Tyrosine-phosphorylated Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Associates with the Adaptor Protein SHC in SRC-transformed Cells*

Helen BarnesDagger §, Brett Larsen||, Mike Tyers, and Peter van der GeerDagger **

From the Dagger  Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0359 and the  Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

Received for publication, December 19, 2000, and in revised form, February 28, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta  chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta -amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein tyrosine phosphorylation is one of several mechanisms that have evolved to mediate signal transduction (1-3). Protein-tyrosine kinases regulate cell division and differentiation in response to extracellular factors. Expression of constitutively active versions of these kinases results in cellular transformation in vitro and tumor formation in vivo (4). Protein-tyrosine kinases come in two varieties, receptor protein-tyrosine kinases and cytoplasmic protein-tyrosine kinases. Receptor protein-tyrosine kinases contain an extracellular ligand binding domain, a single transmembrane domain, and a cytoplasmic kinase domain (5). Upon activation, receptors autophosphorylate on tyrosine residues thereby creating binding sites for phosphotyrosine binding (PTB)1 domain and Src homology 2 (SH2) domain containing signaling proteins. These proteins are activated directly or indirectly as a consequence of their interaction with the receptor (6, 7). In general, association with the receptor precedes tyrosine phosphorylation and activation of receptor substrates (1-3).

The extracellular ligand binding domain is missing from cytoplasmic protein-tyrosine kinases (8). They are often found associated with receptors that lack kinase domains themselves and are thought to function as signal transducing subunits for these receptors (9). Curiously, most cytoplasmic protein-tyrosine kinases also lack the autophosphorylation sites that act as binding sites for their substrates (8). In some cases these sites are located on the associated non-kinase receptors (9, 10). In general, it remains to be established how activated cytoplasmic protein-tyrosine kinases interact with their substrates.

To address this issue, we have investigated tyrosine phosphorylation of Shc in v-Src-transformed cells. Shc is an adaptor protein that is involved in signal transduction by protein-tyrosine kinases (11-13). The shc gene encodes three proteins that all contain an amino-terminal PTB domain, a central region that contains several tyrosine phosphorylation sites, and a carboxyl-terminal SH2 domain (11). PTB domains bind to tyrosine residues in the context of an NPXY motif (14, 15). The Shc PTB domain binds to phosphorylated NPXY motifs preceded by a leucine, isoleucine, or valine residue five residues upstream of the phosphorylated tyrosine (16). The Shc SH2 domain binds to phosphorylated tyrosine residues in the context of acidic or non-polar residues carboxyl-terminal to the tyrosine (17). Tyrosine phosphorylation sites on Shc function as binding sites for other signaling proteins. It is well established that tyrosine-phosphorylated Shc binds to Grb2 (18, 19). It is through the interaction with the Grb2-Sos complex that Shc is thought to be involved in activation of Ras and the mitogen-activated protein kinase pathway (7). Following activation of receptor protein-tyrosine kinases such as the epidermal growth factor receptor or the nerve growth factor receptor, Shc uses either its PTB or its SH2 domain to bind to specific tyrosine phosphorylation sites on the receptor. Binding to the receptor is a prerequisite for tyrosine phosphorylation of Shc, and it is tyrosine-phosphorylated Shc that recruits the Grb2-Sos complex to the receptor at the inner leaf of the plasma membrane, in close proximity to Ras.

v-Src is an activated version of the cytoplasmic protein-tyrosine kinase c-Src. Expression of v-Src results in protein tyrosine phosphorylation and in oncogenic transformation (20, 21). v-Src is localized to cellular membranes through a myristyl group that is attached to its amino terminus (22). Following its unique amino terminus Src contains an SH2 domain, a Src homology 3 (SH3) domain, and a protein-tyrosine kinase domain. The only well characterized autophosphorylation site in v-Src is present within its kinase domain and is known to regulate kinase activity (23, 24). In the wild type protein the SH2 and the SH3 domain are involved in regulation of kinase activity (25, 26). There are some reports that suggest that in addition the SH2 and SH3 domains may play a role in substrate selection. On the other hand, many Src mutants with inactive SH2 and SH3 domains have maintained their transforming ability (27). This suggests that neither the SH2 domain nor the SH3 domain is necessary for the selection of substrates that are critical for cellular transformation. A large number of proteins are phosphorylated on tyrosine in v-Src-transformed cells. It remains unresolved how these proteins are selected as substrates and which of these substrates mediate cellular transformation.

