagenesis (Promega altered sites in vitro mutagenesis system). A SmaI-BamHI DNA fragment of pNM1 was first freed and subcloned into a phagemid, pALTER-1, which contains a tetracycline resistance gene and a mutated ampicillin gene. Recombinant bacteriophage was harvested from an overnight culture and precipitated with 3% polyethylene glycol (PEG8000) and 0.4 M NaCl. Single-stranded DNA template was purified from the phage pellet by phenol chloroform extraction and ethanol precipitation. Two oligonucleotides, a mutagenic one and an ampicillin repairing one, were annealed, and an in vitro polymerase reaction was performed with T4 polymerase. The double-stranded plasmids were recovered by transforming a repair minus strain of Escherichia coli, and ampicillin resistance colonies were selected. Mutations were confirmed by sequencing and then used to swap with the corresponding pNM1 sequence to generate the mutants.

The Tyr-950 mutation and the 13-amino acid deletion were generated by ligating two DNA fragments. A 5`-BglII site-containing oligonucleotide and a 3`-mutagenic oligonucleotide containing the Tyr-950 to Phe-950 mutation and a HgaI site were used with either pNM1 or pUIGFR as a template for polymerase chain reaction to generate the 5`-fragment. The 3`-DNA fragment used for both UF950 and Phe-950 was synthesized using a 5`-mutagenic oligonucleotide containing a HgaI site and a 3`-SphI site-containing oligonucleotide. The two fragments were digested with HgaI and ligated. The ligated product was used in a second round polymerase chain reaction with the BgllI and SphI oligonucleotides to amplify the ligated product. The final fragment was cloned back into pUIGFR or pNM1 using the BglII and SphI sites. The deletion mutant was constructed in a similar manner but using different mutagenic oligonucleotides containing a HgaI site and the sequence flanking the 13-amino acid deletion from tyrosine 943 to valine 956.

Antibodies

Rabbit antiserum anti-IB was raised against the IGFR beta-subunit(17) . Recombinant alkaline phosphatase-conjugated anti-phosphotyrosine antibody, RC20, was purchased from Transduction Laboratories. Rabbit polyclonal anti-PLC antibody was purchased from Upstate Biotechnology INC. Anti-murine annexin II was purchased from Zymed Laboratories. A polyclonal anti-chicken annexin II serum was a gift from Dr. Tony Hunter. Goat anti-rabbit and goat anti-mouse IgG conjugated with alkaline phosphatase were purchased from Boehringer Mannheim.

To prepare the polyclonal rabbit anti-IRS1 antibody, the 3`-sequence coding for rat carboxyl-terminal 271 amino acids. of IRS1 was cloned by polymerase chain reaction and inserted into a glutathione S-transferase bacterial expression vector. Glutathione S-transferase-IRS1 fusion protein was purified with glutathione-agarose beads followed by gel electrophoresis. 250 µg of purified protein was emulsified with an equal volume of complete Freund's adjuvant (Sigma) and used to immunize a New Zealand White rabbit (2.5 kg, female). For boost injections, 150 µg of protein was emulsified with an equal volume of incomplete Freund's adjuvant every 2 weeks. Bleeding was performed weekly starting 10 days after the second boost.

DNA Transfection

CEFs were plated at a density of 1 times 10^6cells/6-cm dish and allowed to recover for 18-20 h at 37 °C; the culture medium was then removed and replaced with 1 ml of fresh medium containing 30 µg/ml polybrene. 20 µg of NM1 or its mutant plasmid DNA, 2 µg of SacI-digested pUR2AV helper virus DNA, were added. The culture was incubated at 37 °C for 6 h with occasional mixing and then subjected to a 27% Me(2)SO shock for 3 min at room temperature. The cells were washed three times with fresh medium and incubated at 37 °C. Cells were overlaid with soft agar medium the day after transfection to enhance the growth of transformed cells.

Protein Assays

[S]Methionine metabolic labeling, in vitro kinase assay, and Western analysis were done according to the procedures described previously(17) . Western analysis was done with the following modifications: after separation of proteins by SDS-PAGE, the gel was immediately placed onto an electrotransferring apparatus and transferred to a nitrocellulose filter without pretreatment with the transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol). The transfer procedure was done for 1 h at 100 V at room temperature. The filter was blocked for 1 h at room temperature with 3% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1% Tween 20 (TBS-Tween) and then subjected to binding of antibody in 3% bovine serum albumin in TBS-Tween at either 4 °C for 6 h or at room temperature for 2 h. After binding, the filter was washed in TBS-Tween three times for 20 min each at room temperature. The washed filter was reacted with an appropriate secondary antibody (either goat anti-rabbit or rabbit anti-mouse immunoglobulin) conjugated with alkaline phosphatase. The filter was then washed extensively to remove nonspecific bound antibody and was developed by adding alkaline phosphatase substrates, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl(2). Alternatively, the filter was labeled with I-protein A, and the protein was visualized by autoradiography.

