Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules.

The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.

The low density lipoprotein receptor-related protein (LRP) 1 is a large endocytic receptor that was initially identified as the molecule responsible for mediating the uptake of ␣ 2 -macroglobulin-protease complexes and apoE-enriched lipoproteins (1)(2)(3)(4)(5)(6). LRP is synthesized as a single chain 600-kDa precursor that is processed by furin into a 515-kDa heavy chain and an 85-kDa light chain (7). The heavy chain of LRP contains multiple clusters of cysteine-rich low density lipoprotein receptor class A repeats that function in ligand recognition, whereas the light chain contains multiple epidermal growth factor repeats, a transmembrane domain, and a cytoplasmic domain. In addition to ␣ 2 -macroglobulin-protease complexes and apoE-containing lipoproteins, LRP also recognizes proteases such as tissuetype plasminogen activator (8) and matrix metalloproteinase-9 (9), serpin-enzyme complexes (10), and matrix proteins such as thrombospondin-1 (11). Upon binding to LRP, ligands are rapidly internalized via coated pit-mediated endocytosis and are subsequently degraded in lysosomes. LRP also plays a role in the phagocytosis of apoptotic cells (12). This may require association of LRP with a specific adaptor protein, CED-6/GULP, as recent data reveal that the ced-6/gulp gene in Drosophila is one of seven genes required for engulfment (13) and that the human homolog of CED-6/GULP binds to the second NPXY motif within the LRP cytoplasmic domain (14).
Although its function as a cargo transporter is well established, evidence is accumulating indicating that LRP may also play important roles in modulating signaling events. Thus, LRP has been implicated as a component of the receptor complex for midkine, a heparin-binding growth factor with migration-promoting and survival-promoting activities (15), and has been suggested to be responsible for the effect that tissue-type plasminogen activator has on late phase long-term potentiation (16). Tissue-type plasminogen activator has also been shown to directly increase vascular permeability in the early stages of blood-brain barrier opening through an LRP-mediated cell signaling event (17). Another LRP ligand, activated ␣ 2 -macroglobulin, mediates calcium influx specifically in neurons present in primary cultures of mouse cortex upon association with LRP (18). LRP is phosphorylated at Tyr 63 within its cytoplasmic domain via platelet-derived growth factor (PDGF) receptor activation (19,20) and modulates the functional activity of the PDGF receptor in atherosclerotic mouse models (21).
Mechanisms that direct LRP to function in cargo transport or in signaling pathways remain unknown, but likely involve the association of specific adaptor proteins with its cytoplasmic tail. This portion of LRP contains two NPXY motifs that are capable of interacting with cytoplasmic adaptor proteins harboring phosphotyrosine-binding (PTB) domains (22,23). Phosphorylation of Tyr 63 , which is located within the terminal NPXY motif in the LRP cytoplasmic domain, provides a recog-nition site for Shc (20,24), an adaptor protein that participates in signaling pathways. In addition to tyrosine phosphorylation initiated by growth factor activation, the LRP cytoplasmic domain is also phosphorylated at serine residues by protein kinase A (25). At this time, however, the role of serine phosphorylation in LRP function remains unknown. Mutation of a critical serine residue in the cytoplasmic tail of an LRP minireceptor slightly delays LRP-mediated endocytosis, suggesting a role for phosphorylation of LRP in endocytosis (25). In this study, we set out to investigate the hypothesis that phosphorylation of the cytoplasmic domain of LRP modulates its function by altering the affinity for cytoplasmic adaptor proteins. Our results reveal that serine and threonine phosphorylation of LRP modulates its phosphorylation at Tyr 63 , where a number of adaptor molecules bind, and modifies its association with adaptor proteins such as Shc, Dab-1 (Disabled-1), and CED-6/GULP.
Expression Constructs-The expression vector of glutathione S-transferase (GST) fusion protein with the LRP cytoplasmic domain was prepared using the pGEX2 vector. The cytoplasmic domain cDNA fragment encoding amino acids 4426 -4525 of human full-length LRP was amplified by PCR using the full-length cDNA of human LRP (28) as a template. For convenience, the first amino acid following the transmembrane domain was numbered 1 as suggested by Li et al. (29). Thr 16 or Thr 28 was replaced with alanine (T16A and T28A, respectively). Mutation of all three serine residues (S73A/S76A/S79A) with T16A is designated as T16A/3S. All mutations/substitutions were performed using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's recommended protocol, and GST fusion protein was expressed and purified following the manufacturer's instructions. LRP-␤ was prepared as described (20) and contains the LRP light chain, the transmembrane domain, and the LRP cytoplasmic tail. Substitutions to generate T16A/3S mutant LRP-␤ were performed using the site-directed mutagenesis kit and were confirmed by sequencing. In this study, we also used minireceptor LRP-␤ with the second NPVY motif mutated to APVA (20). The expression vector of full-length LRP with mutations in the cytoplasmic domain (pcDNA3.1-T16A/3S LRP) was prepared with full-length wild-type LRP protein expression vector pcDNA3.1(Ϫ) (28) using the site-directed mutagenesis kit. All mutations were confirmed by sequencing. mDab-GST was generously provided by Dr. Joachim Herz, and the Shc plasmid was generously provided by Dr. K. S. Ravichandran.
