Association of the Atypical Protein Kinase C-interacting Protein p62/ZIP with Nerve Growth Factor Receptor TrkA Regulates Receptor Trafficking and Erk5 Signaling*

Thangiah Geetha and Marie W. WootenDagger

From the Department of Biological Sciences, Program in Cellular and Molecular Biosciences, Auburn University, Auburn, Alabama 36849

Received for publication, August 19, 2002, and in revised form, December 1, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous work demonstrated an essential role for the atypical protein kinase C interacting protein, p62, in neurotrophin survival and differentiation signaling. Here we show that p62 interacts not only with TrkA but also with TrkB and TrkC, which are the primary receptors for brain-derived neurotrophic factor and neurotrophin-3. The interaction of p62 with TrkA requires the kinase activity of TrkA. Mapping analysis indicates that p62 does not compete with Shc for binding to TrkA, and p62 association was confined to the juxtamembrane region of TrkA, amino acids 472-493. By immunofluorescence the colocalization of p62 and TrkA was observed 30 min post-nerve growth factor treatment within overlapping vesicular structures. Upon subcellular fractionation, activated TrkA colocalized to an endosomal compartment and p62 was coassociated with the receptor post-nerve growth factor stimulation. Moreover, an absence of p62 blocked internalization of TrkA without an effect on phosphorylation of either TrkA or MAPK; however, Erk5 signaling was selectively abrogated. We propose that p62 plays a novel role in connecting receptor signals with the endosomal signaling network required for mediating TrkA-induced differentiation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nerve growth factor (NGF)1 regulates survival, differentiation, and maintenance of neurons. NGF belongs to a family of structurally related neurotrophins such as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) (1). The biological effects of the neurotrophins are mediated by two classes of cell surface receptors, namely a high affinity tyrosine kinase Trk receptor and a low affinity p75NTR receptor. TrkA is the prototype of the Trk family and specifically binds NGF, whereas TrkB and TrkC are the primary receptors for BDNF and NT-3, respectively (2). NGF binding to TrkA stimulates dimerization and autophosphorylation of tyrosine residues Tyr499, Tyr679, Tyr683, Tyr684, and Tyr794 in the intracellular domain of the receptor, thereby creating sites for binding and activation of signaling intermediates (3) such as phospholipase C (PLC)-gamma 1 (4), adapter proteins Shc and FRS2 (5), and phosphatidylinositol 3'-kinase (6) that initiates the intracellular signaling cascades. It has been observed that NGF rapidly induces internalization of TrkA receptor to endocytic vesicles (7-9). The receptors within signaling vesicles remain catalytically active and phosphorylated because they are less accessible to membrane-associated phosphatases (10). Internalized TrkA receptors induce a higher peak level of mitogen-activated protein kinase (ERK/MAPK) activation thereby regulating cell differentiation (11).

Previous work in our laboratory has shown that the atypical protein kinase C-interacting protein, p62, interacts with TrkA and binds tumor necrosis factor receptor-associated factor 6 (TRAF6), which in turn interacts with p75. TRAF6-p62 forms a complex that serves as a bridge to link the common neurotrophin receptor, p75, with TrkA (12). p62 then recruits atypical protein kinase C (aPKC) to phosphorylate Ikappa B kinase and leads to the activation of the transcription factor nuclear factor-kappa B (NF-kappa B). Expression of antisense p62 in PC12 cells inhibits NGF-induced NF-kappa B activation (12). In contrast, it has been shown that a dominant-negative mutant of the Shc adaptor protein effectively blocks TrkA-mediated activation of NF-kappa B (13). p62 also serves as a scaffold for the NF-kappa B pathway through tumor necrosis factor-alpha and interleukin-1 receptor signaling cascades (14, 15). Endogenous and ectopically expressed p62 has been shown to colocalize with lambda /iota PKC and zeta PKC in lysosome-targeted endosomes (16, 17). p62 binds the NGF receptor, TrkA (12), which likewise localizes to late endosomal vesicles. Recent studies have shown that internalized TrkA receptors continue to signal within the endosomal compartment (18) and are required to mediate NGF-induced differentiation (10). Moreover, inhibition of p62 expression has been shown to block NGF-induced neurite outgrowth (17). Hence, it is possible that p62 may be critical for the transport of TrkA from the plasma membrane to the endosome.

In this present study, we observed that the interaction of p62 with TrkA requires the tyrosine phosphorylation of TrkA. Mapping analysis indicates that p62 does not compete with Shc for binding to the TrkA, and p62 association was confined to the juxtamembrane region of TrkA. Both p62 and TrkA were colocalized within similar vesicular structures. We also demonstrate the intracellular localization of activated TrkA along with p62 in an endosomal compartment upon NGF stimulation. In addition, antisense p62 was found to block the internalization of TrkA receptor with a specific effect on the activation of Erk5 pathway. Our observations, viewed in the context of existing knowledge, are compatible with a model where p62 may play a role in trafficking of the TrkA receptor to the endocytic pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Anti-TrkA serum, which recognizes the extracellular domain of TrkA, was obtained from Louis Reichardt, University of California, San Francisco. The anti-pantothenate Trk polyclonal antibody (C-14), anti-Trk monoclonal antibody (B-3), raised against the C-terminal region of TrkA, and monoclonal Trk antibody (E-6) that recognizes Tyr496 phosphorylated TrkA (19) were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine (PY20) anti-p62 was purchased from BD Transduction Laboratories, and anti-Myc, anti-HA, and anti-GST monoclonal antibody and rabbit anti-HA were from Santa Cruz Biotechnology. Erk1/2 and Erk5 antibodies were obtained from Upstate Biotechnology, inc. 2.5 S NGF was obtained from Bioproducts for Science, and 125I (5 mCi) was from PerkinElmer Life Sciences. Reagents for SDS-PAGE and protein molecular weight standards were bought from Bio-Rad. Enhanced chemiluminescence (ECL) reagents, a horseradish peroxidase-conjugated secondary antibody, and hyperfilm were purchased from Amersham Biosciences.