PI 3-Kinase Assay

The PI 3-kinase assay was done according to the published procedure with slight modifications(19) . CEF cells were lysed in Nonidet P-40 buffer (20 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride. The cell lysates were centrifuged at 15,000 times g for 10 min at 4 °C to remove cell debris. The supernatant was incubated with anti-IB or anti-IRS1 for 2 h at 4 °C. Then, 15 µl of protein A-agarose beads was added, and the mixture was further incubated for 1 h at 4 °C. The immunoprecipitates were washed as described above, and the washed beads were resuspended in 25 µl of TGN buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA). 10 µg of phosphatidylinositol (20 µg/µl in dimethyl sulfoxide, purchased from Avanti Polar Lipids, Inc.) was added to the resuspended beads and mixed. The mixture was incubated for 10 min at room temperature to form micelles of PI. [-P]ATP (10 µCi per assay) and MgCl(2) (final concentration, 20 mM) were premixed and added to the immunoprecipitate suspension. The mixture was incubated at room temperature for 10 min. Formation of PI 3-phosphate was linear in this time period. (^2)The reaction was stopped by adding stop solution (chloroform:methanol:12 N HCl = 100:200:2) and extracted with chloroform. The extracted PI 3-phosphate was washed three times with a mixture of methanol and 1 N HCl (1:1). The washed PI 3-phosphate was then dried, resuspended in 15 µl chloroform, and analyzed on a Silica Gel 60 thin layer chromatography plate (Merck) as described previously(19) .

Cytoskeletal Protein Fractionation

Monolayer cells were incubated with 0.5 ml of CSK buffer (10 mM PIPES, pH 6.8, 100 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2). 0.3 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 1% trasylol, 1 mM Na(3)VO(4), 10 µM Na(2)MoO(4), and 1% Triton X-100) on ice for 3 min, and the supernatant was collected. The cells were incubated again with 0.5 ml of CSK buffer for another 1 min, and the supernatant was collect and combined with the previous one. The remaining structure was extracted with Western lysis buffer. The combined supernatants were ethanol precipitated and dissolved in Western lysis buffer. Both factions were analyzed in SDS-PAGE gel, Western blotted, and reacted with appropriate antibodies.


RESULTS

Construction and Expression of Mutant IGFR

To explore the mechanism of cell transformation induced by gag-IGFR, mutants that possess kinase activity but lack transformation ability will be very useful for identifying the essential downstream components for cell transformation by comparing their substrates with those of the parental NM1 protein. Since the phosphorylated tyrosine residues are potentially critical sites for the receptor to interact with its downstream substrates, our initial mutagenesis study focused on the tyrosine residues in the cytoplasmic domain of NM1 gag-IGFR. There are 15 tyrosine residues in the cytoplasmic domain of the IGFR beta subunit, and each tyrosine except Tyr-1316 was substituted with phenylalanine. Our early study has already shown that a mutant, CM2, with deletion of carboxyl 27 amino acids including Tyr-1316 had little effect on the PTK activity and transforming function of the UIGFR-encoded gag-IGFR(19) . In addition, mutants with combinatory mutations of certain tyrosine residues were also constructed. All the mutants were named according to amino acid positions (11) of the mutation (Fig. 1).


Figure 1: Schematic representation of UIGFR and NM1 chimeric receptors. The tyrosine to phenylalanine mutants are indicated by their amino acid positions(11) . The deletion in mutant d950 encompasses tyrosine 943 to valine 956. The CM2 mutant was reported previously(19) . Single-letter amino acid codes are used here and in subsequent figures. F3, Y1131F/Y1135F/Y1136F.



Transforming Activity of the Tyrosine-Phenylalanine Mutants

NM1 or its mutant DNA was cotransfected with 2 µg of SacI-digested UR2AV DNA into CEF as described under ``Experimental Procedures.'' Cells were overlaid with soft agar the next day to select for the transformed cells. Colony formation assay was set up 3 days after transfection and maintained at 42 °C for 2 weeks, after which plates were scored for number of colonies (Table 1). The morphological appearance of monolayer cultures was also monitored and recorded (data not shown). Normal CEF and weak- or non-transforming virus-infected cells had spindle-shaped morphology, while transformed cells showed elongated fusiform morphology and had increased refractility. Most of the mutants have a similar transforming ability as the parental NM1. However, mutants Phe-1221, Phe-1136, double mutant Y1135F/Y1136F, and triple mutant Y1131F/Y1135F/Y1136F (Fig. 1, F3) have either reduced or completely lost the transforming ability (Table 1, Fig. 2). Interestingly, Phe-1136, which contains the third tyrosine mutation in the three-tyrosine cluster, has a significantly reduced cell-transforming and growth-promoting activity (7-fold reduction of colonies in comparison with NM1) despite having a near wild type level of PTK activity (see below). The effect of Tyr-950, the presumed IRS1 interacting site, was addressed using point and deletion mutants. Mutation of Tyr-950 from the highly oncogenic gag-IGFR encoded by NM1 produced little effect, however, when a similar mutation was made in UIGFR (UF950) the transforming activity was greatly reduced. Deletion of a 13-amino acid stretch including the Tyr-950 from NM1, unlike the point mutation, did result in a significant reduction of colony-forming ability (Fig. 2).