Shc is highly phosphorylated in v-Src-transformed cells, and it is thought that it is Shc that mediates the Ras activation that occurs downstream of v-Src (13, 18). Consistent with this model, it is found that in v-Src-transformed cells Shc is associated with Grb-2 and Sos (28). v-Src itself does not bind to Shc, and it remains unclear exactly how v-Src selects Shc for tyrosine phosphorylation. Furthermore, if Shc indeed mediates Ras activation it needs to recruit Grb2-Sos to the plasma membrane, and it remains unresolved how this is accomplished. To address some of these issues, we asked whether Shc relies on the presence of a functional PTB or SH2 domain for phosphorylation by v-Src. Our data show that Shc depends on a functional PTB domain for its phosphorylation in v-Src-expressing cells. The PTB domain binds to a 100-kDa protein that was purified and identified as the low density lipoprotein (LDL) receptor-related protein LRP1. LRP1 is a heterodimer that consists of a 500-kDa extracellular subunit that is non-covalently linked to a smaller (80-100 kDa) subunit. The small subunit contains an extracellular domain, a transmembrane domain, and a short cytoplasmic domain. Two NPXY motifs are present in the cytoplasmic domain of LRP1. LRP1 is thought to function as an import receptor for a variety of proteins including apoE, alpha 2-macroglobulin, and beta -amyloid precursor protein (29). The NPXY motifs were previously thought to mediate receptor internalization (30). Our data suggest that at least one of the NPXY motifs present in LRP1 is involved in a signaling process that depends on protein phosphorylation and PTB domain containing signaling proteins.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- COS-1, 14.30, 14.30R1, and 1430S3 cells were grown in Dulbecco-Vogt's modified Eagle's medium containing 10% calf serum. 14.30 cells are mouse fibroblasts established from a wild type embryo at day 9.5 of development (31). 14.30 cells were transformed in vitro by transfection with mammalian expression constructs containing the v-src and the EJ-Ras genes (31). S7A is a v-Src-transformed Rat-2 cell line (32). S7A cells were grown in Dulbecco's modified Eagle's medium containing 5% calf serum and 5% fetal bovine serum. The hybridoma 11H4 (obtained from ATCC, Manassas, VA) was grown in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 25 mM L-glutamine. A polyclonal anti-Shc serum was raised against a GST-Shc SH2 fusion protein. Anti-phosphotyrosine monoclonal antibody 4G10 and anti-HA monoclonal antibody 12CA5 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and Babco (Richmond, CA).

Transient Expression of Wild Type and Mutant HA-tagged Shc Polypeptides in Mammalian Cells-- Wild type and mutant human Shc cDNAs were cloned into the mammalian expression vector pcDNA3. A Kozak consensus sequence for initiation of protein synthesis and a sequence encoding the hemagglutinin epitope (HA) starting with a methionine were linked in frame by a sequence encoding 13 amino acids to the Shc cDNA starting at the first residue of the 52-kDa isoform. Mutants were generated using a polymerase chain reaction-based strategy. Mutant fragments were sequenced to ensure fidelity and cloned back into the full-length cDNA. COS-1 cells were transfected at 50% confluency with 5 µg of HA-Shc cloned into pcDNA3 with or without 1 µg of v-src cloned into pECE; transfections were carried out using Lipofectin and Opti-MEM (Life Technologies, Inc.) according to the manufacturer's directions. Cells were lysed 72 h after transfection.

GST Fusion Protein-- The GST-Shc PTB fusion protein contains residues 1-225 and the GST-Shc SH2 domain contains residues 387-473. Mutants were generated using a polymerase chain reaction-based strategy. Mutant cDNA fragments were sequenced to ensure fidelity and cloned back into the pGEX constructs. Expression of fusion proteins was induced by incubation in the presence of 100 µM isopropyl-1-thio-beta -D-galactopyranoside for several hours at 30 °C. Bacteria were lysed by sonication in phosphate-buffered saline (PBS) containing 1 mM DTT, 1 mM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Triton X-100 was added to a final concentration of 1%; insoluble material was removed by centrifugation for 10 min at 10,000 × g at 4 °C, and fusion proteins were purified by binding to glutathione-agarose. Glutathione-agarose beads were collected by centrifugation and washed 5 times with PBS containing 1% Triton X-100, 1 mM DTT, 1 mM benzamidine. Fusion proteins were stored bound to beads at 4 °C in PBS containing 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 0.02% sodium azide, and 10% glycerol.