Cell Culture-- Human embryonic kidney 293 (HEK 293) cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum. PC12 cells were grown on culture and were coated with rat tail collagen in DMEM containing 10% heat-inactivated horse serum, 5% heat-inactivated fetal calf serum, 50 µg/ml streptomycin, and 50 units/ml penicillin. For all experiments, 24 h prior to stimulation, the medium was replaced with medium containing reduced serum at a ratio of 1 part complete medium/5 parts serum-free medium and then treated with 50 ng/ml NGF at 37 °C. For inhibition of tyrosine phosphorylation of TrkA, PC12 cells were treated with different concentrations of K252a (Biomol Research Laboratories Inc.) for 60 min before adding NGF. PC12 cells were gently washed with ice-cold phosphate-buffered saline (PBS) and harvested by centrifugation. The cell pellets were incubated on ice for 30 min and then disrupted by sonication for 5 s in 300 µl of lysis buffer (20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 20 mM NaF, 2 mM p-nitrophenyl phosphate, 1 µg/ml leupeptin, and 2 mM sodium orthovanadate). The larger cellular debris was removed by centrifugation at 12,000 × g in a microcentrifuge at 4 °C for 3 min. Protein concentration of the supernatants was determined using the Bio-Rad reagent based on Bradford procedure with bovine serum albumin (BSA) as a standard.

Preparation of GST-Shc Fusion Protein and Subcellular Fractionation-- GST-Shc in glutathione-agarose beads was prepared from a single colony of Escherichia coli cells containing the recombinant GST-Shc plasmid as described previously (17). The protein was released from the beads by adding buffer containing 10 mM glutathione in 50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol and rotated for 2 h at 4 °C. The supernatant containing the GST-Shc protein was collected by centrifugation at 3000 × g at 4 °C. Protein concentration was determined using the Bio-Rad reagent. Subcellular fractions of cytosol, endosomes, Golgi, lysosomes, plasma membrane, small membrane, and nucleus were isolated from PC12 cells as characterized and described previously (17). The protein at the 32/45% sucrose interface has been characterized as endosomes (17) and was used for immunoprecipitation.

DNA Transfections-- Both PC12 and HEK 293 cells were transfected using LipofectAMINE 2000; alternatively, in some experiments HEK 293 cells were transfected by the calcium phosphate procedure (12). After 36 h of transfection, cells were stimulated or not with 50 ng/ml NGF. Cells were harvested and lysed in 1 ml of PD buffer (40 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM beta -glycerophosphate, 10 mM NaF, 10 mM phenyl phosphate, 300 µM Na3VO4, 1 mM benzamide, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM dithiothreitol) for 30 min on ice, followed by centrifugation at 1400 rpm for 15 min at 4 °C to remove the insoluble fraction. The protein concentration of the supernatant was determined by the Bradford method.

Immunoprecipitation and Western Blot Analysis-- Equal volume of whole cell lysates (750 µg) were incubated with 3 µg of appropriate primary antibody for 3 h at 4 °C, followed by 40 µl of agarose-coupled secondary antibody for an additional 2 h at 4 °C. The immunoprecipitates were then washed five times with PD buffer. The proteins were released by boiling for 2 min in SDS-PAGE sample buffer electrophoresed on 10% SDS-PAGE, followed by transfer to nitrocellulose membranes, and subjected to Western blot analysis with the corresponding antibodies (20). In some experiments, whole cell lysates were blotted with antibody to either phosphorylated Erk1/2 or non-phosphorylated Erk5 (21). Erk5 activity was measured by immune complex kinase assay with myelin basic protein as substrate, employing buffers and conditions as described previously (22).

GST Pull-down Assay-- The HEK 293 cell lysates were set up to equal protein concentration (750 µg). GST-ZIP protein in beads was blocked for 1 h at 4 °C with 0.5% BSA in PBS, and the beads were washed 3 times with binding buffer (20 mM Tris, 100 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 2 mM dithiothreitol, 0.05% BSA, and 5% glycerol). 5 µg of GST-ZIP was added to the cell lysates and rotated for 90 min at 4 °C. The samples were washed 5 times with GST wash buffer (2.5 mM Tris, 2.5 mM EDTA, 250 mM NaCl, 0.1% Triton X-100, and 10% glycerol), and SDS-PAGE sample buffer was added, and the samples were boiled for analysis.