Figure 2: Anchorage-independent growth of transfected CEFs. Colony assays were set up 3 days after transfection, and the cultures were maintained at 41 °C. The pictures were taken 10 days after incubation. F3, Y1131F/Y1135F/Y1136F.



Tyrosine Kinase Activity and Intracellular Phosphorylation of the Mutant Receptors

The kinase activity of various mutant gag-IGFR receptors was assessed by comparing their abilities to autophosphorylate in vitro and intracellularly (Fig. 3a). Those activities were compared in parallel with the levels of protein expression. For those transformation-defective mutants, the protein expression levels in long term culture were relatively low; therefore, transiently transfected cells were used for comparison (Fig. 3c). With a few exceptions, most of the mutant proteins exhibited similar levels of in vitro kinase activity and intracellular autophosphorylation as that of NM1. Phe-1095 and Phe-1183 proteins appeared to have 4-5-fold reduction in the in vitro kinase activity; however, their intracellular phosphorylation remained the same as NM1 protein (Fig. 3a). Interestingly, Phe-1136, which had significantly reduced cell-transforming and growth-promoting abilities, retained the in vitro and in vivo autophosphorylation kinase activities similar to those of the NM1 protein. However, its ability to phosphorylate cellular substrates in general is significantly reduced (see below). Surprisingly, the extent of intracellular autophosphorylation of the mutant Phe-1221 and double mutant Y1135F/Y1136F proteins, both of which had dramatically reduced transforming activity, was similar to that of NM1 in the transient transfection assay. However, the in vitro kinase activity of Phe-1221 is greatly reduced. The triple mutant Y1131F/Y1135F/Y1136F, which had lost completely the transforming activity, was devoid of in vitro kinase activity and intracellular autophosphorylation (Fig. 3c). Phe-943 and Phe-950 mutant proteins displayed faster mobility of receptor bands particularly in the antiphosphotyrosine blot, suggesting that these two sites play an important role in the phosphorylation of the receptor (Fig. 4); however, UF950 of UIGFR containing the corresponding Tyr-950 mutation did not exhibit this characteristic mobility shift ( Fig. 3and Fig. 5).


Figure 3: In vitro kinase activity and intracellular phosphorylation of mutant gag-IGFRs. Confluent virus-infected cells were extracted with RIPA buffer, and equivalent protein amounts of total lysates were used in each immunoprecipitation using an anti-IGFR antiserum, anti-IB. Half of the immunoprecipitated proteins was subjected to an in vitro kinase assay, while the other half was Western blotted with anti-IB to monitor the amount of IGFR. A parallel culture was treated with 200 µM Na(3)VO(4) for 4 h and then lysed and immunoprecipitated similarly. The intracellular phosphorylation of the receptor was determined by blotting with an anti-phosphotyrosine antibody, RC20, coupled with alkaline phosphatase (panels a, b, and e). Alternatively, cells were labeled with [S]methionine before lysis, and the relative amounts of gag-IGFR protein were determined by immunoprecipitation, gel analysis, and autoradiography (panel d). The assays in panel c were done with transiently transfected cells, and the protein amount of gag-IGFR was monitored by Western blot. The slower mobility of UIGFR and UF950 proteins is due to the presence of an additional 36 amino acids of the extracellular sequence in comparison with that of NM1 (Fig. 1) and glycosylation of the UIGFR proteins. F3, Y1131F/Y1135F/Y1136F.




Figure 4: Phosphorylation of mutants Phe-943 and Phe-950 proteins. Mutant gag-IGFR-transfected CEFs were treated with Na(3)VO(4) as in Fig. 3and lysed. 200 µg of total cellular proteins was immunoprecipitated with anti-IB and subjected to Western blotting with either anti-IB or anti-P-Tyr (RC20). a, Phe-943 and NM1 proteins were detected by using a goat-anti-rabbit secondary antibody conjugated with alkaline phosphatase and followed by color reaction. b, Phe-950 and NM1 proteins were detected by I-protein A labeling. The extent of tyrosine phosphorylation was detected by alkaline phosphatase-coupled anti-P-Tyr antibody, RC20, in both a and b.




Figure 5: Phosphorylation of cellular proteins by mutant gag-IGFRs. Na(3)VO(4) pretreated cells were lysed in the Western lysis buffer, and 10 µg of total cellular proteins was resolved in the 10% SDS-PAGE gels. The filters were blotted and reacted with anti-P-Tyr (RC20). F3, Y1131F/Y1135F/Y1136F.