Mass Spectrometry-- Protein samples were resolved by SDS-PAGE, and protein bands were excised and digested in the gel as described previously (33, 34). Briefly, proteins were reduced by incubating with 10 mM DTT and alkylated using 50 mM iodoacetamide. Digestion was performed with trypsin at 37 °C, and resulting peptides were extracted from the gel using ammonium bicarbonate, acetonitrile, and formic acid. Samples were dried and desalted using Millipore ZipTips prior to mass spectrometry analysis. Mass spectrometry was performed on a Sciex QSTAR mass spectrometer (MDS-Sciex, Concord, Ontario, Canada), and data were analyzed using PeptideSearch.

Immunoprecipitation-- Cells were grown to subconfluence on 10-cm tissue culture dishes. Cells were rinsed twice with cold PBS and lysed in 1 ml of 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 500 µM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (PLC lysis buffer) per 10-cm tissue culture dish. Lysates were cleared by centrifugation at 10,000 rpm in a microcentrifuge at 4 °C, incubated with 5 µl of polyclonal antiserum or 1 µg of monoclonal antibody for 1 h on ice, and subsequently for 1 h with 100 µl of 10% protein A-Sepharose or anti-mouse IgG-Sepharose for 1 h at 4 °C on an agitator. Sepharose beads were collected by centrifugation and washed 4 times with PLC lysis buffer. Immunoprecipitates were boiled 3 min in 62.5 mM Tris/Cl, pH 6.8, 10% glycerol, 5% beta -mercaptoethanol, 5 mM DTT, 2.3% SDS, and 0.025% bromphenol blue (SDS sample buffer) and resolved by SDS-PAGE.

RIPA (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) lysates were cleared by incubation with excess Pansorbin (Calbiochem) and centrifugation for 10 min in a microcentrifuge at 10,000 rpm at 4 °C. To disrupt previously formed protein-protein complexes, cells from a 10-cm tissue culture dish were collected and boiled for 3 min in 200 µl of 10 mM sodium phosphate, 0.5% SDS, 1 mM EDTA, and 1 mM DTT. After boiling the lysates were diluted in 800 µl of 50 mM sodium phosphate, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, 100 µM sodium orthovanadate. Boiled lysates were cleared by incubation with excess Pansorbin (Calbiochem) and centrifugation for 10 min in a microcentrifuge at 10,000 rpm at 4 °C.

For in vitro binding studies, fibroblast cell lysates were incubated for 1 h at 4 °C on a rocker with 10 µl of glutathione-agarose beads containing 1-2 µg of fusion protein. Beads were washed 4 times with PLC lysis buffer, and bound proteins were analyzed by SDS-PAGE and immunoblotting.

Membrane Isolation-- Cells were lysed by swelling in hypotonic lysis buffer (20 mM Tris/Cl, pH 7.5, 1 mM MgCl2, 2 mM EGTA, 0.1 mM sodium vanadate) on ice and by Dounce homogenization. Crude membranes for protein purification were isolated by centrifugation for 45 min at 25000 × g at 4 °C. For separation into soluble and membrane-bound fractions, nuclei were removed by centrifugation for 10 min at 8000 × g at 4 °C, followed by centrifugation for 60 min at 100,000 × g at 4 °C.

Immunoblotting-- Proteins were transferred to polyvinylidene difluoride membranes using a Bio-Rad semi-dry blotting apparatus at 50 mA per gel for 60 min at room temperature. Membranes were blocked for 1 h at room temperature in 10 mM Tris/Cl, pH 7.4, 150 mM NaCl, 0.2% Tween 20 (TBST) containing 5% dried milk and incubated with 1:200 dilution of polyclonal antisera in TBST 5% milk for 1 h at room temperature. Blots were washed twice for 10 min with TBST and twice for 5 min with 10 mM Tris/Cl, pH 7.4, and 150 mM NaCl (TBS). Membranes were then incubated for 30 min with horseradish peroxidase-protein A or horseradish peroxidase goat anti-mouse (Bio-Rad) diluted 1:10,000 in TBST and washed as before. Reactive proteins were visualized by ECL (Amersham Pharmacia Biotech). For immunoblotting with anti-Tyr(P) serum, membranes were blocked with TBST containing 5% bovine serum albumin followed by an incubation with the antiserum diluted in TBST 5% bovine serum albumin. Washes and detection were done exactly as described for other antisera.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Shc adaptor protein is a major substrate for tyrosine phosphorylation in v-Src-transformed cells (13). It has, however, remained poorly defined exactly how Shc and Src interact. To address this question we developed HA-tagged versions of the 52-kDa isoform of Shc with inactivating mutations in either the PTB (M175) or the SH2 (M401) domain (Fig. 1). These mutant domains have lost the ability to bind with high affinity to their binding sites. A wild type version and a version with mutations in both domains were used as controls (2M) (Fig. 2). To test whether Shc depends on either its PTB domain or its SH2 domain for phosphorylation by v-Src, the HA-tagged mutants were expressed transiently together with v-Src in COS-1 cells. Anti-HA immunoprecipitates were analyzed by anti-Tyr(P) and anti-Shc immunoblotting. The data indicate that v-Src phosphorylates wild type Shc or Shc that lacks a functional SH2 domain (Fig. 2A). Mutation of the PTB domain completely blocks Shc tyrosine phosphorylation in v-Src-transformed cells (compare lanes 2 and 3 or 4 and 5 in Fig. 2A). Mutation of the SH2 domain results in a small decrease in tyrosine phosphorylation. The various Shc proteins were expressed at similar levels (Fig. 2B). We have shown that Shc does not bind directly to Src.2 These observations suggest that Shc uses its PTB domain to bind to an unidentified protein before it can be phosphorylated by v-Src.