Immunofluorescence and Confocal Microscopy-- PC12 cells were grown on coverslips in 24-well plates in DMEM. Cells were serum-starved 24 h before treating with 50 ng/ml NGF at 4 °C for 1 h. Fresh warm serum-free DMEM was added and chased NGF by incubating at 37 °C for different times. Cells were gently washed two times with phosphate-buffered saline (PBS), fixed with methanol for 5 min at 4 °C, and washed three times with PBS. The cells were blocked by adding 3% BSA in PBS for 1 h at room temperature, followed by addition of primary antibody in 0.2% BSA in PBS and incubated overnight at 4 °C. Cells were washed three times in PBS and followed by addition of secondary antibody in 0.2% BSA in PBS and allowed to incubate for 1 h in the dark at room temperature. The cells were finally washed four times in PBS containing 0.05% Tween 20, rinsed in PBS, distilled H2O, blotted dry, mounted, and sealed onto slides for observation by confocal microscopy. The images were captured with an MRC-1024 laser scanning confocal microscope (Bio-Rad) employing five passes with a Kalman filter. The images were processed by Confocal Assistant 4.02 (Bio-Rad) and Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA).

FACS Analysis-- PC12 cells overexpressing TrkA in 60-mm plates were allowed to bind NGF (50 ng/ml) for 1 h at 4 °C. The cells were harvested in PBS and incubated at 37 °C for different times, followed by addition of 500 µl of 1:5000 dilution of anti-TrkA serum, which recognizes the extracellular domain of TrkA (23) in 0.2% BSA in PBS at 4 °C for 1 h. The cells were washed three times with PBS followed by incubation with 500 µl of 1:1000 dilution of fluorescein isothiocyanate in 0.2% BSA in PBS at 4 °C for 30 min. Cells were washed five times with PBS and fixed in 1 ml of 2% paraformaldehyde in PBS at room temperature for 30 min, and 10,000 cells per sample were analyzed in FACScan (Elite model, Beckman-Coulter) equipped with CellQuest software (24, 25).

Iodination of NGF-- Iodination of NGF was performed as described previously (26). The reaction was carried at room temperature with a mixture of 4 mCi of Na125I, 75 µl of 0.1 M phosphate buffer, pH 7.4, 15 µl of lactoperoxidase (30 µg/ml) in the phosphate buffer, 50 µg beta NGF, and 15 µl of 1:104 dilution of H2O2 (30%) in phosphate buffer. After 30 min another 15 µl of H2O2 was added, and the reaction was allowed for an additional 1 h. Iodinated NGF was separated from free 125I through a Sephadex G-50 column. Fractions of 500 µl were collected, and aliquots of 5 µl were counted on a gamma -counter (Topcount NXT microplate scintillation and luminescence counter, Packard Instruments), and a molecular weight of 125I-NGF was estimated by SDS-PAGE. The peak fractions recovered from the column were used in subsequent receptor binding assays.

Ligand-Receptor Complex Internalization Assay-- The acid-wash technique (27) was used to determine the kinetics of NGF-induced internalization of TrkA. PC12 cells expressing TrkA were allowed to bind 0.4 µCi/ml 125I-NGF alone, or with excess NGF (200 ng/ml), and 0.1 mg/ml BSA at 4 °C for 1 h. The medium was subsequently removed and fresh warm serum-free DMEM was added and incubated at 37 °C for the indicated time. The cells were washed with 1 ml of ice-cold 0.2 M acetic acid and 0.5 M NaCl for 6 min to remove the surface 125I-NGF. The cells were then washed once with 1 ml of ice-cold PBS and lysed with 1 ml of 1% SDS with 0.1 N NaOH. The radioactive cell lysate was then counted on a gamma -counter, and specific internalization (internalized 125I-NGF minus internalized 125I-NGF with 200 ng/ml NGF) was assessed for each time point.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p62 Directly Binds the Neurotrophin Trk Receptors in a Phosphotyrosine-dependent Manner-- As a preliminary step, we analyzed the ability of p62 to interact with various Trk receptors. HEK cells were cotransfected with cDNAs encoding HA-tagged TrkA, TrkB, or TrkC along with Myc-tagged p62 followed by stimulation with or without NGF, BDNF, or NT-3 respectively. The lysates were immunoprecipitated with anti-HA or anti-Myc antibody and probed with anti-HA. In control immunoprecipitates (anti-HA), all three neurotrophin receptors were equally expressed; p62 coprecipitated with all three Trk receptors. Moreover, the association of p62 with Trk was dependent upon the stimulation with neurotrophin (Fig. 1). However, BNDF treatment consistently resulted in enhanced association of p62 with TrkB. By comparison, TrkA or -C both possessed a large degree of p62 associated with the receptor in the basal unstimulated state. Collectively, these results reveal that p62 interacts not only with the NGF receptor TrkA, but also with TrkB and TrkC, which are the primary receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).


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Fig. 1.   Interaction of p62 with TrkA, -B, and -C. HEK 293 cells were cotransfected with HA-tagged TrkA, -B, or -C along with Myc-tagged p62 followed by stimulation with or without NGF, BDNF, and NT-3 (50 ng/ml) for 15 min as shown. The interaction was determined by immunoprecipitation (IP) of the cell lysates (750 µg) with anti-HA and anti-Myc and Western blotting (WB) with anti-HA as shown. As control, a fraction of the lysate (40 µg) was blotted with anti-HA or Myc to check for the expression of Trks and p62. This experiment is representative of three separate experiments.

To determine whether the association of p62 with TrkA receptor required the activation of TrkA, HEK 293 cells were cotransfected with wild-type or kinase-inactive form of TrkA (K538A) and Myc-tagged p62, followed by NGF treatment. TrkA was immunoprecipitated from lysates and Western blotted with anti-phosphotyrosine and Myc antibody. We observed that wild-type TrkA was autophosphorylated and interacted with p62 on NGF stimulation (Fig. 2A, 3rd lane), whereas the kinase inactive form was not tyrosine-phosphorylated and poorly interacted with p62 (Fig. 2A, 4th lane).