Phosphorylation of Cellular Proteins

To evaluate tyrosine phosphorylation of cellular substrates by the mutant proteins, total cell lysates of different mutant-transfected CEFs were analyzed by immunoblotting with RC20, a recombinant antiphosphotyrosine antibody conjugated with alkaline phosphatase (Fig. 5). The overall tyrosine phosphorylation of cellular proteins was remarkably increased over normal CEF in cells transfected by all the mutants except the weak and non-transforming variants, namely Phe-1221, Phe-1136, Y1135F/Y1136F, and Y1131F/Y1135F/Y1136F. There was no obvious difference when compared among the strong transforming mutants and NM1. The Phe-950, UF950, and d950 did not appear to have a difference in the pattern and extent of substrate phosphorylation, despite the fact that both UF950 and d950 have a reduced transforming activity from their respective parental viruses UIGFR and NM1. Despite the Phe-1136 protein being as efficiently autophosphorylated as that of the NM1 protein (Fig. 3a), it has a significantly reduced ability to phosphorylate cellular proteins in general. By comparing the substrate pattern of Phe-1136 protein with those of strong transforming mutants and NM1, the mutant Phe-1136 protein failed to induce tyrosine phosphorylation of a 36-kDa protein, in particular, which was strongly phosphorylated in NM1- and other strong transforming mutant-infected cells. In other weak transforming or non-transforming mutant-infected CEFs, this 36-kDa protein was not phosphorylated either. Annexin II, a 36-kDa phospholipid binding protein, was reported to be tyrosine phosphorylated in src-transformed cells(20) . A monoclonal antibody against annexin II was used to identify this 36-kDa protein in the mutant and NM1-transformed cells. The protein band detected by the monoclonal antibody superimposed with the tyrosine-phosphorylated 36-kDa protein detected with the anti-P-Tyr antibody (Fig. 6a). To examine subcellular localization of the 36-kDa protein, NM1-transformed cell lysates were fractionated into soluble and CSK fractions. The proteins from the two fractions were resolved in SDS-PAGE gel, blotted, and reacted with RC20 or the anti-annexin II antibody (Fig. 6b). The result showed that the 36-kDa protein and annexin II were colocalized in the CSK fraction. To further confirm that annexin II is phosphorylated in NM1-transformed cells, NM1-infected cells were lysed in RIPA buffer containing 0.1% SDS and immunoprecipitated with a polyclonal anti-annexin II serum. Annexin II was strongly phosphorylated in NM1-infected cells but not in normal CEF (Fig. 6c). However, we cannot conclude that annexin II is the sole 36-kDa protein observed since it remains possible that there is more than one protein comigrating at this position. It is concluded that the overall tyrosine phosphorylation of transforming virus-infected cells is remarkably higher than that of non- or weak-transforming virus-infected cells, and annexin II is tyrosine phosphorylated in the transformed cells but not in the normal or weakly transformed cells.


Figure 6: Phosphorylation of annexin II in mutant gag-IGFR transformed cells. a, Na(3)VO(4) pretreated cells were lysed in the Western lysis buffer, and 10 µg of total cell lysate was analyzed in the SDS-PAGE gel. The filter was first blotted with anti-annexin II to detect the annexin II protein (lower panel). The same filter was stripped off the antibody and further reacted with an anti-P-Tyr monoclonal antibody (PT22) followed by I-protein A labeling. b, Na(3)VO(4) treated NM1-transformed cells were fractionated into soluble (Sol.) and cytoskeletal fractions as described under ``Experimental Procedures.'' The soluble fraction was precipitated with 70% ethanol and redissolved in Western lysis buffer. Both fractions were analyzed in the gel followed by Western blotting with either RC20 or anti-annexin II. The far right NM1 lane is the total cell extract. c, NM1-transformed cells were treated with Na(3)VO(4). In lanes 1 and 2, cells were lysed with RIPA buffer containing 0.1% SDS, and the lysate was immunoprecipitated with the anti-annexin II. In lanes 3 and 4, cells were lysed in the Western lysis buffer. The anti-annexin II precipitates and direct Western buffer extracts were analyzed in the same gel and Western blotted. The filter was reacted with RC20.