Wild-type and Mutant LRP-expressing Stable Chinese Hamster Ovary Cell Lines-LRP-null CHO13-5-1 cells were grown to 30 -40% confluency in 150-mm plates and transfected with cDNA from fulllength wild-type LRP (cloned into pcDNA3.1(Ϫ)) or full-length mutant T16A/3S LRP (cloned into pcDNA3.1(Ϫ)) using FuGENE TM 6 transfection reagent (Roche Applied Science) following the manufacturer's instructions. 48 h after transfection, the cells were placed under selection pressure (600 g/ml G418 or hygromycin); and 2 weeks later, 100 colonies were picked for both wild-type and mutant LRPs, and positive clones were subcloned twice before being used for the experiments.
PKC Assay-In vitro phosphorylation of purified full-length wildtype LRP and wild-type and mutant LRP cytoplasmic tail-GST fusion proteins was carried out using the BIOTRAK TM PKC assay system (Amersham Biosciences) following the manufacturer's instructions. Reactions were carried out at 37°C for 15 min using 7.5 g of GST fusion protein, 0.2 g of human recombinant PKC␣, and 2.5 Ci of [␥-32 P]ATP (Amersham Biosciences) in 1.2 mM ATP buffer with calcium, lipid, and dithiothreitol buffers. As a positive control, the PKC substrate peptide provided in the kit was used; and as a negative control, purified GST was used. For quantitative analysis, reactions were terminated with stop reagent (300 mM orthophosphoric acid containing carmosine red), spotted on phosphocellulose membrane, washed, and assayed using a scintillation counter. For qualitative analysis, the kinase reactions were terminated by adding 5ϫ SDS sample buffer containing ␤-mercaptoethanol, separated by 4 -20% SDS-PAGE, transferred to nitrocellulose membrane stained with Ponceau S, exposed, and analyzed using a PhosphorImager (Amersham Biosciences).
In Vivo Phosphorylation and Phosphoamino Acid Analysis-PAC-1 cells or WI-38 fibroblasts were grown to 70 -80% confluency in 150-mm plates, washed twice with Dulbecco's modified Eagle's medium (DMEM), preincubated in phosphate-free DMEM for 3-4 h, and labeled for 30 min to 1 h with 1 mCi/plate [ 32 P]orthophosphate (Amersham Biosciences) with or without kinase inhibitors and PMA. Similarly, LRP-null CHO13-5-1 cells stably transfected with full-length wild-type or T16A/3S mutant LRP were washed and preincubated in phosphatefree DMEM with serum for 3-4 h and labeled for 1 h with 1 mCi/plate [ 32 P]orthophosphate. Labeling was terminated by placing the culture plates on ice and washing the cell layers three times with ice-cold phosphate-free DMEM. Cell extracts were made in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, and 1% Nonidet P-40) containing protease (Roche Applied Science) and phosphatase inhibitors (EMD Biosciences). Cell lysates were subjected to immunoprecipitation with anti-LRP polyclonal antibody R2629 as described below. Immunoprecipitates were washed and boiled in 2ϫ nonreducing SDS-PAGE sample buffer for 10 min. Proteins were then separated by 4 -12% SDS-PAGE, transferred to nitrocellulose membranes, and exposed Biomax-ML film or measured using a PhosphorImager. After evaluating LRP phosphorylation, membranes were blocked and then probed with anti-LRP light chain antibody 5A6 (0.5 g/ml) to measure total LRP expression. For COS-1 cells transiently transfected with Myc-tagged wild-type or T16A/3S mutant LRP-␤, 48 h after transfection, cell layers were preincubated in phosphate-free medium with serum and labeled with [ 32 P]orthophosphate as described above. Cell lysates were prepared and immunoprecipitated with antibody 9E10 (10 g/ml). After assessing LRP-␤ phosphorylation, the membranes were probed with anti-Myc IgG polyclonal antibody. To determine whether 32 P was incorporated into serine, threonine, or tyrosine residues, immunoprecipitates from 32 P-labeled WI-38 fibroblasts were separated by 4 -12% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The band corresponding to phosphorylated LRP was excised, washed, and hydrolyzed with 6 N HCl at 110°C for 60 min. The liquid hydrolysates were dried, resuspended in 10 l of water, and spotted along with nonradioactive phosphoamino acid standard mixture on glass-backed cellulose TLC plates. One-dimensional electrophoresis was carried out using the Hunter thin-layer peptide mapping electrophoresis system (HTLE-7002, CBS Scientific, Del Mar, CA). After visualizing the phosphoamino acid standards with 0.25% ninhydrin in acetone, the TLC plates were exposed to Biomax-MS film with an intensifying screen at Ϫ70°C to detect 32 P incorporated into serine, threonine, or tyrosine residues.
