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

2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês

10.1074/jbc.m208468200

1083-351X

Thangiah Geetha, Marie W. Wooten,

Virus-based gene therapy research

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. 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. Nerve growth factor (NGF) 1The abbreviations used are: NGF, nerve growth factor; FACS, fluorescence-activated cell sorter; GST, glutathioneS-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-κB, nuclear factor-κB; 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 1The abbreviations used are: NGF, nerve growth factor; FACS, fluorescence-activated cell sorter; GST, glutathioneS-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-κB, nuclear factor-κB; 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 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) (1Chao M.V. J. Neurosci. Res. 2000; 59: 353-355Crossref PubMed Scopus (87) Google Scholar). 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 (2Patapoutian A. Reichardt L.F. Curr. Opin. Neurobiol. 2001; 11: 272-280Crossref PubMed Scopus (906) Google Scholar). 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 (3Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (470) Google Scholar) such as phospholipase C (PLC)-γ1 (4Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (544) Google Scholar), adapter proteins Shc and FRS2 (5Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar), and phosphatidylinositol 3′-kinase (6Holgado-Madruga M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar) that initiates the intracellular signaling cascades. It has been observed that NGF rapidly induces internalization of TrkA receptor to endocytic vesicles (7Grimes M.L. Zhou J. Beattie E. Yuen E. Hall D. Valletta J. Topp K. LaVail J. Bunnett N. Mobley W.C. J. Neurosci. 1996; 16: 7950-7964Crossref PubMed Google Scholar, 8Grimes M.L. Beattie E. Mobley W.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9909-9914Crossref PubMed Scopus (147) Google Scholar, 9Ricco A. Pierchala B.A. Ciarallo C.L. Ginty D.D. Science. 1997; 277: 1097-1100Crossref PubMed Scopus (363) Google Scholar). The receptors within signaling vesicles remain catalytically active and phosphorylated because they are less accessible to membrane-associated phosphatases (10Tisi M. Xie Y. Yeo T. Longo F. J. Neurobiol. 2000; 42: 477-486Crossref PubMed Scopus (40) Google Scholar). Internalized TrkA receptors induce a higher peak level of mitogen-activated protein kinase (ERK/MAPK) activation thereby regulating cell differentiation (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar).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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). p62 then recruits atypical protein kinase C (aPKC) to phosphorylate IκB kinase and leads to the activation of the transcription factor nuclear factor-κB (NF-κB). Expression of antisense p62 in PC12 cells inhibits NGF-induced NF-κB activation (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In contrast, it has been shown that a dominant-negative mutant of the Shc adaptor protein effectively blocks TrkA-mediated activation of NF-κB (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). p62 also serves as a scaffold for the NF-κB pathway through tumor necrosis factor-α and interleukin-1 receptor signaling cascades (14Sanz L. Sanchez P. Lallena M.J. Diaz-Meco M.T. Moscat J. EMBO J. 1999; 18: 3044-3053Crossref PubMed Scopus (323) Google Scholar, 15Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). Endogenous and ectopically expressed p62 has been shown to colocalize with λ/ιPKC and ζPKC in lysosome-targeted endosomes (16Sanchez P. De Carcer G. Sandoval I.V. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1998; 18: 3069-3080Crossref PubMed Scopus (197) Google Scholar, 17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). p62 binds the NGF receptor, TrkA (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), which likewise localizes to late endosomal vesicles. Recent studies have shown that internalized TrkA receptors continue to signal within the endosomal compartment (18Howe C.L. Valletta J.S. Rusnak A.S. Mobley W.C. Neuron. 2001; 32: 801-814Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and are required to mediate NGF-induced differentiation (10Tisi M. Xie Y. Yeo T. Longo F. J. Neurobiol. 2000; 42: 477-486Crossref PubMed Scopus (40) Google Scholar). Moreover, inhibition of p62 expression has been shown to block NGF-induced neurite outgrowth (17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). 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.DISCUSSIONOur 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 of125I-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 (32Buxser S. Decker D. Ruppel P. J. Biol. Chem. 1990; 265: 12701-12710Abstract Full Text PDF PubMed Google Scholar, 33Zapf-Colby A. Olefsky J.M. Endocrinology. 1998; 139: 3232-3240Crossref PubMed Scopus (57) Google Scholar). We have shown previously (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The kinetics of binding between p62 and p75 is an early rapid event, taking place between 1 and 5 min (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Recent studies (24Jullien J. Guili V. Reichardt L.F. Rudkin B.B. J. Biol. Chem. 2002; 277: 38700-38708Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) 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.Antisense p62 has been shown to block NGF-mediated differentiation of PC12 cells (17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). These data shed light on the underlying mechanism whereby p62 can affect NGF-mediated differentiation. Previous studies (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar) 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 (30Meakin S.O. MacDonald J.I.S. J. Neurochem. 