IRS1 Phosphorylation and PI 3-Kinase Activity

IRS1 is a major phosphorylation substrate of IR and IGFR and is believed to be important in the recruitment of other signaling molecules such as PI 3-kinase, Grb2bulletSos complexes, and Syp. To determine the ability of those mutant proteins to phosphorylate IRS1, the infected cell lysates were immunoprecipitated with anti-IRS1 serum and Western blotted with antiphosphotyrosine antibody. The phosphorylation of IRS1 was not significantly changed in most mutant-infected cells in repeated experiments (Fig. 7). Surprisingly, UF950 and Phe-950 proteins, in which the putative IRS1 docking site (21) has been mutated, phosphorylate IRS1 as efficiently as their parental UIGFR and NM1 proteins. Even the deletion mutant d950 protein retains the ability to phosphorylate IRS1 though at a slightly lower level. Association of IRS1 with PI 3-kinase results in activation of its activity(22, 23) . To examine the activation of PI 3-kinase, its activity was assayed in the receptor or IRS1 immunoprecipitates of cell extracts prepared from mutant receptor plasmid-transfected CEF in comparison with those from normal CEF and NM1 transfectants (Fig. 7). For most mutants, receptor- and IRS1-associated PI 3-kinase activity levels were not affected. The Phe-950 mutant retained a wild type level of receptor- and IRS1-associated PI 3-kinase activity, but UF950 and d950 proteins exhibited a decrease in the associated PI 3-kinase activity when compared to their respective parental proteins. The basal level of receptor-associated PI 3-kinase is largely due to the cross-reactivity of anti-IB for chicken endogenous IGFR. IRS1-associated PI 3-kinase activity was approximately 4-5-fold higher than the receptor-associated PI 3-kinase activity. This could be due to the possibility that the IRS1bulletPI 3-kinase complex is more stable than the receptorbulletIRS1bulletPI 3-kinase triple complex, or alternatively the receptor-IRS1 interaction is much more transient. Overall, except for the kinase-inactive mutants, UF950 and d950, no significant difference in either gag-IGFR- or IRS1-associated PI 3-kinase activity was observed.


Figure 7: Mutant gag-IGFR- and IRS1-associated PI 3-kinase activity and phosphorylation of IRS1. Cells pretreated or untreated with Na(3)VO(4) were lysed in RIPA or Nonidet P-40 buffer and used for detecting IRS1 tyrosine phosphorylation or PI 3-kinase assay, respectively, after immunoprecipitation with either anti-IRS1 or anti-IB. For PI 3-kinase assay, 300 µg of lysate was used, 1 mg was used for IRS1 tyrosine phosphorylation, and 50 µg was used for monitoring the gag-IGFR protein. The reduced IRS1-associated PI 3-kinase activity of Phe-1162 was due to experimental fluctuation, and repeated experiments showed no difference from NM1.



Phosphorylation of PLC

An anti-PLC antibody was used to examine PLC phosphorylation in the cells infected with various mutants including a carboxyl deletion mutant, CM2, which lacks the carboxyl 27 amino acids including Tyr-1316 in the YAHM sequence, which is considered a potential motif for interacting with Src homology 2-containing proteins. CM2 protein is active in PTK and cell-transforming activities(19) . All transforming mutants are capable of inducing PLC tyrosine phosphorylation while CM2 is unable to (Fig. 8). This result suggests that tyrosine 1316 together with its neighboring sequence may be involved in the association and/or phosphorylation of PLC. However, phosphorylation of PLC appears not to be essential for CEF transformation. 70- and 180-kDa tyrosine-phosphorylated proteins were reproducibly co-immunoprecipitated with PLC from NM1-infected but not from UR2-infected cells. The identities of those proteins are unknown.


Figure 8: Tyrosine phosphorylation of PLC. Various mutant gag-IGFR-transformed cells were treated with Na(3)VO(4) and lysed with RIPA. 1 mg of total cellular protein was used in each immunoprecipitation with anti-PLC. the immunoprecipitates were analyzed in the 10% SDS-PAGE gel and followed by immunoblotting with RC20.