Enzyme-linked Immunosorbent Assays-Ligands (PKC␣ and bovine serum albumin (BSA)) were coated on 96-well flat-bottom microtiter plates at 1 g/ml in Tris-buffered saline (TBS; 50 mM Tris and 150 mM NaCl) with 2 mM CaCl 2 overnight at 4°C and blocked with 1% BSA in TBS and 2 mM CaCl 2 for 1 h at room temperature. The wells were then incubated with increasing concentrations of purified human LRP, wildtype LRP cytoplasmic tail-GST fusion protein, or GST in TBS, 2 mM CaCl 2 , and 0.05% Tween 20 overnight at 4°C. Bound LRP was detected with antibody 8G1 (0.5 g/ml) and horseradish peroxidase-conjugated goat anti-mouse IgG antibody, and bound wild-type LRP cytoplasmic tail-GST was detected using polyclonal antibody R704 (1 g/ml) and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Wells were developed using tetramethylbenzidine peroxidase substrate (KPL, Gaithersburg, MD), and absorbance was measured at 590 nm.
Measurement of the Endocytic Rate Constant-LRP-null CHO13-5-1 cells stably transfected with full-length wild-type or T16A/3S mutant LRP were grown to 70% confluency in 6-well culture plates. The endocytic rate constant was measured by internalized/surface analysis as described by Wiley and Cunningham (30). Cells expressing wild-type or T16A/3S LRP were incubated with 1 ml of 125 I-labeled antibody 8G1 (specific activity of 2-10 Ci; 1.5 g/ml to 10 nM) with or without unlabeled antibody 8G1 (75 g/ml to 500 nM) in prewarmed assay medium (Ham's F-12 medium) containing 20 mM HEPES and 0.5% BSA at 37°C for 2, 4, 6, and 8 min. At the indicated times, the culture dishes were placed on ice, and cells were extensively washed with chilled buffers (twice with Dulbecco's phosphate-buffered saline (DPBS) and 0.5% BSA and twice with DPBS). Cells were treated with trypsin (0.5 ml/well) and proteinase K (50 g/ml) for 10 min on ice, and the detached cells were collected by centrifugation at 6000 rpm for 7 min. Radioactivity associated with the supernatant (proteinase K-released cell-surface 125 I-labeled antibody 8G1) and cell pellet (internalized 125 I-labeled antibody 8G1) was quantified. The endocytic rate (k e ) was calculated from the slope of the internalized/surface versus time plot.
Co-immunoprecipitation and Immunoblot Analysis-Rat pulmonary artery smooth muscle cells were grown to 70% confluency in M199 medium containing 10% fetal calf serum with antibiotics and glutamine in 150-mm culture plates. For treatment with PMA (150 nM), staurosporine (7 nM), or Gö 6976 (10 nM), cell layers were washed and treated with 1% Nutridoma TM NS (Roche Applied Science) in M199 medium for 1 h. Cell layers were washed twice with cold DPBS and lysed on ice in 1.0 ml of lysis buffer containing protease and phosphatase inhibitors as described above. After preclearing with rabbit IgG and protein G-Sepharose, the lysates were immunoprecipitated with antibody R2629 and protein G-Sepharose overnight at 4°C. Immunoprecipitates were washed three times with lysis buffer containing protease and phosphatase inhibitors and boiled in 2ϫ nonreducing SDS-PAGE sample buffer for 10 min. Samples were separated by 4 -12% SDS-PAGE and transferred to nitrocellulose membranes for immunoblot analysis. Membranes were blocked with 5% nonfat dry milk in TBS for 1 h at room temperature and incubated with antibody 5A6 (0.2-0.4 g/ml) in 5% milk in TBS and 0.05% Tween 20 for 1 h. After 3 washes, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:10,000) in 5% milk in TBS and 0.05% Tween 20 for 1 h, washed three times, and developed with chemiluminescent reagent (Pierce). The bands were visualized with Biomax Light film (Eastman Kodak Co.). For visualizing PKC␣, the membranes were stripped using a Re-blot Western blot recycling kit (Chemicon International, Inc., Temecula, CA) and probed with anti-PKC␣ monoclonal antibody as described above.
GSH Pull-down Assay-GSH-Sepharose was prepared by coupling glutathione to epoxy-activated Sepharose. COS-1 cells were grown to 40% confluency in 150-mm plates and transfected with 10 g of expression vector containing Myc-tagged wild-type or T16A/3S mutant LRP-␤ using FuGENE 6 in DMEM and 10% serum. 48 h after transfection, the cell layers were washed twice with cold DPBS, and lysates were prepared in lysis buffer containing protease and phosphatase inhibitors as described above. After preclearing with 75 l of a 1:1 slurry of GSH-Sepharose for 2-3 h at 4°C, wild-type and mutant cell lysates were incubated with varying concentrations (3, 1.5, and 0.75 g) of Dab-1-GST fusion protein or control GST protein (3 g) and 75 l of a 1:1 slurry of GSH-Sepharose overnight at 4°C. Following the incubation, Sepharose beads were washed three times with lysis buffer containing protease and phosphatase inhibitors and boiled in 2ϫ nonreducing SDS-PAGE sample buffer for 10 min. Samples were separated by 4 -12% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes for immunoblot analysis. Membranes were blocked with 5% nonfat dry milk in TBS for 1 h at room temperature and incubated with anti-Myc monoclonal antibody 9E10 (1 g/ml) in 5% milk in TBS and 0.1% Tween 20 for 1 h. After 3 washes, membranes were incubated with horseradish peroxidase-conjugated goat antimouse IgG antibody (1:10,000) in 5% milk in TBS and 0.1% Tween 20 for 1 h, washed three times, and developed with chemiluminescent reagent (Pierce). The bands were visualized with Biomax Light film.