1998; 71: 1875-1888Crossref PubMed Scopus (34) Google Scholar). Interestingly, another signaling adapter GIPC binds to TrkA employing the same amino acids (19Lou X. Yano H. Lee F. Chao M.V. Farquhar M.G. Mol. Biol. Cell. 2001; 12: 615-627Crossref PubMed Scopus (130) Google Scholar). We have examined whether p62 binds to GIPC and have failed to observe a direct interaction. 2T. Geetha and M. W. Wooten, unpublished data. 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 (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar, 21Watson F.L. Heerssen H.M. Bhattacharya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (382) Google Scholar). Moreover, TrkA internalization and retrograde transport has been shown to activate specifically the Erk5 pathway (21Watson F.L. Heerssen H.M. Bhattacharya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (382) Google Scholar). 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 (22Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Interestingly, MEK5 contains an aPKC interaction domain, and deletion of this region impairs formation of a complex between aPKC and MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar). Moreover, it has been shown that aPKC recruitment into a complex triggers the activation of ErK5 in a manner dependent on MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar); 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 (20Wooten M.W. Vandenplas M.L. Seibenhener M.L. Geetha T. Diaz-Meco M.T. Mol. Cell. Biol. 2001; 21: 8414-8427Crossref PubMed Scopus (75) Google Scholar), followed by activation of MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar), leading to activation of Erk5 (Fig. 9). It is interesting to note that a newly isolated protein, Pincher (36Shao Y. Akmentin W. Toledo-Aral J. Rosenbaum J. Valdez G. Cabot J. Hilbush B. Halegoua S. J. Cell Biol. 2002; 157: 679-691Crossref PubMed Scopus (141) Google Scholar), 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar,37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) 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-κB pathway (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). With respect to TrkA, it has been shown that Shc is required for this pathway (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar, 34Raffioni S. Thomas D. Foehr E.D. Thompson L.M. Bradshaw R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7178-7183Crossref PubMed Scopus (68) Google Scholar, 38Thomas D. Bradshaw R.A. J. Biol. Chem. 1997; 272: 22293-22299Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The failure of Shc to compete for with p62 for binding to TrkA reveals that p62 activates the κB 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 κB signal is attenuated (37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); inhibition of TrkA by K252a enhances survival signaling, impairs TrkA internalization, and promotes activation of NF-κB. Alternatively, overexpression of p62 can enhance survival signaling and activation of NF-κB (37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, our data support a model whereby survival signaling occurs predominantly from NGF bound at the cell surface (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). 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-κB (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) 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-κB activation in neurons. Nerve growth factor (NGF) 1The abbreviations used are: NGF, nerve growth factor; FACS, fluorescence-activated cell sorter; GST, glutathioneS-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-κB, nuclear factor-κB; 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 1The abbreviations used are: NGF, nerve growth factor; FACS, fluorescence-activated cell sorter; GST, glutathioneS-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-κB, nuclear factor-κB; 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 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) (1Chao M.V. J. Neurosci. Res. 2000; 59: 353-355Crossref PubMed Scopus (87) Google Scholar). 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 (2Patapoutian A. Reichardt L.F. Curr. Opin. Neurobiol. 2001; 11: 272-280Crossref PubMed Scopus (906) Google Scholar). 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 (3Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (470) Google Scholar) such as phospholipase C (PLC)-γ1 (4Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (544) Google Scholar), adapter proteins Shc and FRS2 (5Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar), and phosphatidylinositol 3′-kinase (6Holgado-Madruga M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar) that initiates the intracellular signaling cascades. It has been observed that NGF rapidly induces internalization of TrkA receptor to endocytic vesicles (7Grimes M.L. Zhou J. Beattie E. Yuen E. Hall D. Valletta J. Topp K. LaVail J. Bunnett N. Mobley W.C. J. Neurosci. 1996; 16: 7950-7964Crossref PubMed Google Scholar, 8Grimes M.L. Beattie E. Mobley W.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9909-9914Crossref PubMed Scopus (147) Google Scholar, 9Ricco A. Pierchala B.A. Ciarallo C.L. Ginty D.D. Science. 