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Fig. 1.   Shc proteins. An HA-tagged version of the p52 isoform of Shc was cloned into pcDNA3. To inactivate the PTB domain Arg-175 was mutated to Met (M175), and to inactivate the SH2 domain Arg-401 was mutated to Met (M401). A mutant in which both residues were mutated (2M) was also used. The PTB domain (residues 1-225) and the SH2 domain (residues 387-473) were expressed separately as GST fusion proteins in Escherichia coli. M175 and M401 mutant GST fusion proteins were used as controls.


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Fig. 2.   Shc depends on a functional PTB domain for phosphorylation by the v-Src protein-tyrosine kinase. Wild type or mutant HA-tagged Shc proteins were expressed transiently together with v-Src in COS-1 cells. Anti-HA immunoprecipitates were analyzed by anti-Tyr(P) (A) and anti-Shc (B) immunoblotting. COS-1 cells were transfected with v-Src and a control vector, lane 1; v-Src and wild type HA-Shc, lane 2; v-Src and M175 HA-Shc, lane 3; v-Src and M401 HA-Shc, lane 4; v-Src and 2M HA-Shc, lane 5.

To investigate further the role of the PTB domain during phosphorylation of Shc in v-Src-transformed cells, we used GST fusion proteins containing the PTB domain (Fig. 1) to look for the presence of PTB domain binding proteins. Cell lysates of Src-transformed fibroblasts (14.30S3) were incubated with fusion proteins that were immobilized on glutathione-agarose beads, and bound proteins were analyzed by anti-Tyr(P) blotting. As a control, lysates of Ras-transformed fibroblasts (14.30R1) were also incubated with these fusion proteins. Shc immunoprecipitates were analyzed in parallel. This experiment confirms that Shc is highly tyrosine-phosphorylated in Src-transformed cells. Tyrosine phosphorylation of all three isoforms of Shc (p46, p52 and p66) can be detected (Fig. 3, lane 4). Several unidentified, tyrosine-phosphorylated proteins are present in Shc immunoprecipitates from Src-transformed cells (Fig. 3, lane 4). As expected, Shc is not tyrosine-phosphorylated or associated with tyrosine-phosphorylated proteins in Ras-transformed cells (Fig. 3, lanes 1-3). Several tyrosine-phosphorylated proteins present in lysates of Src-transformed cells were able to bind to either the PTB or the SH2 domain of Shc (Fig. 3, lanes 5 and 6). Most of these proteins also appeared to be present in the Shc immunoprecipitate, suggesting that they represent true Shc-interacting proteins. Since Shc depends on its PTB domain for phosphorylation by v-Src, we focused on a 100-kDa protein (p100) that was present in Shc immunoprecipitates and that co-migrates with a protein that bound to the PTB domain (Fig. 3, lanes 4 and 5). P100 did not bind to the GST-SH2 domain fusion protein.


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Fig. 3.   A 100-kDa Shc PTB-binding protein co-immunoprecipitates with Shc from v-Src-transformed cells. Anti-Shc immunoprecipitates, GST-Shc PTB domain binding proteins, and GST-Shc SH2 domain binding proteins from v-Src-transformed (14.30S3, lanes 4-6) and Ras-transformed (14.30R1, lanes 1-3) fibroblasts were analyzed by anti-Tyr(P) blotting. Anti-Shc immunoprecipitates, lanes 1 and 4; GST-PTB-binding proteins, lanes 2 and 5; GST-SH2-binding proteins lanes 3 and 6.