Transient Transfection of Shc, HA-GULP, and LRP-␤ in COS Cells for Co-immunoprecipitation Experiments-COS-1 cells were grown to 30 -40% confluency in 150-mm plates and transfected with 7.5 g of Shc or HA-GULP plasmid and 7.5 g of empty vector (pSec-Tag) or Myc-tagged wild-type, T16A/3S mutant, or NPVY/APVA mutant LRP-␤. 48 h after transfection, the cell layers were washed twice with cold DPBS, and lysates were prepared in lysis buffer containing protease and phosphatase inhibitors as described above. Cell lysates were precleared with non-immune IgG-protein G complex for 2-3 h at 4°C. Shc-and LRP-␤-expressing cell lysates were immunoprecipitated with either anti-Shc monoclonal antibody (5 g/ml) or antibody 9E10 (10 g/ml). Similarly, HA-GULP-and LRP-␤-expressing cell lysates were immunoprecipitated with either anti-HA IgG polyclonal antibody (5 g/ml) or anti-Myc IgG polyclonal antibody (5 g/ml). Immunoprecipitates were washed three times with lysis buffer containing protease and phosphatase inhibitors and boiled in 2ϫ nonreducing SDS-PAGE sample buffer for 10 min. Samples were separated by 4 -12% SDS-PAGE and transferred to nitrocellulose membranes for immunoblot analysis.

LRP Is Phosphorylated at Serine, Threonine, and Tyrosine
Residues-Previous studies have reported that LRP is phosphorylated at serine (25) and tyrosine (20) residues within its cytoplasmic domain. We sought to fully characterize the phosphorylation of LRP and to study the role this may play in LRP function. To this end, human WI-38 fibroblasts, which express high levels of LRP, were metabolically labeled by incubation with [ 32 P]H 3 PO 4 for 1 h at 37°C. Cell extracts were then subjected to immunoprecipitation with either affinity-purified anti-LRP IgG antibody R2629 or preimmune IgG as a control. The results of this experiment (Fig. 1A) demonstrate that a phosphorylated protein with an apparent mass of 85 kDa was immunoprecipitated by anti-LRP IgG antibody (lane 1), but not by preimmune IgG (lane 2). The phosphorylated band shown in lane 1 has an identical mobility on SDS-PAGE as the light chain of LRP as revealed by immunoblotting the immunoprecipitates with monoclonal antibody 5A6, which is specific for the LRP ␤-subunit (data not shown).
To identify the types of amino acid residues within the LRP cytoplasmic domain that are phosphorylated, LRP metabolically labeled with [ 32 P]H 3 PO 4 was subjected to immunoprecipitation and SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The 32 P-labeled 85-kDa ␤-subunit was next excised from the membranes and subjected to acid hydrolysis, and the phosphoamino acid content was determined by thin-layer cellulose electrophoresis. The results shown in Fig.  1B reveal that LRP was phosphorylated at serine, threonine, and tyrosine residues. Furthermore, the results reveal that under normal cell culture conditions, the extent of tyrosine phosphorylation was relatively low compared with the levels of serine and threonine phosphorylation.
Kinase Inhibitors Suggest That Protein Kinase A and PKC␣ Phosphorylate the LRP Cytoplasmic Domain-Our next experiments were aimed at identifying the kinase class responsible for LRP phosphorylation. WI-38 fibroblasts were incubated for 1 h at 37°C with [ 32 P]H 3 PO 4 in the presence of one of the following broad-spectrum serine/threonine kinase inhibitors: staurosporine, K-252a, H-89, and the tyrosine kinase inhibitor genistein. Following incubation, cell extracts were prepared, subjected to immunoprecipitation with anti-LRP IgG antibody, and analyzed by SDS-PAGE and autoradiography. The results of this experiment demonstrate that staurosporine and K-252a dramatically reduced the extent of LRP phosphorylation (Fig.  1C). As reported previously (25), H-89, the potent protein kinase A inhibitor, also reduced LRP phosphorylation. On the other hand, LRP phosphorylation was not noticeably affected by the tyrosine kinase inhibitor genistein, consistent with the data in Fig. 1B, indicating that only a small amount of tyrosine phosphorylation was detected on the LRP light chain. We also examined the phosphorylation of LRP in a rat pulmonary smooth muscle cell line and found that the selective PKC␣ inhibitor Ro-32-0432 significantly reduced the extent of LRP phosphorylation, implicating a role for PKC␣ activity in LRP phosphorylation (Fig. 1D).