1997; 277: 1097-1100Crossref PubMed Scopus (363) Google Scholar). The receptors within signaling vesicles remain catalytically active and phosphorylated because they are less accessible to membrane-associated phosphatases (10Tisi M. Xie Y. Yeo T. Longo F. J. Neurobiol. 2000; 42: 477-486Crossref PubMed Scopus (40) Google Scholar). Internalized TrkA receptors induce a higher peak level of mitogen-activated protein kinase (ERK/MAPK) activation thereby regulating cell differentiation (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). p62 then recruits atypical protein kinase C (aPKC) to phosphorylate IκB kinase and leads to the activation of the transcription factor nuclear factor-κB (NF-κB). Expression of antisense p62 in PC12 cells inhibits NGF-induced NF-κB activation (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In contrast, it has been shown that a dominant-negative mutant of the Shc adaptor protein effectively blocks TrkA-mediated activation of NF-κB (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). p62 also serves as a scaffold for the NF-κB pathway through tumor necrosis factor-α and interleukin-1 receptor signaling cascades (14Sanz L. Sanchez P. Lallena M.J. Diaz-Meco M.T. Moscat J. EMBO J. 1999; 18: 3044-3053Crossref PubMed Scopus (323) Google Scholar, 15Sanz L. Diaz-Meco M.T. Nakano H. Moscat J. EMBO J. 2000; 19: 1576-1586Crossref PubMed Google Scholar). Endogenous and ectopically expressed p62 has been shown to colocalize with λ/ιPKC and ζPKC in lysosome-targeted endosomes (16Sanchez P. De Carcer G. Sandoval I.V. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1998; 18: 3069-3080Crossref PubMed Scopus (197) Google Scholar, 17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). p62 binds the NGF receptor, TrkA (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), which likewise localizes to late endosomal vesicles. Recent studies have shown that internalized TrkA receptors continue to signal within the endosomal compartment (18Howe C.L. Valletta J.S. Rusnak A.S. Mobley W.C. Neuron. 2001; 32: 801-814Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and are required to mediate NGF-induced differentiation (10Tisi M. Xie Y. Yeo T. Longo F. J. Neurobiol. 2000; 42: 477-486Crossref PubMed Scopus (40) Google Scholar). Moreover, inhibition of p62 expression has been shown to block NGF-induced neurite outgrowth (17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). 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. DISCUSSIONOur 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 of125I-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 (32Buxser S. Decker D. Ruppel P. J. Biol. Chem. 1990; 265: 12701-12710Abstract Full Text PDF PubMed Google Scholar, 33Zapf-Colby A. Olefsky J.M. Endocrinology. 1998; 139: 3232-3240Crossref PubMed Scopus (57) Google Scholar). We have shown previously (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The kinetics of binding between p62 and p75 is an early rapid event, taking place between 1 and 5 min (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Recent studies (24Jullien J. Guili V. Reichardt L.F. Rudkin B.B. J. Biol. Chem. 2002; 277: 38700-38708Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) 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.Antisense p62 has been shown to block NGF-mediated differentiation of PC12 cells (17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). These data shed light on the underlying mechanism whereby p62 can affect NGF-mediated differentiation. Previous studies (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar) 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 (30Meakin S.O. MacDonald J.I.S. J. Neurochem. 1998; 71: 1875-1888Crossref PubMed Scopus (34) Google Scholar). Interestingly, another signaling adapter GIPC binds to TrkA employing the same amino acids (19Lou X. Yano H. Lee F. Chao M.V. Farquhar M.G. Mol. Biol. Cell. 2001; 12: 615-627Crossref PubMed Scopus (130) Google Scholar). We have examined whether p62 binds to GIPC and have failed to observe a direct interaction. 2T. Geetha and M. W. Wooten, unpublished data. 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 (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar, 21Watson F.L. Heerssen H.M. Bhattacharya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (382) Google Scholar). Moreover, TrkA internalization and retrograde transport has been shown to activate specifically the Erk5 pathway (21Watson F.L. Heerssen H.M. Bhattacharya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (382) Google Scholar). 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 (22Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Interestingly, MEK5 contains an aPKC interaction domain, and deletion of this region impairs formation of a complex between aPKC and MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar). Moreover, it has been shown that aPKC recruitment into a complex triggers the activation of ErK5 in a manner dependent on MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar); 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 (20Wooten M.W. Vandenplas M.L. Seibenhener M.L. Geetha T. Diaz-Meco M.T. Mol. Cell. Biol. 2001; 21: 8414-8427Crossref PubMed Scopus (75) Google Scholar), followed by activation of MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar), leading to activation of Erk5 (Fig. 9). It is interesting to note that a newly isolated protein, Pincher (36Shao Y. Akmentin W. Toledo-Aral J. Rosenbaum J. Valdez G. Cabot J. Hilbush