DISCUSSION

Our results show that mutation of tyrosine residues of the gag-IGFR fusion receptor can affect its kinase activity and transforming ability. The cluster of the three tyrosines (Y1131F/Y1135F/Y1136F) plays an important role in regulating the PTK activity of the receptor. Those three tyrosines correspond to the tyrosine residues 1158, 1162, and 1163 of insulin receptor shown to be located at the gate of the kinase catalytic center(24, 25) . It was reported earlier that the autophosphorylation sites of the insulin receptor are tyrosines 965, 972, 1158, 1162, 1163, 1328, and 1334 with tyrosines 1158, 1162, and 1163 being the main regulators of the enzymatic activity for insulin receptor(26, 27, 28) . Mutation at each site reduces insulin-stimulated autophosphorylation by 45-60% of that of the wild type receptor. Double mutation reduces autophosphorylation by 70%, and replacement of all three tyrosines with phenylalanines almost abolishes the kinase activity(29) . Our study indicates that single mutation at position 1135 or 1136 produces little effect on the kinase activity of the chimeric receptor. However, mutation at position 1136 drastically reduced cell-transforming and growth-promoting activities without affecting significantly the receptor kinase autophosphorylation activity in vitro and intracellularly. The Phe-1136 mutation does significantly affect its ability to phosphorylate cellular substrates in general and the 36-kDa annexin II protein in particular. This property correlates well with its reduced biological function. These observations suggest that mutation of this tyrosine may change the receptor conformation or that phosphorylation of this residue is important for substrate recognition. Interestingly, mutation of the Tyr-1136 corresponding tyrosines in the two closely related RPTKs, IR and Ros, also results in significantly reduced transforming activity without affecting their PTK activity. (^3)However, no global decrease in substrate phosphorylation like Phe-1136 was observed in the IR and Ros mutants, although reduction in interaction with specific substrates was observed. Double mutation of Tyr-1135 and Tyr-1136 did not produce the drastic effect as that of Tyr-1162 and Tyr-1163 in native IR. It is possible that the NM1 gag-IGFR is constitutively and highly activated such that it is less sensitive to changes in those sites. Mutations at positions 943 and 950 result in faster mobility of the receptor proteins, indicating that these two sites play important roles in the IGFR phosphorylation. The downshift in the autophosphorylation products in vitro and missing of the upper band in the anti-P-Tyr blot of those mutant proteins suggest that Tyr-950 and Tyr-943 are the prominent autophosphorylation sites. This is in agreement with an earlier study showing that the juxtamembrane tyrosines of IR are the preferred autophosphorylation sites in an in vitro assay at low ATP concentrations(30) . The two corresponding tyrosines in IR are tyrosines 965 and 972, which have been shown to be in vivo autophosphorylation sites(27) . The Tyr-972 mutation of a gag-IR also resulted in a faster mobility of the protein in anti-P-Tyr immunoblot. (^4)Surprisingly, when the reported major phosphorylation sites tyrosines 1135 and 1136 were substituted with phenylalanines, no significant change in the receptor protein mobility was observed. It is possible that tyrosine 943 and 950 mutations may indirectly affect serine/threonine phosphorylation as well, resulting in a more pronounced effect. Our observation suggests that, similar to IR, IGFR also contains several potential tyrosine autophosphorylation sites, and mutation of any of them, except Tyr-943 and Tyr-950, does not significantly affect the overall phosphorylation of the receptor.

IRS1 is a key component in the IR signal transduction. It has been shown that tyrosine 972 is critical for the binding of IRS1 to IR. Point mutation of this tyrosine results in impaired phosphorylation of IRS1 upon insulin stimulation(7, 8) . From sequence alignment, tyrosine 950 in IGFR could be the interacting site for IRS1. However, no clear experimental evidence has been provided(21) . Mutation of tyrosine 950 in the highly activated and oncogenic NM1 gag-IGFR does not appear to affect its ability to induce IRS1 phosphorylation or to promote association of PI 3-kinase with IRS1, but when the mutation is generated in the less active and moderate transforming gag-IGFR, encoded by UIGFR, there is a significant decrease in both cell-transforming ability and IRS1-associated PI 3-kinase activity, while tyrosine phosphorylation of IRS1 remains unchanged. Deletion of 13 amino acids flanking tyrosine 950(943-956) of NM1 gag-IGFR, however, impacts on all three activities, namely causing a decrease in transformation, IRS1 phosphorylation, and PI 3-kinase activity. While our result with UIGFR is consistent with the notion that Tyr-950 of IGFR is involved in interaction of IGFR with IRS1, the data with NM1 are not in complete agreement. There are three possible explanations for these paradoxical results. First, tyrosine 950 may not be the only site for the IRS1 interaction with IGFR, and IRS1 may have another redundant binding site. Redundancy in substrate binding sites has been shown in other RPTKs such as PDGFR(31) . In PDGFR, Shc has four binding sites, which are tyrosines 579, 740, 751, and 771. The second explanation is that IRS1 is phosphorylated by other tyrosine kinases activated by IGFR. Interactions of RPTKs with non-receptor tyrosine kinase has been reported. Src can be activated by PDGFR, and the binding sites for Src in PDGFR have been identified as tyrosines 579 and 581(31) . Furthermore, IGFR is activated in v-src transformed cells(32) . The highly activated receptor, such as NM1 gag-IGFR in particular, may have a higher potential of cross-activating other PTKs. The third possibility is that this mutation can be compensated for by either overexpression or hyperactivation of the gag-IGFR. Recent studies suggested that the phosphotyrosine binding domain (6, 7) and the pleckstrin homology domain (33) of IRS1 may be involved in its interaction and tyrosine phosphorylation with IR. However, the pleckstrin homology domain apparently is not the direct binding site of IRS1 with IR(33) . Phosphorylation of IRS1 can be abolished if the pleckstrin homology domain is deleted; however, it can be restored by overexpression of IR. (^5)It has also been shown that overexpression of full-length IR containing Phe-972 can rescue the phosphorylation of IRS1 despite the mutated interaction site.^5 By using the yeast two-hybrid system, a recent study showed that the amino-terminal region from amino acids 160 to 516 of IRS1 is sufficient for its interaction with IGFR and that phosphorylation of Tyr-950 of IGFR is essential for its interaction with IRS1 and Shc but not with p85(34) . However, it is not known from their study whether IRS1 can be phosphorylated by the IGFR with the Phe-950 mutation. In our case, the constitutively activated NM1 gag-IGFR may have compensated for the Tyr-950 mutation with its high level expression and catalytic activity, which may result in cross-activation of other IRS1-interacting PTKs as mentioned above. Our result with d950 suggests that the sequence surrounding the site is involved in the interaction with IRS1, but Tyr-950 is not critical in the highly activated receptor. IRS1 is apparently phosphorylated to a similar extent in all mutant-infected cells. However, it remains possible that different sites are phosphorylated, which may account for the reduced PI 3-kinase activity in d950 and UF950.