PKC␣ Coprecipitates with LRP following PMA Treatment-Since inhibitor studies suggested that PKC␣ mediates LRP phosphorylation, we initiated experiments to determine whether PKC␣ can associate with LRP. For these experiments, a rat pulmonary smooth muscle cell line (PAC-1) was cultured in the absence (control) or presence of PMA. Following treatment, the cells were lysed, and LRP was immunoprecipitated with anti-LRP IgG antibody. The immunoprecipitates were subjected to immunoblot analysis using anti-PKC␣ antibody, and the results demonstrate that PKC␣ co-immunoprecipitated with LRP, especially following activation by PMA ( Fig. 2A). To determine whether PKC␣ can directly bind to LRP, solid-phase assays were performed to measure the binding of full-length LRP (Fig. 2B) or the LRP cytoplasmic domain (Fig. 2C) to microtiter wells coated with PKC␣. Both LRP and the GSTfused LRP cytoplasmic domain demonstrated dose-dependent binding to immobilized PKC␣ with apparent K d values of 18 and 224 nM, respectively.
PKC␣ Phosphorylates LRP in Vitro-To confirm that PKC␣ is capable of phosphorylating LRP, in vitro phosphorylation experiments were conducted by incubating purified full-length LRP with recombinant PKC␣ in the presence of [␥-32 P]ATP. Following incubation, LRP was analyzed by SDS-PAGE and autoradiography. The results indicate that the LRP light chain was readily phosphorylated by PKC␣ in vitro (data not shown), confirming that PKC␣ is capable of phosphorylating sites on the LRP ␤-subunit. Phosphoamino acid analysis of the LRP light chain confirmed that both serine and threonine residues were phosphorylated by PKC␣ (data not shown).
To identify the specific amino acids phosphorylated by PKC␣, we employed the LRP cytoplasmic domain expressed as a fusion protein with GST. When incubated with [␥-32 P]ATP in the presence of PKC␣, the GST-fused LRP cytoplasmic domain was phosphorylated, whereas under identical conditions, GST itself was not phosphorylated, and this was confirmed by qualitative (Fig. 3, B and C) as well as quantitative (Fig. 3D) kinase assays. These experiments confirm that the LRP cytoplasmic domain contains phosphorylation sites that act as substrates for PKC␣. Similar results were obtained when cell extracts from WI-38 fibroblasts were utilized as a source of kinases present in fibroblast extracts (data not shown).
Having confirmed that the LRP cytoplasmic domain is a target for PKC␣-mediated phosphorylation, we next set out to identify potential phosphorylation sites. The LRP cytoplasmic domain contains a consensus PKC phosphorylation site at Thr 28 that was targeted for mutation (Fig. 3A). Another threonine (Thr 16 ) that is upstream of the first NPXY motif was also targeted for mutation. The basis for selecting Thr 16 as a potential phosphorylation site was derived from studies on another transmembrane protein, the amyloid precursor protein. The amyloid precursor protein contains a threonine residue (Thr 668 ) that lies upstream from the NPXY motif in its cytoplasmic domain and that is phosphorylated (31). Phosphorylation of this residue modulates the conformation of its cytoplasmic domain and its association with adaptor proteins that bind to the NPXY motif (32). Based on these prior studies, we selected Thr 16 in the LRP cytoplasmic domain for mutation as well. However, we found that PKC␣ readily phosphorylated mutant molecules in which either Thr 16 or Thr 28 was replaced with alanine (T16A and T28A, respectively). Since we know that LRP contains phosphorylated serine residues and since prior work found that Ser 76 is phosphorylated by protein kinase A (25), we mutated all three serine residues within the LRP cytoplasmic domain to alanines (S73A/S76A/S79A) and found that these mutations reduced the extent of phosphorylation only slightly. However, mutation of Thr 16  or BSA (f). Bound LRP was detected using anti-LRP monoclonal antibody 8G1 and horseradish peroxidase-conjugated goat anti-mouse IgG antibody. Each data point represents the average of triplicate values, and the solid curves represent the best fit to a binding isotherm determined by nonlinear regression analysis. C, increasing concentrations of the GST-fused LRP intracellular domain (GST-LRP-CD; q and E) or GST alone (f and Ⅺ) were incubated with microtiter wells coated with PKC␣ (q and f) or BSA (E and Ⅺ). Bound protein was detected using the C terminus-specific anti-LRP polyclonal antibody R704 and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Each data point represents the average of triplicate values, and the solid curves represent the best fit to a binding isotherm determined by nonlinear regression analysis.

FIG. 3. PKC␣ phosphorylates the LRP cytoplasmic tail expressed as a GST fusion protein.
A, shown is a schematic diagram of the LRP cytoplasmic domain with key amino acids highlighted. For convenience, the first amino acid following the transmembrane domain is numbered 1 as suggested by Li et al. (29). B and C, shown are the results from phosphorylation analysis of wild-type (WT) and mutant (T28A, S73A/S76A/S79A (3S), T16A/3S, and T16A) LRP cytoplasmic domain molecules expressed as fusion proteins with GST. Phosphorylation was accomplished by incubating LRP cytoplasmic domains with human recombinant PKC␣ at 37°C for 15 min using the BIOTRAK TM PKC assay system (7.5 g/reaction). Purified GST was used as a negative control. After 15 min, reactions were terminated by adding 5ϫ reducing Laemmli sample buffer, separated by 4 -20% SDS-PAGE, and transferred to nitrocellulose membrane. Membranes were stained with Ponceau S (B) and detected by phosphorimaging (C). D, for quantitative analysis, reactions were terminated by adding stop buffer containing orthophosphoric acid, spotted on phosphocellulose membrane, washed with 5% acetic acid, and counted. WT LRPCT, wild-type LRP cytoplasmic tail.