To find out whether p100 is truly a specific Shc-PTB domain binding protein we tested the ability of an inactive GST-Shc PTB domain fusion protein to bind to p100 (Fig. 4). In addition, Shc immunoprecipitates from Src-transformed cells lysed either in PLC lysis buffer, in RIPA, or by boiling in 0.5% SDS were tested for the presence of p100 (Fig. 4). PLC lysis buffer is a mild lysis buffer that contains 1.0% Triton X-100. RIPA is a more stringent lysis buffer that contains 1.0% Nonidet P-40, 1.0% deoxycholate, and 0.1% SDS. During boiling lysis all proteins are denatured by boiling in a small volume of 0.5% SDS, and the lysate is subsequently diluted in RIPA without SDS. Protein-protein interactions that depend on the presence of functional protein-protein interaction domains are broken up. The data show that mutation of Arg-175 in the phosphotyrosine-binding site to Met results in a loss of p100 binding (Fig. 4, lane 1). Furthermore, most proteins that co-immunoprecipitate with Shc in PLC lysis buffer or RIPA, including p100, are lost upon boiling of the lysate (Fig. 4, lane 5). These data indicate that p100 specifically binds to Shc in v-Src-transformed cells and that binding depends on the presence of a functional PTB domain.


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Fig. 4.   The association between Shc and p100 depends on a functional PTB domain. Anti-Tyr(P) blot of proteins bound to the wild type or the inactive M175 mutant PTB domain analyzed in parallel with Shc immunoprecipitates from v-Src-transformed cells lysed in PLC lysis buffer, lysed in RIPA, or lysed by boiling in 0.5% SDS. Proteins bound to the M175 mutant PTB domain, lane 1; proteins bound to the wild type PTB domain, lane 2; Shc immunoprecipitate from Src-transformed cells lysed in PLC lysis buffer, lane 3; Shc immunoprecipitate from Src-transformed cells lysed in RIPA, lane 4; Shc immunoprecipitate from Src-transformed cells lysed by boiling in 0.5% SDS, lane 5.

To find out where in the cell p100 is localized, v-Src-transformed cells were lysed by swelling in hypotonic lysis buffer and Dounce homogenization. Nuclei were removed by centrifugation, and the lysate was separated into a soluble and a particulate fraction by centrifugation for 1 h at 100,000 × g. Both fractions were suspended in PLC lysis buffer and tested for the presence of PTB-binding proteins by incubation with the immobilized fusion protein. p100 was found to be present in the particulate fraction (Fig. 5A). This experiment indicates that p100 is associated with cellular membranes. In parallel, we have analyzed Shc immune precipitates and found that 20-30% of cellular Shc is present in the particulate fraction in v-Src-transformed cells (Fig. 5B).


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Fig. 5.   P100 and Shc are present in the particulate fraction. S7A cells were lysed in hypotonic lysis buffer and separated by centrifugation into a particulate and a soluble fraction. A, GST-PTB-binding proteins present in the particulate fraction (P100, lane 1) and soluble fraction (S100, lane 2) fractions were detected by anti-Tyr(P) blotting. B, Shc immunoprecipitates from the particulate fraction (P100, lane 1) and soluble fraction (S100, lane 2) fractions were analyzed by anti-Shc immunoblotting.

In order to establish its identity, p100 was purified from membranes of v-Src-transformed S7A cells based on its ability to bind to the PTB domain. To increase recovery of p100, cells were treated before lysis with orthovanadate and peroxide, which inhibit cellular protein-tyrosine phosphatases. In a mid-sized experiment, membranes obtained from 2 × 108 cells were resuspended in 20 ml of PLC lysis buffer and incubated batchwise with ~2 mg of GST-PTB fusion protein immobilized on 100 µl of glutathione-agarose beads. The beads were washed, poured into a small column, and bound proteins were eluted with 100 mM triethylamine at pH 11.5. Six 50-µl fractions were collected and analyzed by anti-Tyr(P) blotting (Fig. 6A). Tyrosine-phosphorylated p100 was eluted efficiently from the PTB column. As a control, the purification was repeated with the inactive M175 mutant GST-PTB fusion protein, and the peak fractions eluted from the PTB and M175 PTB column were compared by anti-Tyr(P) immunoblotting and silver staining (Fig. 6, B and C). No p100 could be detected when the M175 PTB domain was used (Fig. 6, B and C). A silver-stainable band with a molecular mass of ~100 kDa was present in the material eluted from the PTB column (Fig. 6C). As expected, this band was missing in the material eluted from the M175 mutant PTB column.