Annexin II is a tetrameric Ca-dependent phospholipid binding protein and is associated with the cytoskeleton(35) . However, other reports showed that annexin II was mainly found in the membrane-associated fraction(36) . Annexin II was first shown to be a substrate of v-src in its transformed cells(20) . Tyrosine phosphorylation of annexin II can also be induced by PDGF receptor after treatment of PDGF(37) . Annexin II in addition to being a substrate for protein-tyrosine kinases can also be a substrate for protein kinase C(38) . Overexpression of annexin II was reported in human pancreatic carcinoma cells and primary pancreatic cancers, as well as in multidrug-resistant small lung cancer(39, 40) . Study of the RAW large lymphoma cells showed that depletion of cell surface annexin II could inhibit RAW cells from adhering to the liver microvessel endothelial cells, suggesting that annexin II might be involved in metastasis(41) . Moreover, annexin II was shown to be the receptor of tenascin-C, indicating that annexin II may mediate cellular response to soluble tenascin-C in the extracellular matrix(42) . Furthermore, annexin II is one of the fos target genes(43) . However, the exact function of annexin II remains unknown. We have identified that annexin II is a substrate for the gag-IGFR, and its phosphorylation correlates with the extent of cell transformation by the mutant gag-IGFRs, raising the possibility that it may play a role in the cell transformation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA55054. 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.

§
To whom correspondence should be addressed: Tel.: 212-241-3795; Fax: 212-534-1684.

(^1)
The abbreviations used are: RPTK, receptor protein-tyrosine kinase; PTK, protein-tyrosine kinase; PLC, phospholipase C-; PDGFR, platelet-derived growth factor receptor; PAGE, polyacrylamide gel electrophoresis; IGFR, insulin-like growth factor I receptor; PI 3, phosphatidylinositol 3-kinase; IRS1, insulin receptor substrate 1; PIPES, 1,4-piperazinediethanesulfonic acid; CEF, chicken embryo fibroblast; CSK, cytoskeletal.

(^2)
L.-H. Wang, unpublished data.

(^3)
C. Zong, J. Chan, and L.-H. Wang, unpublished results.

(^4)
J. Chan and L.-H. Wang, unpublished results.

(^5)
M. White, private communication.


ACKNOWLEDGEMENTS

We thank Dr. T. Hunter for anti-annexin II antibody.