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Fig. 2.   Kinase activity of TrkA is required for interaction with p62. A, subconfluent cultures of HEK 293 cells were cotransfected with wild-type TrkA or kinase-inactive K538A TrkA and Myc-tagged p62 followed by treatment with NGF (50 ng/ml) for 15 min. The lysates (750 µg) were immunoprecipitated (IP) with anti-TrkA (B-3) and followed by Western blot (WB) analysis with anti-phosphotyrosine antibody, PY20 or Myc. The blot was reprobed with anti-TrkA (C-14) antibody. The lysates (40 µg) were Western-blotted with anti-TrkA (C-14) or Myc to verify the protein expression levels. B, PC12 cells were pretreated with K252a (0-300 nM) or not (-) for 1 h prior to stimulation with 50 ng/ml NGF for 15 min. Tyrosine phosphorylation of TrkA was determined by immunoprecipitation with anti-TrkA (E-6) followed by Western blotting with anti-phosphotyrosine antibody, PY20, or p62. The blot was reprobed with anti-TrkA (C-14) antibody. Cell lysate was analyzed by blotting with anti-TrkA (B-3) or p62. This experiment is representative of three separate experiments.

We next conducted a parallel experiment in PC12 cells treated with the kinase inhibitor K252a, which prevents the tyrosine phosphorylation of TrkA upon NGF stimulation (28, 29). When immunoprecipitation was carried out with anti-TrkA followed by Western blotting for anti-phosphotyrosine or anti-p62 antibody, we observed that K252a treatment prevented the interaction between p62 and TrkA (Fig. 2B, 2nd to 4th lanes), when compared with cells stimulated with NGF, which were not treated with K252a (Fig. 2B, 2nd lane). Thus, p62 interacts with TrkA in a phosphotyrosine-dependent manner.

Mapping the Interaction of p62 with TrkA-- To map the binding site within TrkA that mediates p62 interaction, HA-tagged TrkA receptor mutants: S17 (Delta 450KFG452), S3 (Delta 493IMENP497), S8 (Y499F), and S9 (Y794F) that affect specific signaling pathways were used. Shc binds Tyr499; PLCgamma -1 binds Tyr794; both 493IMENP497 and 450KFG452 stimulate NGF-dependent cell cycle arrest or neuronal differentiation, and 450KFG452 is also involved in phosphorylation of FRS2 (30, 31). TrkA mutants were coexpressed with Myc-tagged p62 in HEK 293 cells, followed by immunoprecipitation with anti-HA and Western blotting for HA-TrkA and Myc-p62. Wild-type TrkA and all the TrkA point mutants (S17, S3, S8, and S9) coprecipitated with Myc-p62 (data not shown). These results suggest that these amino acids of TrkA were not required for its interaction with p62.

p62 interacts with TrkA and recruits aPKC, which leads to phosphorylation of Ikappa B kinase leading to the activation of NF-kappa B (12). The adapter protein Shc has also been shown to bind TrkA on NGF stimulation (31). Moreover, a dominant-negative mutant of Shc inhibits NF-kappa B activation (13). As both p62 and Shc are involved in NF-kappa B pathway, we also examined whether p62 might compete for Shc binding to TrkA. We transfected HEK 293 cells with HA-tagged TrkA and Myc-tagged p62 cDNAs, followed by immunoprecipitation with anti-HA in the presence of increasing concentrations of GST-Shc, and we tested for the interaction of p62 or Shc with TrkA. We observed that as the concentration of GST-Shc increases, the interaction of p62 with TrkA was not affected, thus confirming that p62 does not compete with the Shc-binding site for interaction with TrkA (data not shown).

To explore further the p62-interacting region within TrkA, a series of deletion mutants were employed; TrkA-(1-452), TrkA-(1-472), TrkA-(1-493), TrkA-(1-501), and TrkA-(1-522) (Fig. 3A) were transfected in HEK 293 cells. After NGF stimulation the cell lysates were immunoprecipitated with anti-p62 (Fig. 3B). Alternatively the lysates were also subjected to in vitro GST-p62 pull-down assay (Fig. 3C), followed by Western blotting for TrkA employing a TrkA antibody that recognizes the extracellular domain of TrkA (23) to detect TrkA complexed with p62. Full-length TrkA and TrkA-(1-522) coprecipitated endogenous p62 (lanes 2 and 3), whereas TrkA-(1-501) and TrkA-(1-493) showed slight interaction with p62 (4th and 5th lanes); however, no interaction was observed with TrkA-(1-472) or TrkA-(1-452) (lanes 6 and 7) by either coimmunoprecipitation or GST pull-down assay (Fig. 3, B and C). These findings reveal that amino acids 472-493 in the juxtamembrane region play a role in the interaction of p62 with TrkA.


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Fig. 3.   Mapping the p62 interacting region in TrkA. A, schematic representation of the TrkA deletion mutants TrkA-(1-452), TrkA-(1-472), TrkA-(1-493), TrkA-(1-501), and TrkA-(1-522). B, TrkA, wild-type and mutants were expressed in HEK 293 cells followed by NGF (50 ng/ml) stimulation for 15 min. The interaction was determined by immunoprecipitation (IP) of the cell lysates (750 µg) with anti-p62 and Western blotting (WB) with anti-TrkA, which recognizes the extracellular domain of TrkA or p62. C, HEK 293 cell lysates (500 µg) expressing various TrkA mutants were interacted with an equivalent amount of GST-p62 in a pull-down assay. The association of TrkA was determined by Western blotting with TrkA antibody. These experiments are representative of three separate experiments.