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Fig. 6.   Purification of P100 using the GST-Shc PTB domain as an affinity reagent. 2 × 108 Src-transformed fibroblasts were lysed by Dounce homogenization in hypotonic lysis buffer. Membranes were isolated by high speed centrifugation, resuspended in PLC lysis buffer, and incubated with GST-PTB or M175 mutant GST-PTB domain fusion proteins, immobilized on glutathione-agarose. Proteins were eluted with 100 mM triethylamine, pH 11.5, and fractions were analyzed by anti-Tyr(P) blotting. A, 0.2% of the first six 50-µl fractions collected from the PTB column were analyzed by anti-Tyr(P) blotting. B and C, fractions 3 from the PTB column and the M175 PTB column were analyzed in parallel by anti-Tyr(P) blotting (B) and by silver staining (C).

To obtain sufficient material for identification of p100, the purification was scaled up 5-fold. Approximately 100 ng of p100 was purified and digested with trypsin, and fragments were analyzed by mass spectrometry. Several fragments were selected for fragmentation, and the resultant mass spectra were used to search the data base. The data for five peptides matched the small subunit of the LDL receptor-related protein LRP1 (Fig. 7).


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Fig. 7.   P100 is identical to the LDL-related protein LRP1. Approximately 100 ng of P100 was purified from 109 cells and subjected to tryptic digestion. Tryptic peptides were identified by mass spectrometry. The sequence of the LRP1 beta  chain and the tryptic peptide fragments identified by mass spectroscopy are shown. Amino acids are numbered starting at the first residue of the beta  chain. The transmembrane sequence is underlined, and the two NPXY motifs are boxed.

To confirm that p100 is indeed LRP1, GST-PTB and GST-M175 PTB-bound proteins from control and orthovanadate/peroxide-treated cells were analyzed in parallel with anti-LRP1 immunoprecipitates by anti-Tyr(P) and anti-LRP1 immunoblotting. LRP1 immunoprecipitated from v-Src-transformed cells appeared to be tyrosine-phosphorylated (Fig. 8A, lanes 5 and 6). Tyrosine-phosphorylated LRP1 comigrated with p100 (Fig. 8A, lanes 4-6). Orthovanadate/peroxide treatment increased tyrosine phosphorylation of LRP1 (Fig. 8A, lanes 5 and 6) and resulted in a small decrease in mobility during SDS-PAGE (Fig. 8B, lanes 5 and 6). p100 could be detected by immunoblotting with the anti-LRP1 monoclonal antibody (Fig. 8B). No LRP1 was bound to the M175 mutant GST-PTB fusion protein.


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Fig. 8.   LRP1 binds to the Shc PTB domain in vitro. Lysates from control and vanadate/peroxide-treated v-Src-transformed fibroblasts were incubated with wild type GST-Shc PTB and M175 mutant GST-Shc PTB fusion protein immobilized on glutathione-agarose. Bound proteins were analyzed by anti-Tyr(P) immunoblotting (A) and anti-LRP1 immunoblotting (B). Anti-LRP1 immunoprecipitates were analyzed in parallel.

To test if Shc binds to LRP1 in cells, Shc and LRP1 immunoprecipitates were analyzed in parallel by anti-Tyr(P) and anti-LRP1 immunoblotting (Fig. 9). p100 present in Shc immunoprecipitates comigrates with tyrosine-phosphorylated LRP1 and can be detected by immunoblotting with the anti-LRP1 antibody (Fig. 9). No LRP1 was present in immunoprecipitates with a pre-immune serum. These data clearly establish that LRP1 becomes tyrosine-phosphorylated in cells containing the activated v-Src protein-tyrosine kinase and that tyrosine-phosphorylated LRP1 associates with the adaptor protein Shc. This association is mediated by the PTB domain of Shc.


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Fig. 9.   Shc binds to LRP1 in Src-transformed cells. Anti-Shc and anti-LRP1 immunoprecipitates from control and vanadate/peroxide-treated v-Src-transformed cells were analyzed by anti-Tyr(P) (A) and anti-LRP1 (B) immunoblotting. Immunoprecipitates with a control preimmune serum were analyzed in parallel.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been well established that Shc proteins are highly tyrosine-phosphorylated in cells transformed by the v-Src protein-tyrosine kinase (13). Tyrosine phosphorylation in v-Src-transformed fibroblasts results in association of Shc with Grb2 and Sos (18). This suggests that Shc may function as a link between Src and the Ras-mitogen-activated protein kinase pathway. It remains unclear exactly how this link is established. In order to play a role in Ras activation Shc needs to localize to the membrane. How translocation to the membrane is accomplished during v-Src transformation remains unresolved. Furthermore, there are no reports in the literature that indicate whether or not the PTB or the SH2 domain are involved in this process. Finally, it remains to be established exactly how Src and Shc interact.