REFERENCES

  1. Williams, L. T. (1989) Science 243, 1564-1570 [Medline] [Order article via Infotrieve]
  2. Yarden, Y., and Ullrich, A. (1988) Annu. Rev. Biochem. 57, 443-478 [CrossRef][Medline] [Order article via Infotrieve]
  3. Yarden, Y., and Schlessinger, J. (1987) Biochemistry 26, 1443-1457 [Medline] [Order article via Infotrieve]
  4. Cohen, G. B., Ren, R., and Baltimore, D. (1995) Cell 80, 237-248 [Medline] [Order article via Infotrieve]
  5. Pawson, T., and Gish, G. D. (1992) Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  6. Kavanaugh, W. M., and Williams, L. T. (1994) Science 266, 1862-1865 [Medline] [Order article via Infotrieve]
  7. Gustafson, T., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508 [Abstract]
  8. White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988) Cell 54, 641-649 [Medline] [Order article via Infotrieve]
  9. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A., Cahill, D. A., Goldstein, B. J., and White, M. F. (1991) Nature 352, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  10. Pronk, G. J., McGlade, J., Pelicci, G., Pawson, T., and Bos, J. L. (1993) J. Biol. Chem. 268, 5748-5753 [Abstract/Free Full Text]
  11. Ullrich, A., Gray, A., Tam, A. W., Yang-Feng, T., Tsubokawa, M., Jacobs, S., Francke, U., Ramachandran, J., and Fujita-Yamaguchi, Y. (1986) EMBO J. 5, 2503-2512 [Abstract]
  12. Randszzo, P. A., Morey, A., Polishook, A. K., and Jaret, L. (1990) Exp. Cell. Res. 190, 25-30 [Medline] [Order article via Infotrieve]
  13. Pekonen, F., Partane S., Makinen, T., and Rutanen, E.-M. (1988) Cancer Res. 48, 1343-1347 [Abstract]
  14. Prager, D., Li, H. L., Asa, S., and Melmed, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2181-2185 [Abstract]
  15. Resnicoff, M., Coppola, D., Sell, C., Rubin, R., Ferron, S., and Baserga, R. (1994) Cancer Res. 54, 4848-4850 [Abstract]
  16. Coppola, D., Ferber, A., Miura, M., Sell, C., D'Ambrosio, C., Rubin, R., and Baserga, R. (1994) Mol. Cell. Biol. 14, 4588-4589 [Abstract]
  17. Liu, D., Rutter, W. J., and Wang, L. H. (1992) J. Virol. 66, 374-385 [Abstract]
  18. Liu, D., Rutter, W. J., and Wang, L. H. (1993) J. Virol. 67, 9-18 [Abstract]
  19. Liu, D., Zong, C., and Wang, L. H. (1993) J. Virol. 67, 6835-6840 [Abstract]
  20. Cooper, J., and Hunter, T. (1981) Mol. Cell. Biol. 1, 394-407 [Medline] [Order article via Infotrieve]
  21. Keegan, A. D., Nelms, K., White, M., Wang, L. M., Pierce, J. H., and Paul, W. E. (1994) Cell 76, 811-820 [Medline] [Order article via Infotrieve]
  22. Backer, J. M., Schroeder, G. G., Kahn, C. R., Myers, M. G., Jr., Wilden, P. A., Cahill, D. A., and White, M. F. (1992) J. Biol. Chem. 267, 1367-1374 [Abstract/Free Full Text]
  23. Myers, M. G., Sun, X. J., Cheatham, B., Jachna, B. R., Glasheen, E. M., Backer, J. M., and White, M. F. (1993) Endocrinology 132, 1421-1430 [Abstract]
  24. Hubbard, S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994) Nature 372, 726 [Medline] [Order article via Infotrieve]
  25. Wei, L., Hubbard, S. R., Hendrickson, W. A., and Ellis, L. (1995) J. Biol. Chem. 270, 8122-8130 [Abstract/Free Full Text]
  26. Kohanski, R. (1993) Biochemistry 32, 5773-5780 [Medline] [Order article via Infotrieve]
  27. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., and Rutter, W. J. (1986) Cell 45, 721-732 [Medline] [Order article via Infotrieve]
  28. Zhang, B., Tavare, J. M., Ellis, L., and Roth, R. A. (1991) J. Biol. Chem. 266, 990-996 [Abstract/Free Full Text]
  29. Smith, J. E., Sheng, Z. F., and Kallen, R. G. (1994) DNA Cell Biol. 13, 593-604 [Medline] [Order article via Infotrieve]
  30. Kohanski, R. A. (1993) Biochemistry 32, 5766-5772 [Medline] [Order article via Infotrieve]
  31. Marshall, C. J. (1995) Cell 80, 179-185 [Medline] [Order article via Infotrieve]
  32. Peterson, J. E., Jelinek, T., Kaleko, M., Siddle, K., and Weber, M. J. (1994) J. Biol. Chem. 269, 27315-27321 [Abstract/Free Full Text]
  33. Voliovitch, H., Schindler, D. G., Hadari, Y. R., Taylor, S. I., Accili, D., and Zick, Y. (1995) J. Biol. Chem. 270, 18083-18087 [Abstract/Free Full Text]
  34. Craparo, A., O'Neill, T., and Gustafson, T. (1995) J. Biol. Chem. 270, 15639-15643 [Abstract/Free Full Text]
  35. Ma, A. S., Bystol, M. E., and Tranvan, A. (1994) In Vitro Cell & Dev. Biol. Anim. 30, 329-335
  36. Drust, D. S., and Creutz, C. E. (1991) J. Neurochem. 56, 469-478 [Medline] [Order article via Infotrieve]
  37. Brambilla, R., Zippel, R., Sturani, E., Morello, L., Peres, A., and Alberghina, L. (1991) Biochem. J. 278, 447-452 [Medline] [Order article via Infotrieve]
  38. Johnstone, S. A., Hubaishy, I., and Waisman, D. M. (1992) J. Biol. Chem. 267, 25976-25981 [Abstract/Free Full Text]
  39. Cole, S. P., Pinkoski, M. J., Bhardwaj, G., and Deely, R. G. (1992) Br. J. Cancer 65, 498-502 [Medline] [Order article via Infotrieve]
  40. Tressler, R. J., Updyke, T. V., Yeatman, T., and Nicolson, G. L. (1993) J. Cell. Biochem. 53, 265-276 [Medline] [Order article via Infotrieve]
  41. Vishwanatha, J. K., Chiang, Y., Kumble, K. D., Hollingsworth, M. A., and Pour, P. M. (1993) Carcinogenesis 14, 2575-2579 [Abstract]
  42. Chung, C. Y., and Erickson, H. P. (1994) J. Cell. Biol. 126, 539-548 [Abstract]
  43. Braselman, S., Bergers, G., Wrighton, C., Graninger, P., Superti, G., and Busslinger, M. (1992) J. Cell Sci. (Suppl.) 16, 97-109 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.