Colocalization of p62 with TrkA in Endocytic Vesicles-- Because p62 interacts with the TrkA receptor, which is subsequently internalized to an endosomal compartment (18), we next analyzed whether p62 colocalizes with TrkA within vesicular structures by double immunofluorescence and confocal microscopy (Fig. 4). To examine the intracellular colocalization of p62 with TrkA, PC12 cells overexpressing TrkA were treated with NGF for 1 h at 4 °C to allow binding at that temperature which inhibits membrane trafficking (7). The unbound NGF was extensively washed, and the cells were warmed for different times at 37 °C to permit endocytosis. Both p62 and TrkA showed a similar punctate staining pattern within endocytic vesicles (Fig. 4) throughout the course of NGF treatment. By 30 min, there was increased colocalization of p62 with TrkA in a majority of the vesicles. All of the p62 containing vesicular structures colocalized with internalized TrkA; however, there were numerous TrkA containing vesicles that did not colocalize with p62. It thus appears that p62 only associates with a fraction of the intracellular receptor pool of activated TrkA receptors. In addition, as a fraction of the TrkA pool was observed to recycle to the cell membrane (60 min), p62 containing vesicles remained coassociated with intracellular vesicles. Taken together, these results provide evidence for the presence of p62 in the same intracellular sites of trafficking as TrkA receptor.


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Fig. 4.   Confocal microscopic analysis of the codistribution of p62 and TrkA in PC12 cells overexpressing TrkA. Cells were treated with NGF (50 ng/ml) for 1 h at 4 °C and chased at 37 °C for 0, 10, 30, and 60 min, followed by fixation and staining with both anti-p62 and anti-TrkA (B-3) antibody. The extent of colocalization was assessed by superimposing red (p62, Texas Red) and green (TrkA, Oregon Green) signals. Areas of colocalization are noted by the presence of arrows. These experiments are representative of three separate experiments.

Interaction of Activated TrkA with p62 in Endosomes-- Previous studies have shown that p62 colocalizes with aPKC in endosomes (16), and also p62 acts as a shuttling protein involved in routing activated aPKC to an endosomal compartment (16, 17). In PC12 cells, p62 has been purified as a component of the late endosomal compartment and colocalizes with specific marker proteins of the endosomal-lysosomal network (17). Upon NGF treatment most of the activated TrkA has been shown to localize to the endosomal network (18, 24). Because p62 colocalizes with internalized TrkA in endocytic vesicles (Fig. 4), it is possible that upon NGF stimulation p62 may be routed along with activated TrkA to the endosomes and may play a role in degradation of TrkA. In order to test this possibility, we employed a previously characterized subcellular fractionation procedure that had been adapted for the subcellular fractionation of membranes employing a sucrose step gradient (17). PC12 cells overexpressing TrkA were allowed to bind NGF at 4 °C for 1 h and chased at 37 °C for 0 and 15 min, followed by subcellular fractionation. When the equivalent protein of various fractions (small membrane, plasma membrane, cytoplasm, nucleus, lysosomes, endosomes, and Golgi) were Western-blotted with an anti-TrkA antibody (E-6) that recognizes only phosphorylated and activated TrkA (19), an increased level of phosphorylated TrkA was observed in both endosomes and nucleus at 15 min of NGF stimulation (Fig. 5A, lanes N and E). It is likely that the increase in the nucleus reflects contamination of the nuclear pellet with the membrane fraction because the fractionation procedure produces a crude nuclear pellet (17). The presence of activated TrkA within the endosomal compartment in resting cells is consistent with other studies that have observed a significant amount of activated TrkA within the endosomes of resting PC12 cells (24). As a measure of purity for the separation of the vesicular network, an aliquot of the separated fractions were Western-blotted with affinity-purified Rab7 antibody, a protein that is enriched in the late endosomes (16, 17). The 18/32% interface collected as the endosomal fraction was enriched in Rab7 (Fig. 5B). To determine whether p62 and TrkA are cotrafficked and remain coassociated within the endosome, equivalent protein from the endosomal fraction was immunoprecipitated with anti-TrkA and Western-blotted with anti-phosphotyrosine (PY20) or p62 antibody. We observed that TrkA in the endosome was tyrosine-phosphorylated and hence confirmed the sequestration of activated TrkA receptor in the endosome (Fig. 5B, lane P). Moreover, TrkA complexes contained p62, and the interaction of p62 with TrkA was dependent upon treatment with NGF (Fig. 5C, 2nd lane). Collectively, these findings reveal that p62 associates with activated TrkA and is cotrafficked to an endosomal compartment along with the receptor.