In an attempt to address some of these issues, we tested whether either the PTB or the SH2 domain of Shc is essential for phosphorylation to occur in v-Src-expressing cells. The PTB domain was found to be absolutely required for phosphorylation of Shc by v-Src in cells. Mutation of the SH2 domain results in a small but reproducible decrease in phosphorylation. Our data are consistent with a model in which phosphorylation of Shc by v-Src depends on the association of Shc through its PTB domain with a tyrosine-phosphorylated membrane protein (Fig. 10). The SH2 domain plays some role during Shc phosphorylation but appears not to be essential.


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Fig. 10.   Model for the role of LRP1 during transformation by v-Src. v-Src is present at the plasma membrane in close proximity to LRP1. As a consequence, LRP1 becomes phosphorylated on tyrosine residues, and this creates a binding site for the Shc PTB domain. Recruitment of Shc to the membrane results in its tyrosine phosphorylation and possibly the recruitment of additional signaling proteins that mediate the activation of downstream signal transduction pathways.

The PTB domain was found to bind to a 100-kDa protein that was also present in Shc immunoprecipitates. This protein was purified from Src-transformed Rat fibroblasts and identified by mass spectrometry as the small subunit of LRP1. This observation identifies LRP1 as a novel substrate for the v-Src protein-tyrosine kinase. LRP1 tyrosine phosphorylation may be important for signal transduction downstream of v-Src.

To test whether LRP1 is the only 100-kDa protein that associates with Shc, we tried, unsuccessfully, to remove the 100-kDa tyrosine-phosphorylated protein by repeated immune precipitation with an anti-LRP1 antibody. This suggests that there might be a second 100-kDa protein that associates with Shc in v-Src-transformed cells. Alternatively, this may indicate that the epitope for the anti-LRP1 monoclonal antibody used in these studies is less accessible when LRP1 is tyrosine-phosphorylated.

LRP1 is a member of a small family of proteins that are related to the LDL receptor (29). Members of the LDL receptor family are involved in the receptor-mediated endocytosis of proteins, lipoproteins, and protein complexes (29). To accomplish this, these receptors cycle continuously between the cell surface and cytoplasmic vesicles. LRP1 is composed of a large 500-kDa and a small 80-90-kDa subunit (35, 36). The large subunit is entirely extracellular and is linked non-covalently to the small subunit. The small subunit contains an extracellular domain, a single membrane spanning domain, and a cytoplasmic domain of 100 amino acids (35, 36). The cytoplasmic domain contains two NPXY motifs (residues 527-530 and 561-564, Fig. 7). NPXY motifs are present in all members of the LDL receptor family and were originally identified as internalization signals (30).

More recently NPXY motifs were characterized as binding sites for PTB domains (16, 37). A number of PTB domains have been identified, some of which bind phosphorylated NPXY motifs, whereas others bind unphosphorylated NPXY motifs (15). Binding specificities are further defined by residues upstream of the NPXY sequence (38-40). The Shc PTB domain binds specifically to the sequence phiXNPXpY, in which phi is Val, Leu, or Ile (16) and pY is Tyr(P). The first NPXY motif in the cytoplasmic domain of LRP1 conforms to this consensus (35). The notion that the Shc PTB domain binds only to phosphorylated NPXY motifs is in agreement with our observation that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells. All data are consistent with a model in which LRP1 phosphorylation plays an important role during cellular transformation by v-Src (Fig. 10). LRP1 is present at the membrane, presumably in proximity to v-Src. Upon phosphorylation of LRP1, Shc is recruited to the membrane where it becomes a substrate for the v-Src protein-tyrosine kinase. Phosphorylation by v-Src creates a binding site for Grb2 and results in the recruitment of Grb2-Sos to the membrane. In this model LRP1 acts as an anchor for Shc and Grb2-Sos at the membrane (Fig. 10). Translocation of Sos to the membrane is a prerequisite for Ras activation (41, 42). Our data are in conflict with a previous study that failed to detect an interaction between the Shc PTB domain and LRP1 (40). It is most likely that unphosphorylated recombinant LRP1 was used in this study. Our results indicate that association between LRP1 and Shc depends on LRP1 tyrosine phosphorylation and that the association is mediated by the Shc PTB domain.

We have failed to detect LRP1 tyrosine phosphorylation in fibroblast cell lines following activation of a variety of receptor protein-tyrosine kinase, and it appears that LRP1 is specifically phosphorylated by cytoplasmic protein-tyrosine kinases.2 Our data suggest that LRP1 can become phosphorylated on tyrosine residues and that tyrosine-phosphorylated LRP1 is involved in signal transduction with the help of phosphotyrosine-binding adaptor proteins.

