The Atypical Protein Kinase C-interacting Protein p62 Is a Scaffold for NF-κB Activation by Nerve Growth Factor

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor κB (NF-κB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-κB pathway, which mediates NGF survival and differentiation responses. Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor κB (NF-κB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-κB pathway, which mediates NGF survival and differentiation responses. nerve growth factor nuclear factor κB IκB kinase atypical protein kinase C human embryonic kidney cells glutathione S-transferase hemagglutinin The biological responses to neurotrophins such as NGF1 include neuronal survival and differentiation (1Klesse L.J. Parada L.F. Microsc. Res. Tech. 1999; 45: 210-216Crossref PubMed Scopus (136) Google Scholar). Two receptors, TrkA and p75, participate in the formation of the high affinity NGF binding site (2Casaccia-Bonnefil P. Kong H. Chao M.V. Cell Death Differ. 1998; 5: 357-364Crossref PubMed Scopus (120) Google Scholar). TrkA enhances both NGF responsiveness and cell survival (3Kaplan D.R. Prog. Brain Res. 1998; 117: 35-46Crossref PubMed Google Scholar). The transcription factor nuclear factor κB (NF-κB) is activated by both TrkA and p75 receptor components (4Foehr E.D. Lin X. O'Mahony A. Geleziunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). Moreover, p75 has been shown to interact with TRAF6 (5Khursigara G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), a critical adapter in the activation of NF-κB by interleukin-1 and other cytokines (6Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (517) Google Scholar). In addition, inhibition of NF-κB increases p75-mediated apoptosis in this system (7Ye X. Mehlen P. Rabizadeh S. Van Arsdale T. Zhang H. Shin H. Wang J.J.L. Leo E. Zapata J. Hauser C.A. Reed J.C. Bredesen D.E. J. Biol. Chem. 1999; 274: 30202-30208Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), demonstrating a prosurvival requirement for this transcription factor (8Gentry J.J., C.- Bonnefil P. Carter B.D. J. Biol. Chem. 2000; 275: 7558-7565Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 9Feng Z. Porter A.G. J. Biol. Chem. 1999; 274: 30341-30344Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Furthermore, mice deficient in IKK, the enzyme that phosphorylates and targets the inhibitory molecule IκB leading to NF−κB activation, leads to a defect in neureulation (10Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733Crossref PubMed Google Scholar). Similarly, TRAF6-deficient mice also display a failure of neural tube closure and exencephaly (11Lomaga M.A. Henderson J.T. Elia A.J. Robertson J. Noyce R.S. Yeh W.C. Mak T.W. J. Neurosci. 2000; 20: 7384-7393Crossref PubMed Google Scholar). Collectively, these findings underscore the importance of NF−κB in the nervous system. The activation of IKK and NF-κB has been shown to require atypical protein kinase C (aPKC) in both neuronal and non-neuronal systems (reviewed in Ref. 12Wooten M.W. J. Neurosci. Res. 1999; 58: 607-611Crossref PubMed Scopus (77) Google Scholar). Moreover, aPKC over-expression enhances NGF prosurvival signaling through up-regulation of NF-κB (13Wooten M.W. Seibenhener M.L. Zhou G. Vandenplas M.L. Tan T.H. Cell Death Differ. 1999; 6: 753-764Crossref PubMed Scopus (58) Google Scholar). In contrast, proapoptotic signaling inhibits aPKC and blocks NF-κB (14Wang Y. Seibenhener M.L. Vandenplas M.L. Wooten M.W. J. Neurosci. Res. 1999; 55: 293-302Crossref PubMed Scopus (123) Google Scholar). Additionally, the selective aPKC-binding protein, p62 (15Puls A. Schmidt S. Grawe F. Stabel S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6191Crossref PubMed Scopus (194) Google Scholar, 16Sanchez P. Carcer G. Sandoval I.V. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1998; 18: 3069-3080Crossref PubMed Scopus (200) Google Scholar), has been shown to interact with TRAF6 and to be essential during interleukin-1 signaling to NF-κB (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). Here we report that p62 plays a novel role as a scaffold for the activation of NF-κB by nerve growth factor, linking both p75 and TrkA receptor components. Cultures of human embryonic kidney 293 (HEK 293) or NIH-3T3 cells were maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Subconfluent cells were transfected by the calcium phosphate method. PC12 cells were grown on cultureware coated with rat tail collagen in RPMI containing 10% horse serum and 5% fetal calf serum and antibiotics (50 units/ml penicillin and 50 mg/ml streptomycin). PC12 or NIH-3T3 cells were routinely transfected using LipofectAMINE 2000 (Life Technologies, Inc.). 