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Fig. 5.   Colocalization of activated TrkA with p62 in endosomes. A, PC12 cells were treated with NGF (50 ng/ml) for 1 h at 4 °C and chased at 37 °C for 0 and 15 min. Cells were fractionated into small membrane (S), plasma membrane (P), cytoplasm (C), nuclei (N), lysosomes (L), endosomes (E), and Golgi (G). Equivalent protein from each fraction (30 µg) was Western-blotted (WB) with anti-TrkA (E-6), which recognizes the activated TrkA receptor. B, the separated fractions were also Western-blotted with affinity-purified Rab7 antibody. C, the endosomal fraction (350 µg) was immunoprecipitated (IP) with anti-TrkA (C-14) followed by Western blotting with anti-phosphotyrosine antibody, PY20 or p62. The TrkA blot was stripped and reprobed with anti-TrKA (C-14) antibody.

p62 Influences TrkA Trafficking-- Because p62 localized with TrkA to the endosomal network, and remained associated with a fraction of activated TrkA along the endocytic route (Fig. 4), we sought to investigate whether p62 may affect trafficking of TrkA receptor. PC12 cells were transfected with Myc-tagged p62 (OEp62) or a full-length antisense construct of p62 (ASp62), which has been shown to deplete the levels of p62 upon transfection (12, 15). As control, the expression levels of TrkA and p62 were examined employing equal protein concentration of whole cell lysate (Fig. 6A). The level of TrkA was the same in all the lysates, but p62 expression increased in cells transfected with Myc-tagged p62 (2nd lane) and was significantly decreased in cells transfected with ASp62 (3rd lane).


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Fig. 6.   p62 influences TrkA trafficking. A, PC12 cells overexpressing TrkA were transfected with Myc-tagged p62 (OE) or ASp62. 60 µg of protein from the cell lysates were Western-blotted (WB) with anti-TrkA (B-3) and anti-p62 antibody to check the expression level of each protein. B, PC12 cells expressing TrkA were transfected with or without antisense (AS) p62. Cells were treated with NGF (50 ng/ml) for 1 h at 4 °C and chased at 37 °C for 30 min followed by staining for anti-p62 and anti-TrkA (B-3) antibody and examined by confocal microscopy. C, PC12 cells expressing TrkA were transiently transfected with ASp62 or Myc-tagged p62. The cells were allowed to bind NGF (50 ng/ml) for 1 h at 4 °C and chased at 37 °C for 0, 10, 30, and 60 min. Thereafter, the cells were incubated with rabbit anti-TrkA serum which recognizes the extracellular domain of TrkA at 4 °C for 1 h, washed, and incubated with fluorescein isothiocyanate-labeled goat anti-rabbit at 4 °C for 30 min. The cells were washed, fixed, and subjected to FACS analysis. The graph represents the percentage of cell surface TrkA receptor at different time points. D, the cells were transfected as above and were incubated with 0.4 µCi/ml 125I-NGF alone or with excess unlabeled NGF (200 ng/ml) for 1 h at 4 °C and chased at 37 °C for 0, 10, 30, and 60 min. The cell surface 125I-NGF was removed by acid wash, followed by lysis with 1% SDS in 0.1 N NaOH. Specific internalized counts (125I-NGF internalized minus 125I-NGF internalized in presence of excess NGF) were determined by gamma -counting. The data shown are mean ± S.D. of the internalized NGF receptor for three different experiments. The data were analyzed using Student's t test (#, p < 0.005; *, p < 0.001).

To examine the effect of p62 upon trafficking of TrkA, the cells were transfected with a full-length antisense p62 and treated with NGF for 1 h at 4 °C and chased at 37 °C for 30 min. Transfection of antisense (AS) p62 blocked trafficking of TrkA into the vesicular punctate structures. The majority of TrkA staining was at the cell surface, and little or no p62 staining was observed when compared with the control non-transfected cells (Fig. 6B), thus confirming decrease in p62 expression induced by ASp62 construct.

As a separate independent measure, the effect of p62 upon NGF receptor internalization was examined by employing fluorescence-activated cell sorting (FACS) to detect cell surface TrkA employing an antibody, which recognizes the extracellular domain of TrkA (23) (Fig. 6C). The cell surface expression of TrkA was measured in control, cells transfected with antisense p62, and in cells overexpressing p62. PC12 cells overexpressing TrkA were treated with NGF for 1 h at 4 °C to allow binding and were chased for different times at 37 °C to permit internalization. As shown in Fig. 6C, 30 min post-warming, NGF reduced the expression of TrkA at the cell surface in both control and in cells overexpressing p62, representing the internalization of TrkA in these cells. On incubation at 37 °C for 60 min, there was a significant increase in the expression of TrkA at the cell surface. However, no change in the expression of cell surface TrkA receptor was observed in cells transfected with antisense p62.

In addition, the extent of TrkA internalized was also assessed by 125I-NGF binding assay in control, PC12 cells transfected with antisense p62 and in cells overexpressing p62. Cells were incubated with 125I-NGF alone or with excess unlabeled NGF for 1 h at 4 °C, and then warmed at 37 °C for different times. The cell surface 125I-NGF was eliminated by acid washing; the cells were lysed, and internalized 125I-NGF was estimated in a gamma -counter. Cells transfected with antisense p62 did not internalize 125I-NGF during any period of NGF treatment. In cells overexpressing p62, there was a significant (p < 0.005) increase in internalization of 125I-NGF at 30 min of NGF incubation at 37 °C when compared with control (Fig. 6D). When cells were incubated for 60 min at 37 °C, a significant proportion of the 125I-NGF appeared on the cell surface in both control and cells overexpressing p62. These findings reveal that a significant amount of 125I-NGF can bypass a lysosomal/degradative pathway in the cell and recirculates to the cell surface after internalization, which is consistent with previous study on internalization/trafficking of TrkA (32, 33). Altogether these findings reveal that p62 may play an important role in trafficking of the cell surface TrkA receptor to the signaling vesicle.