Our observations are consistent with recent reports that indicate that two other members of the LDL receptor family are also involved in signal transduction (43). Disruption of the genes for the VLDL receptor and the ApoE receptor 2 results in defective development of the cerebral cortex and the cerebellum in mice (44). The cerebral cortex and the cerebellum have a characteristic layering of neurons that is disorganized in the mutant mice, most likely as a consequence of defects in cell migration (44). This suggests that the VLDL receptor and ApoE receptor 2 are essential for processing of positional information by migrating neurons. Interestingly, the phenotype of the VLDLR-/-ApoE-/- mice closely resembles the phenotypes encountered in mice that lack the genes for either Reelin or Disabled (45, 46). Reelin is a large glycoprotein that is secreted in particular regions of the brain and is thought to provide positional information to migrating neurons (47). Disabled is a cytoplasmic PTB-containing protein that can be phosphorylated by a variety of protein-tyrosine kinases. The current model is that the VLDL receptor and the ApoE receptor 2 act as signaling receptors that relay the presence of Reelin on the outside of the cell to Disabled on the inside of the cell (47). Thus far there is no evidence that either the VLDL receptor or the ApoE receptor 2 themselves are phosphorylated on tyrosine residues.

The second NPXY motif in LRP1 is present in the context of a phenylalanine five residues upstream of the tyrosine (35). An (Y/F)XNPXY motif is present in al members of the LDL receptor family (29). There is evidence that this sequence binds in vitro and in vivo to the Disabled PTB domain (39, 48). The Disabled PTB domain, in contrast to the Shc PTB domain, binds to unphosphorylated NPXY motifs that contain an aromatic amino acid five residues upstream of the tyrosine (39). This suggests that the second NPXY motif in LRP1 could function as a binding site for Disabled. The hypothesis that NPXY motifs in LRP1 are involved in interactions with signaling molecules is consistent with the recent observation that these motifs are dispensable for LRP1 internalization (49).

We detected several different forms of the LRP1 beta  chain that differ from each other in mobility during SDS-PAGE (Figs. 8 and 9). All beta  chains migrate as the high molecular weight isoform upon treatment of the cells with vanadate and peroxide, which inhibits protein-tyrosine phosphatases. This suggests that the different isoforms represent phosphorylation state isomers and indicates that there are multiple phosphorylation sites present in LRP1. Our data are in agreement with the previously reported observation that LRP1 can be labeled in vivo with [32P]orthophosphate (50). If tyrosine phosphorylation of LRP1 occurs on both NPXY tyrosines, activation of Src could inhibit the association with Disabled and increase the association with Shc. This would likely result in a change in signaling output due to the shift in the association with adaptor proteins, from Disabled to Shc.

Shc depends on its PTB domain for tyrosine phosphorylation in v-Src-expressing cells. We failed to detect a direct interaction between Src and Shc; this is consistent with the absence of a PTB domain binding site in Src. The Shc PTB domain binds in vivo and in vitro to the LRP1 beta  subunit. This interaction is most likely mediated by the first NPXY motif in the cytoplasmic domain of LRP1 beta  chain. This suggests that LRP1 is an important target for tyrosine phosphorylation in v-Src-transformed cells. Following tyrosine phosphorylation LRP1 becomes an anchor for Shc at the plasma membrane, and this might prove essential for Shc tyrosine phosphorylation, recruitment of the Grb2-Sos complex, and Ras activation. The data further suggest that LRP1 may have a signaling function that involves cytoplasmic protein-tyrosine kinases and PTB domain-containing adaptor proteins. These observations are relevant to understanding the physiological function of LRP1, its role during signaling by the v-Src kinase, and its role during neurodegeneration.

    FOOTNOTES

* This work was supported in part by Grant 1R29 CA78629 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health Training Grant DK 07233.

|| Current address: MDS/Ocata, Suite 401, 480 University Ave., Toronto, Ontario, M5G 1V2 Canada.

** To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0359. Tel.: 858-822-2024; Fax: 858-534-6174; E-mail: geer@ucsd.edu.

Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M011437200

2 H. Barnes and P. van der Geer, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PTB, phosphotyrosine binding; LDL, low density lipoprotein; PBS, phosphate-buffered saline; SH2, Src homology-2; SH3, Src homology 3; LRP1, LDL receptor-related protein 1; HA, hemagglutinin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; VLDL, very low density lipoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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