was not effectively phosphorylated in these cell lines compared with wild-type LRP (Fig. 4A). Similar results were obtained when COS-1 cells were transiently transfected with LRP-␤ and a mutant version of this minireceptor containing the T16A/3S mutations (Fig. 4B). LRP-␤ is an LRP minireceptor that represents the 85-kDa ␤-subunit of LRP containing the cytoplasmic domain, the transmembrane domain, and a portion of the ectodomain and is fully functional in endocytosis (20).
We next wanted to determine whether the LRP mutant is internalized at the same rate as the wild-type molecule. To measure this, we compared the endocytic rate constants for full-length wild-type and mutant LRPs using 125 I-labeled antibody 8G1 as the ligand. The results reveal that the LRP mutant has a higher endocytic rate constant (k e ϭ 0.192 min Ϫ1 ) than the wild-type molecule (k e ϭ 0.144 min Ϫ1 ), revealing that lack of serine and threonine phosphorylation does not inhibit receptor internalization (Fig. 5). In fact, the data suggest that phosphorylation of the LRP cytoplasmic domain generates a molecule in which the rate of internalization is decreased by 25%.
LRP Serine/Threonine Phosphorylation Is Required for Optimal Association with Shc-The LRP cytoplasmic domain is phosphorylated at Tyr 63 by PDGF receptor-␤ activation (20). This residue is located within the second NPXY motif of the LRP cytoplasmic domain and is a site where a number of cytoplasmic adaptor proteins bind. Phosphorylation of Tyr 63 within this motif generates a docking site for Shc (20,24,33), an adaptor protein that contains a PTB domain that recognizes phosphorylated tyrosine residues in the context of NPXY motifs.
Previous experiments have shown that co-transfection of COS-1 cells with Shc and LRP-␤ results in increased phospho- FIG. 4. T16A/3S mutant LRP is not effectively phosphorylated in Chinese hamster ovary cells or in COS-1 cells. A, LRP-null CHO13-5-1 cells were stably transfected with full-length wild-type (WT) or T16A/3S mutant LRP. Following treatment, the cells were metabolically labeled with [ 32 P]orthophosphate (1 mCi/plate) for 1 h in phosphate-free medium and immunoprecipitated with anti-LRP antibody R2629. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The incorporation of 32 P into LRP was analyzed using a PhosphorImager (upper panel), and total LRP was detected by immunoblotting with anti-LRP monoclonal antibody 5A6 (lower panel). B, COS-1 cells were transiently transfected with empty vector (Vector), Myc-tagged wild-type LRP-␤ (WT), or the T16A/3S mutant were immunoprecipitated with anti-Myc monoclonal antibody 9E10. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The incorporation of 32 P into LRP-␤ was analyzed using phosphorimager (upper panel), and total LRP was detected by immunoblotting with anti-Myc polyclonal antibody and by chemiluminescence (lower panel).
FIG. 5. T16A/3S mutant LRP is internalized more rapidly than wild-type LRP. CHO13-5-1 cell lines stably transfected with either wild-type (WT; q) or mutant (E) LRP were grown to 70% confluency. The endocytic rate constant was measured by incubating the cells with 125 I-labeled antibody 8G1 (1.5 g/ml) at 37°C for the indicated time periods. Following incubation, the cells were placed on ice, and the amount of ligand bound to the surface (B) and internalized (I) was measured. Nonspecific binding and internalization were determined by incubating parallel cultures with 50-fold excess unlabeled antibody 8G1 and were subtracted from the total to determine specific binding and internalization.  Following transfer to nitrocellulose membranes, the extracts were probed with antibody 9E10 to confirm expression of wild-type and mutant LRP-␤. B, varying concentrations of purified Dab-1-GST (3, 1.5, and 0.75 g) or GST (3 g) along with 75 l of GSH-Sepharose were added to wild-type or T16A/3S cell lysates and incubated overnight at 4°C. Proteins bound to the GSH-Sepharose beads was separated by 4 -12% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Membranes were then probed with antibody 9E10 to detect Myc-LRP-␤. rylation of Tyr 63 within the LRP cytoplasmic domain, leading to binding of Shc to LRP (20). To determine whether phosphorylation of serine and threonine residues in LRP influences the tyrosine phosphorylation of LRP and its association with Shc, COS-1 cells were co-transfected with Shc and wild-type LRP-␤ or various mutant LRP-␤ molecules. Analysis of LRP-␤ for the presence of phosphotyrosine residues demonstrated that the T16A/3S mutant was not effectively phosphorylated at Tyr 63 (Fig. 6). Furthermore, mutant LRP-␤ demonstrated a reduced interaction with Shc as assessed by co-immunoprecipitation analysis when either LRP (Fig. 6A) or Shc (Fig. 6B) was immunoprecipitated. These results indicate that elimination of serine and threonine phosphorylation sites in the LRP cytoplasmic domain reduces the extent of Tyr 63 phosphorylation and leads to impaired association with the adaptor protein Shc.