Effects of p62 on TrkA Receptor Signaling-- NGF binding to the TrkA receptor plays a role in the activation of two distinct signaling cascades, MAPK/Erk kinase 1/2 (11) and the Erk5 pathways (21). Both pathways are dependent upon TrkA kinase activity, as K252a pretreatment abolishes NGF induced activation of these pathways. However, Erk5 activation is also dependent upon internalization of TrkA (21), and likewise TrkA delivery to the signaling endosome is required for NGF-mediated differentiation (11). Because p62 modulated internalization of TrkA (Fig. 6, A-D), as well as NGF-mediated differentiation (17), we set out to examine whether p62 expression affected TrkA signaling of either MAPK or Erk5 (Fig. 7). PC12 cells were transfected with either ASp62 or Myc-tagged p62 (OEp62). The cells were stimulated with NGF for different times followed by immunoprecipitation of TrkA. NGF induced transient phosphorylation of TrkA, which coincided with both MAPK and Erk5 activation (Fig. 7A). Overexpression of p62 was without any effect on either the phosphorylation of TrkA or downstream signaling targets. However, the removal of p62 with the antisense construct slightly enhanced MAPK signaling when compared with either the control cells or those overexpressing p62. We next set out to examine the effect of p62 removal on the activation of Erk5. By employing two separate methods, the examination of Erk5 phosphorylation by Western blot analysis (Fig. 7A) and immune complex kinase assay (Fig. 7B) demonstrated that depletion of p62 severely abrogated NGF-induced activation of Erk5, whereas overexpression of p62 enhanced Erk5 activity compared with control. Altogether these findings reveal that p62 connects the trafficking/internalization of TrkA to the Erk5 pathway.


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Fig. 7.   Effects of p62 on TrkA receptor signaling. A, PC12 cells overexpressing TrkA were transfected with either ASp62 or Myc-tagged p62 (OEp62). The transfected cells were treated with 50 ng/ml NGF for 0, 10, 30, and 60 min at 37 °C. Equivalent cell lysate (750 µg) was immunoprecipitated (IP) with either anti-TrkA (C-14) or anti-Erk5 and Western-blotted (WB) with anti-phosphotyrosine antibody, PY20. The blot was stripped and reprobed with anti-TrkA (B-3) or anti-Erk5 antibody. Equivalent protein from cell lysates (60 µg) was Western-blotted with anti-p-MAPK antibody. The p- MAPK blot were stripped and reprobed with non-phospho-MAPK antibody as shown. B, lysates from control and transfected cells were Western-blotted with anti-Erk5. Alternatively, lysates (750 µg) were immunoprecipitated with anti-Erk5 antibody and subjected to immune complex kinase assay. Shown is the activity of Erk5 as measured employing myelin basic protein (MBP) as substrate. The autoradiogram was scanned, and relative activity of Erk5 was plotted. These findings are representative of two independent experiments with similar results.

Intracellular Colocalization of TrkA and p62 in Differentiated Cells-- Because transport of NGF is required for mediating the longer term effects of NGF on differentiation of PC12 cells (11), we sought to examine whether p62 and TrkA were colocalized in NGF-differentiated PC12 cells. Hence PC12 cells were allowed to differentiate and form neurites by treatment with NGF. Both p62 and TrkA showed punctate staining pattern in the cell body (×63) and along the neurites (×100) by double labeling as shown in Fig. 8. These findings suggest that cotransport of p62 with TrkA may play a role in prolonged signaling required for NGF differentiation via moving signals from axon terminals to neuronal cell bodies.


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Fig. 8.   Immunofluorescence analysis of TrkA and p62 in NGF-differentiated cells. PC12 cells stably overexpressing TrkA were treated with 50 ng/ml NGF for 4 days to stimulate differentiation and outgrowth of neurites. The cells were fixed with methanol and double-labeled with anti-p62 and anti-TrkA (B-3) antibody, and the extent of colocalization was examined by confocal microscopy.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our findings support a model where p62 interaction with TrkA regulates the internalization and localization of the receptor (Fig. 9). Results obtained by coexpression/immunoprecipitation, native coassociation, and immunofluorescence confirm the interaction and colocalization of p62 with TrkA. Moreover, the coassociation between the two proteins is retained upon trafficking of the receptor to the endosomal compartment. Interestingly, an absence of p62 blocks receptor internalization as measured by three independent means: immunofluorescence staining, FACS analysis of cell surface TrkA expression, or association of 125I-NGF within the cell. In this study we observed a significant amount of TrkA or 125I-NGF recirculation to the cell surface. The kinetics and reappearance of TrkA at the cell surface correlate well with earlier more extensive studies on receptor internalization/recycling (32, 33). We have shown previously (12) that the interaction between p62 and TrkA peaks at 15 min post-NGF treatment, whereas internalization of NGF peaks at 30 min. The kinetics are thus consistent with a model whereby p62 binding precedes the internalization of TrkA. On the other hand, p62 can also bind the common neurotropin receptor p75 through the signaling adapter TRAF6 (12). The kinetics of binding between p62 and p75 is an early rapid event, taking place between 1 and 5 min (12). Recent studies (24) suggest that p75 is capable of modulating TrkA internalization. In the context of p62, we speculate that p75-TRAF6-p62 may prime the complex causing a conformational change thereby allowing for optimal interaction between p62/TrkA. Further studies will be needed to explore this possibility.


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Fig. 9.   Model depicting the role of p62 in trafficking. In the presence of p62 the NGF-TrkA receptor complex is endocytosed to the signaling vesicle, whereas in the absence of p62 blocks NGF-TrkA receptor internalization and downstream Erk5 activation.