2.5 S NGF was purchased from Bioproducts for Science (Indianapolis, IN). The monoclonal 12CA5 anti-HA and anti-Flag antibodies were from Sigma. The rabbit anti-Myc, anti-TRAF6, anti-TrkA, and anti-IKK antibodies were from Santa Cruz Biotechnology. The monoclonal anti-p62 was obtained from BD Transduction Laboratories. To detect endogenous proteins in PC12 cells or those cotransfected into HEK cells, lysates were prepared from subconfluent cultures of cells grown on 100-mm dishes. Typically cells were transfected for 36–42 h with 5–10 μg of construct and pcDNA3 plasmid to give 30 μg of total DNA. After transfection, cells were stimulated or not with 50 ng/ml NGF. Cells were then harvested and lysed in PD buffer (40 mm Tris-HCl pH 8.0, 500 mm NaCl, 0.1% Nonidet P-40, 6 mm EDTA, 6 mm EGTA, 10 mmβ-glycerophosphate, 10 mm NaF, 10 mm phenyl phosphate, 300 μm Na3V04, 1 mm benzamidine, 2 mm phenylmethysulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mm dithiothreitol). Extracts were centrifuged at 15,000 × g for 15 min. Protein was determined, and equal amounts of whole-cell lysate were diluted in PD buffer and incubated with antibody as indicated for 2 h. Then protein A or G beads were added for an additional hour at 4 °C. The immunoprecipitates were then washed five times with PD buffer. As control an aliquot of the cell lysate (1/10) volume was also analyzed by immunoblotting. Samples were fractionated on 7.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to Western blot analysis with the corresponding antibodies. Proteins were detected with ECL reagents (Amersham Pharmacia Biotech). NF-κB activation was measured using a reporter gene assay. HEK, 3T3, or PC12 cells were transfected with a κB-luciferase reporter gene plasmid, 3EconA-Luc (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). After 24 or 48 h, the cells were stimulated with NGF and activity determined using a Promega luciferase assay kit. Individual constructs were transfected in duplicate and each assay measured in triplicate. Values are reported as the mean ± S.E. of three individual experiments. Because aPKC is critically involved in the NGF prosurvival signaling pathway (13Wooten M.W. Seibenhener M.L. Zhou G. Vandenplas M.L. Tan T.H. Cell Death Differ. 1999; 6: 753-764Crossref PubMed Scopus (58) Google Scholar, 18Wooten M.W. Seibenhener M.L. Neidigh K.B. Vandenplas M.L. Mol. Cell. Biol. 2000; 20: 4494-4504Crossref PubMed Scopus (70) Google Scholar), we decided to investigate whether neurotrophin binding would stimulate the formation of a complex between NGF receptor components, TrkA and p75, and p62. PC12 cell lysates were prepared from NGF-treated cells followed by pull-down assays (19Yamashita T. Tucker K.L. Barde Y.A. Neuron. 2000; 24: 585-593Abstract Full Text Full Text PDF Scopus (445) Google Scholar) employing GST-TrkA or GST-p75 (Fig.1 A). p62 associated with TrkA but not with p75. To confirm this finding in a more in vivosetting, p62 was coexpressed with either TrkA or p75 in HEK cells (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar), and the coprecipitation of p62 with either receptor was examined. The results, shown in Fig. 1 B, confirmed that p62 selectively associates with TrkA but not with p75. As in this experiment both TrkA and p62 were over-expressed, their interaction took place even in the absence of any stimulus. To demonstrate that endogenous p62 and TrkA interact in vivo and that this interaction may be induced in response to NGF, PC12 cells were stimulated with NGF for different times after which the coprecipitation of endogenous p62 with TrkA was examined. The results shown in Fig. 1 C demonstrate that there is a significant portion of p62 bound to TrkA under basal conditions but that this interaction was reproducibly enhanced in the NGF-treated cells (Fig. 1 C), indicating that this is an NGF-regulated process. To establish which region of p62 is required for interaction with TrkA, deletion mutants of p62 were transfected into HEK cells along with HA-tagged TrkA, and their association was determined by immunoprecipitation as above (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). The region encompassing amino acids 266–446 of p62 binds TrkA, whereas the binding of p62 to TRAF6 has been mapped to amino acids 225–251 (Ref.17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar and Table I). This indicates that p62 may accommodate both TrkA and TRAF6 simultaneously. Deletion of the TRAF6 binding site did not effect TrkA binding to p62, thus further strengthening the notion that p62 interacts with TrkA and TRAF6 through two independent binding domains (Table I).Table IMapping of p62-TrkA interaction domainsMyc-tagged p62 constructs were prepared as described previously (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar) and coexpressed in HEK cells with HA-tagged TrkA. Interaction between p62 and TrkA was determined by coimmunoprecipitation (21Lallena M.J. Diaz-Meco M.T. Bren G. Paya C. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). For comparison, the binding of p62 to TrkA was mapped relative to aPKC and TRAF6 (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). AID, acidic sequence and atypical PKC interacting domain; ZZ, zinc-finger domain. Open table in a new tab Myc-tagged p62 constructs were prepared as described previously (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar) and coexpressed in HEK cells with HA-tagged TrkA. Interaction between p62 and TrkA was determined by coimmunoprecipitation (21Lallena M.J. Diaz-Meco M.T. Bren G. Paya C. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). For comparison, the binding of p62 to TrkA was mapped relative to aPKC and TRAF6 (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). AID, acidic sequence and atypical PKC interacting domain; ZZ, zinc-finger domain. TRAF6 has been reported to interact with p75 (5Khursigara G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). To determine whether TRAF6 interacts with TrkA as well, both p75 and TrkA were coexpressed in HEK cells along with Flag-TRAF6. Whereas p75 associated with TRAF6 in an NGF-dependent manner (Fig.2 A) as previously reported (5Khursigara G. Orlinick J.R. Chao M.V. J. Biol. Chem. 1999; 274: 2597-2600Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), TRAF6 failed to associate directly with TrkA. The interaction of p75 with TRAF6 was mapped to the C-proximal TRAF-C domain, a region that also accommodates p62 (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). Coexpression experiments in HEK cells revealed that TRAF6 can bind both p75 and p62 simultaneously (not shown). Because p62 interacts with TRAF6 (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar), it is conceivable that p62 may be brought into a p75 complex via TRAF6 serving as a bridge. If this model is correct we should be able to coimmunoprecipitate p62 with p75 only in the presence of TRAF6. The results shown in Fig.2 B strongly suggest that these predictions are correct. Thus, in HEK cells transfected with different expression vectors, a small amount of p75 was found to associate with p62, likely through endogenous TRAF6. Upon coexpression of TRAF6, recruitment of p75 into the p62 complex was significantly enhanced (∼2.5-fold). The ability of TrkA to coassociate with TRAF6 was dramatically and consistently enhanced by the presence of exogenous p62 (Fig.2 B). We next determined whether NGF could stimulate the formation of an endogenous TRAF6-p62 complex in PC12 cells (Fig.2 C). In the absence of NGF little or no association of TRAF6 with p62 or aPKC could be detected. However, the addition of NGF resulted in a rapid interaction of TRAF6 with p62 and consequently with aPKC. Close examination revealed that the kinetics of association between TRAF6 and p62 (maximum 1–5 min) occurs prior to the recruitment of p62 to the TrkA receptor (Fig. 1 C, peaks at 15 min), suggesting that it is a two-step process. Collectively, these results reveal that p62 interacts with TRAF6 in response to NGF and may likely serve as a bridge between both p75 and TrkA receptor components. As TRAF6 interaction with p75 results in activation of NF-κB (7Ye X. Mehlen P. Rabizadeh S. Van Arsdale T. Zhang H. Shin H. Wang J.J.L. Leo E. Zapata J. Hauser C.A. Reed J.C. Bredesen D.E. J. Biol. Chem. 1999; 274: 30202-30208Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), it was of interest to determine whether the down-regulation of p62 with a p62 antisense construct (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar) would block the induction of NF-κB as measured by a luciferase reporter system. The results shown in Fig.3 A demonstrate that this is the case, because there was a dramatic reduction of NF-κB activation by the expression of p75 and TRAF6 in cells transfected with the p62 antisense construct as compared with the nontransfected cells. On the other hand, the transfection of a p62 expression vector, that by itself does not activate NF-κB in this system (17Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar), dramatically enhanced p75-TRAF6 or TrkA-TRAF6 activation of NF-κB (Fig.3 B). In contrast, p62/ZIP2, which lacks the TRAF6 binding site (16Sanchez P. Carcer G. Sandoval I.V. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1998; 18: 3069-3080Crossref PubMed Scopus (200) Google Scholar), failed to activate NF-κB (Fig. 3 B). Altogether this indicates that the recruitment of p62 to the NGF receptor signaling complex is critical for the activation of NF-κB. Consistent with this notion, overexpression of p62 in PC12 cells resulted in a dose-dependent enhancement of both basal as well as NGF-stimulated activation of NF-κB (Fig. 3 C). We next conducted experiments to investigate whether the antisense construct of p62 would block NGF-induced activation of NF-κB in cells expressing either one or both NGF receptors. Interestingly, antisense p62 blocked NGF-induced activation of NF-κB in PC12 cells expressing both receptors (Fig. 3 D). Likewise, p62 down-regulation in NIH-3T3 cells expressing either p75 or TrkA receptor (20Jiang H. Takeda K. Lazarovici P. Katagiri Y., Yu, Z. Dickens G. Chabuk A. Liu X. Ferrans V. Guroff G. J. Biol. Chem. 1999; 274: 26209-26216Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) (Fig.3 D) also abrogated NGF-induced activation of NF-κB. Collectively these findings demonstrate that p62, like aPKC (13Wooten M.W. Seibenhener M.L. Zhou G. Vandenplas M.L. Tan T.H. Cell Death Differ. 1999; 6: 753-764Crossref PubMed Scopus (58) Google Scholar), is essential in the activation of NF-κB by NGF and that it serves to scaffold proximal NGF receptor components in this pathway. The functional relevance of the presence of p62 in these complexes was addressed further in the following series of experiments. Overexpression of p75 in HEK cells results in ligand-independent cell death that is prevented by TRAF6 (7Ye X. Mehlen P. Rabizadeh S. Van Arsdale T. Zhang H. Shin H. Wang J.J.L. Leo E. Zapata J. Hauser C.A. Reed J.C. Bredesen D.E. J. Biol. Chem. 1999; 274: 30202-30208Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 8Gentry J.J., C.- Bonnefil P. Carter B.D. J. Biol. Chem. 2000; 275: 7558-7565Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Consistent with the role of NF-κB in cell survival signaling (4Foehr E.D. Lin X. O'Mahony A. Geleziunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar), the expression of antisense p62 enhanced p75 mediated-cell death (Fig.4 A), whereas expression of TRAF6 or p62 blocked cell death. The activation of NF-κB is likewise required for neuronal differentiation (9Feng Z. Porter A.G. J. Biol. Chem. 1999; 274: 30341-30344Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and TrkA responsiveness (4Foehr E.D. Lin X. O'Mahony A. Geleziunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). Transfection of antisense p62 significantly impaired NGF-induced neurite outgrowth, whereas overexpression of p62 enhanced NGF responsiveness (Fig. 4 A). The mechanism whereby p62 regulates activation of NF−κB likely involves recruitment of TRAF6 and aPKC onto the p62 scaffold, thus enabling aPKC-mediated phosphorylation of IKK (21Lallena M.J. Diaz-Meco M.T. Bren G. Paya C. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). To provide evidence for the involvement of p62 in this process, we assessed the activity of endogenous IKK activity in an in vitro kinase assay using GST-Iκβα as the substrate (4Foehr E.D. Lin X. O'Mahony A. Geleziunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar, 21Lallena M.J. Diaz-Meco M.T. Bren G. Paya C. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). Transfection of antisense p62 suppressed NGF-stimulated activation of IKK (Fig. 4 B). The findings reported here provide new insight into the proximal components of the NGF/NF-κB pathway and demonstrate formation of a p62 bridge that scaffolds together both p75 and TrkA receptors for the activation of NF-κB (Fig. 4 C). Our results stress the role of p62 as a common and critical intermediary that channels different signaling pathways toward IKK activation. Understanding the mechanism whereby the TRAF6-p62 complex is regulated in vivois an area of ongoing study. Together, these findings underscore the importance of p62 as a scaffold for NF-κB and as a common platform for communication of both p75 and TrkA receptor signals. We are indebted to Moses Chao and Rick Dobrosky for constructs, Gordon Guroff for support and cell lines, Laura Sanz for advice, and Esther Garcia for technical assistance.

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