LRP Serine/Threonine Phosphorylation Is Required for Optimal Association with mDab and CED-6/GULP-LRP is known to interact with a number of other adaptor proteins such as Fe65 and mDab (22) and CED-6/GULP (14). To determine whether T16A/3S mutant LRP has altered interactions with other adaptor proteins, the ability of the wild-type and mutant receptors to bind mDab was evaluated. For these experiments, increasing concentrations of the mDab PTB domain fused to GST were incubated with extracts of cells expressing either wild-type or T16A/3S mutant LRP-␤. Following incubation, the Dab-1-GST fusion protein was absorbed onto GSH-Sepharose, and the amount of coprecipitated LRP-␤ was measured. The results of these experiments are shown in Fig. 7 and reveal that the wild-type LRP intracellular domain interacted avidly with Dab-1-GST, even at relatively low concentrations of the fusion protein. In contrast, mutant LRP-␤ did not interact as tightly with Dab-1-GST as did the wild-type receptor. These results indicate that phosphorylation of the LRP intracellular domain increases its affinity for Dab.
We also utilized co-immunoprecipitation experiments to evaluate the effect of the T16A/3S mutations on association of the LRP cytoplasmic domain with the adaptor protein CED-6/GULP. In these experiments, CED-6/GULP was cotransfected with LRP-␤ in COS-1 cells, and co-immunoprecipitation experiments were performed. The results show reduced association of CED-6/ GULP with mutant LRP both when LRP was immunoprecipitated ( Fig. 8A) and when CED-6/GULP was immunoprecipitated (Fig. 8B). CED-6/GULP did not associate with LRP when the second NPXY motif in LRP was mutated to APVA. DISCUSSION The trafficking and functional properties of receptor tyrosine kinases (34 -36) and G-protein-coupled receptors (35,37,38) are regulated by phosphorylation of their cytoplasmic domains. In contrast to our understanding of how phosphorylation modulates the function of these signaling receptors, the role of phosphorylation in the function of endocytic receptors is much less defined. Thus, although the low density lipoprotein receptor (39,40) and LRP (25) are phosphorylated at serine residues within their cytoplasmic domains, the functional significance of this phosphorylation has not been understood until now. LRP functions in endocytosis, in the phagocytosis of necrotic cells, and in modulating signaling pathways. Presumably, these varied activities are regulated by association of LRP with distinct cytoplasmic adaptor proteins (22,23). We hypothesized that phosphorylation of the LRP cytoplasmic domain may modulate its interaction with adaptor proteins; and to test this hypothesis, we set out to investigate LRP phosphorylation in more detail.
Our initial studies employed inhibitors to identify the kinase class responsible for phosphorylating the LRP cytoplasmic domain. This work revealed that inhibitors of protein kinase A and PKC␣ reduced the extent of LRP phosphorylation. Protein kinase A was previously reported to mediate phosphorylation of the LRP light chain (25); and in this study, in vitro experiments demonstrated that PKC␣ readily phosphorylated the cytoplasmic domain of LRP. Furthermore, our experiments indicated that PKC␣ coprecipitated with LRP in smooth muscle cells treated with PMA. PKC␣ is a ubiquitously expressed serine/threonine kinase that is activated by a variety of stimuli, including tyrosine kinase receptors, and plays an important role in cellular proliferation, apoptosis, differentiation and motility. Upon activation, PKC␣ translocates to the membrane and associates with numerous receptors, including syndecan-4 (41,42) as well as integrins (43,44). Inhibition of PKC␣ activity or expression of a PKC␣ dominant-negative mutant suppresses focal adhesion formation and cell migration mediated by ␣ 5 ␤ 1 integrin in cooperation with syndecan-4 in a human metastatic melanoma cell line (43). Furthermore, in a squamous carcinoma cell line, activation of PKC␣ and the subsequent phosphorylation of the ␤ 4 integrin subunit at serine residues are required for mobilization of ␣ 6 ␤ 4 integrin from hemidesmosomes and its redistribution to cell protrusions (44). The association of PKC␣ with the LRP cytoplasmic domain and its subsequent phosphorylation of LRP indicate a new role for this kinase in modulating LRP function.
Phosphoamino acid analysis of metabolically labeled LRP revealed that serine, threonine, and tyrosine residues are phosphorylated. Mutational analysis demonstrated that conversion of Thr 16 along with Ser 73 , Ser 76 , and Ser 79 to alanine generated an LRP molecule in which the extent of phosphorylation was greatly diminished. By comparing the properties of mutant LRP with that of the wild-type receptor, we gained insight into the contribution of serine and threonine phosphorylation to LRP function. First, we observed that mutant LRP was internalized 25% more rapidly than the wild-type phosphorylated receptor. This was confirmed by measurements of the endocytic rate constant, a reliable measure of receptor activity and func- tion. Thus, serine and threonine phosphorylation of the LRP cytoplasmic domain appears to diminish its association with adaptors of the endocytic machinery, which, in the case of LRP, is the adaptor protein complex AP-2 (45). AP-2 is a multifunctional heterotetramer that contains ␣-, ␤ 2 -, 2 -, and 2 -subunits. The 2 -subunit interacts with the cytoplasmic domain of membrane-bound receptors containing a variety of tyrosinebased internalization motifs, although the motifs present in the LRP cytoplasmic tail do not conform exactly to the preferred sequences recognized by the 2 -subunit.