Antisense p62 has been shown to block NGF-mediated differentiation of PC12 cells (17). These data shed light on the underlying mechanism whereby p62 can affect NGF-mediated differentiation. Previous studies (11) have shown that internalized TrkA receptors within the signaling endosome are the site of the differentiation signal for NGF/TrkA. The region where p62 binds TrkA lies within amino acids 472-493, and the TrkA juxtamembrane domain has been implicated previously as critical for TrkA-mediated differentiation (30). Interestingly, another signaling adapter GIPC binds to TrkA employing the same amino acids (19). We have examined whether p62 binds to GIPC and have failed to observe a direct interaction.2 Thus, we propose that p62 interacts directly with TrkA. A conserved domain search employing amino acids 472-493 of TrkA reveals no domain or motif specific to these amino acids. Because TrkA phosphorylation is required for optimal interaction with p62, we favor a model whereby the interaction domain for p62 within TrkA is unmasked by conformational changes that follow receptor activation and autophosphorylation.

Binding of neurotrophins to Trk receptors is known to stimulate activation of both MAP kinase (Erk1/2) and Erk5, which have been shown to be critical to both the differentiation and survival of PC12 cells (11, 21). Moreover, TrkA internalization and retrograde transport has been shown to activate specifically the Erk5 pathway (21). Depletion of p62 by use of the antisense p62 construct was without any effect upon TrkA phosphorylation or the MAP kinases (Erk1/2); however, NGF-induced Erk5 activity was specifically affected. Our findings suggest that it may not be activation of TrkA which drives Erk5 phosphorylation/activation, but rather, it is the coupling of TrkA to p62 that may control Erk5 activation. Upstream MEK5 has been shown to be a direct and specific activator of Erk5 (22). Interestingly, MEK5 contains an aPKC interaction domain, and deletion of this region impairs formation of a complex between aPKC and MEK5 (35). Moreover, it has been shown that aPKC recruitment into a complex triggers the activation of ErK5 in a manner dependent on MEK5 (35); thus, activation of aPKC is capable of triggering the ErK5 cascade. We propose a model whereby TrkA activation results in binding of p62 scaffold to the receptor and recruitment of aPKC (20), followed by activation of MEK5 (35), leading to activation of Erk5 (Fig. 9). It is interesting to note that a newly isolated protein, Pincher (36), has been shown to participate in clathrin-independent internalization. A dominant-negative mutant form of Pincher inhibited NGF-induced endocytosis of TrkA and selectively blocked TrkA-mediated signaling of Erk5 but not Erk1/2 kinases. Given the similarities between p62 and Pincher in their ability to modulate Erk5 and TrkA internalization, it is possible that p62 may connect with the Pincher-mediated internalization pathway. Altogether, these findings underscore the role of p62 as a critical regulator of not only NGF survival signaling (12, 37) but also in intracellular signaling of activated TrkA. We propose that p62 is a component of a targeting pathway leading to delivery of TrkA and subsequently degradation of TrkA in the lysosomes.

TrkA as well as p75 independently activate the NF-kappa B pathway (13). With respect to TrkA, it has been shown that Shc is required for this pathway (13, 34, 38). The failure of Shc to compete for with p62 for binding to TrkA reveals that p62 activates the kappa B pathway independently of this site. In addition, the p62-binding site within TrkA-(472-493) lies outside the Shc-binding site (493-497). When both receptors, p75 and TrkA, are coexpressed, NGF-activated kappa B signal is attenuated (37); inhibition of TrkA by K252a enhances survival signaling, impairs TrkA internalization, and promotes activation of NF-kappa B. Alternatively, overexpression of p62 can enhance survival signaling and activation of NF-kappa B (37, 12). Thus, our data support a model whereby survival signaling occurs predominantly from NGF bound at the cell surface (11). Collectively, these findings underscore the critical nature p62 plays in mediating the balance in survival signaling versus differentiation signaling, by providing a scaffold for selective activation of NF-kappa B (12, 37) as well as modulating internalization and signaling of activated TrkA. Given the colocalization of p62 with TrkA along the neurite of differentiating cells, we propose that p62 may be a possible target for mediating retrograde NF-kappa B activation in neurons.

    ACKNOWLEDGEMENTS

We are indebted to Susan Meakin and Moses Chao for TrkA mutants, Maria T. Diaz-Meco and Jorge Moscat for providing us with p62 constructs, and Louis Reichardt for anti-TrkA serum. We thank Michael Miller for helping us carry out the confocal microscopy analysis, Lamar Seibenhener for assistance in graphics, and Randy White for assistance with flow cytometry.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant NS 33661.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.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Biological Sciences, 331 Funchess Hall, Auburn University, AL 36849. Tel.: 334-844-9245; Fax: 334-844-9234; E-mail: mwwooten@acesag.auburn.edu.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M208468200

2 T. Geetha and M. W. Wooten, unpublished data.

    ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; FACS, fluorescence-activated cell sorter; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-related protein kinase; PKC, protein kinase C; ASp62, antisense p62; OEp62, overexpressed p62; p-TrkA, phosphorylated TrkA; NF-kappa B, nuclear factor-kappa B; aPKC, atypical protein kinase C; BDNF, brain-derived neurotrophic factor; NT, neurotrophin; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TRAF6, tumor necrosis factor receptor-associated factor 6.

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RESULTS
DISCUSSION
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