Second, we found that mutant LRP was less prone to undergo tyrosine phosphorylation in Shc-transfected COS-1 cells. We observed in a previous study that in COS-1 cells transfected with both Shc and an LRP "minireceptor," the cytoplasmic domain of the LRP minireceptor is phosphorylated at Tyr 63 (20). In WI-38 fibroblasts and smooth muscle cells, tyrosine phosphorylation of LRP is mediated by PDGF receptor-␤ activation (20) and also occurs at Tyr 63 . This residue is located within the terminal NPXY motif of LRP, which is a binding site for a number of adaptor proteins, including Fe65, Dab-1, Shc, and CED-6/GULP (14,22,23). In addition to binding adaptor proteins that are involved in a variety of cellular processes, Tyr 63 is also a component of the YXXL internalization sequence in LRP (29).
We noted that impaired serine and threonine phosphorylation of LRP dramatically impacted its ability to bind Shc. Shc is an adaptor protein that is involved in signal transduction by protein-tyrosine kinases (46) and contains a C-terminal Src homology-2 domain that binds with weak affinity to several phosphorylated tyrosine residues on the PDGF receptor (47) and a PTB domain that recognizes phosphorylated LRP (20,24,33). Phosphorylation of Shc at Tyr 317 allows for Grb2 binding (48), thereby activating the Ras pathway, whereas phosphorylation of Shc at Tyr 239 and Tyr 240 initiates a second signaling pathway involving the induction of c-myc (49). Thus, Shc interfaces LRP with both the Ras and c-Myc signaling pathways. Our studies revealing that serine/threonine phosphorylation is required for Shc association indicate that phosphorylation events switch LRP from an endocytic receptor to one capable of participating in signaling pathways.
In addition, we also found that serine/threonine phosphorylation of LRP also modulated its interaction with other adaptor proteins such as Dab-1 and CED-6/GULP. Dab is an adaptor protein that binds to the cytoplasmic domains of low density lipoprotein receptor family members (especially the very low density lipoprotein receptor and apoE receptor-2) and plays a key role in migration of neurons during brain development as a component of the Reelin signaling pathway (50,51). The N terminus of Dab-1 contains a PTB domain that interacts with NPXY consensus sequences within the cytoplasmic domains of several receptors, including LRP. CED-6/GULP, which contains an Nterminal PTB domain and a proline-rich C-terminal domain, is the product of one of seven genes in Caenorhabditis elegans that are involved in the clearance of apoptotic cells (52,53). Although the initial studies characterizing the PTB domain of Shc found a requirement for phosphorylation of tyrosine residues in order for Shc to bind, both CED-6/GULP (14) and Dab (54) do not require phosphorylated tyrosine residues to interact with proteins containing NPXY motifs. In this study, mutant LRP, which was not phosphorylated at serine or threonine residues, interacted more weakly with these two adaptor proteins, and it is not clear if this is related to the reduction in tyrosine phosphorylation noted in mutant LRP or to a preference of these adaptor proteins for phosphorylated serine/threonine residues.
The results of this study revealing that serine and threonine phosphorylation increased the extent of tyrosine phosphorylation of the LRP cytoplasmic domain are reminiscent of regula-tory events that occur in the insulin signaling pathway. The binding of insulin to its cellular receptor stimulates the intrinsic tyrosine kinase activity of the insulin receptor, which in turn phosphorylates several select proteins, including the family of insulin receptor substrate proteins. Serine/threonine phosphorylation of insulin receptor substrate proteins serves to modulate the insulin signaling responses. Thus, phosphorylation of serine residues within the PTB domain of insulin receptor substrate-1 protects this protein from the rapid action of phosphotyrosine phosphatases and enables insulin receptor substrate-1 proteins to maintain their tyrosine-phosphorylated active conformation (55). In a similar manner, serine and threonine phosphorylation of LRP may protect the tyrosine-phosphorylated form of this receptor.
In summary, the results of this study reveal that interaction of LRP with cytoplasmic adaptor proteins can be regulated by phosphorylation of its cytoplasmic domain. Serine and threonine phosphorylation, mediated by PKC␣ and protein kinase A, promotes the tyrosine phosphorylation of LRP and its association with Shc, thus enabling this receptor to participate in signaling pathways. Furthermore, serine and threonine phosphorylation of the LRP cytoplasmic domain generates a receptor that has substantially increased affinity for other adaptor proteins such as Dab-1 and CED-6/GULP, which are involved in signaling pathways and phagocytosis, respectively. Serine and threonine phosphorylation also impacts the internalization rate of LRP by reducing the endocytic rate constant by 25%. Thus, phosphorylation of LRP represents a molecular mechanism to switch LRP function from that of an endocytic receptor to that of a signaling receptor by modulating the class and type of adaptor proteins that associate with LRP.