B. Halegoua S. J. Cell Biol. 2002; 157: 679-691Crossref PubMed Scopus (141) Google Scholar), 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar,37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) 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-κB pathway (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). With respect to TrkA, it has been shown that Shc is required for this pathway (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar, 34Raffioni S. Thomas D. Foehr E.D. Thompson L.M. Bradshaw R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7178-7183Crossref PubMed Scopus (68) Google Scholar, 38Thomas D. Bradshaw R.A. J. Biol. Chem. 1997; 272: 22293-22299Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The failure of Shc to compete for with p62 for binding to TrkA reveals that p62 activates the κB 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 κB signal is attenuated (37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); inhibition of TrkA by K252a enhances survival signaling, impairs TrkA internalization, and promotes activation of NF-κB. Alternatively, overexpression of p62 can enhance survival signaling and activation of NF-κB (37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, our data support a model whereby survival signaling occurs predominantly from NGF bound at the cell surface (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). 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-κB (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) 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-κB activation in neurons. 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 of125I-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 (32Buxser S. Decker D. Ruppel P. J. Biol. Chem. 1990; 265: 12701-12710Abstract Full Text PDF PubMed Google Scholar, 33Zapf-Colby A. Olefsky J.M. Endocrinology. 1998; 139: 3232-3240Crossref PubMed Scopus (57) Google Scholar). We have shown previously (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The kinetics of binding between p62 and p75 is an early rapid event, taking place between 1 and 5 min (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Recent studies (24Jullien J. Guili V. Reichardt L.F. Rudkin B.B. J. Biol. Chem. 2002; 277: 38700-38708Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) 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. Antisense p62 has been shown to block NGF-mediated differentiation of PC12 cells (17Samuels I.S. Seibenhener M.L. Neidigh K.B.W. Wooten M.W. J. Cell. Biochem. 2001; 82: 452-466Crossref PubMed Scopus (39) Google Scholar). These data shed light on the underlying mechanism whereby p62 can affect NGF-mediated differentiation. Previous studies (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar) 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 (30Meakin S.O. MacDonald J.I.S. J. Neurochem. 1998; 71: 1875-1888Crossref PubMed Scopus (34) Google Scholar). Interestingly, another signaling adapter GIPC binds to TrkA employing the same amino acids (19Lou X. Yano H. Lee F. Chao M.V. Farquhar M.G. Mol. Biol. Cell. 2001; 12: 615-627Crossref PubMed Scopus (130) Google Scholar). We have examined whether p62 binds to GIPC and have failed to observe a direct interaction. 2T. Geetha and M. W. Wooten, unpublished data. 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 (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar, 21Watson F.L. Heerssen H.M. Bhattacharya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (382) Google Scholar). Moreover, TrkA internalization and retrograde transport has been shown to activate specifically the Erk5 pathway (21Watson F.L. Heerssen H.M. Bhattacharya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (382) Google Scholar). 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 (22Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Interestingly, MEK5 contains an aPKC interaction domain, and deletion of this region impairs formation of a complex between aPKC and MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar). Moreover, it has been shown that aPKC recruitment into a complex triggers the activation of ErK5 in a manner dependent on MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar); 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 (20Wooten M.W. Vandenplas M.L. Seibenhener M.L. Geetha T. Diaz-Meco M.T. Mol. Cell. Biol. 2001; 21: 8414-8427Crossref PubMed Scopus (75) Google Scholar), followed by activation of MEK5 (35Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 2001; 21: 1218-1227Crossref PubMed Scopus (60) Google Scholar), leading to activation of Erk5 (Fig. 9). It is interesting to note that a newly isolated protein, Pincher (36Shao Y. Akmentin W. Toledo-Aral J. Rosenbaum J. Valdez G. Cabot J. Hilbush B. Halegoua S. J. Cell Biol. 2002; 157: 679-691Crossref PubMed Scopus (141) Google Scholar), 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 (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar,37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) 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-κB pathway (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar). With respect to TrkA, it has been shown that Shc is required for this pathway (13Foehr E.D. Lin X. O'Mahony A. Gelezinunas R. Bradshaw R.A. Greene W.C. J. Neurosci. 2000; 20: 7556-7563Crossref PubMed Google Scholar, 34Raffioni S. Thomas D. Foehr E.D. Thompson L.M. Bradshaw R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7178-7183Crossref PubMed Scopus (68) Google Scholar, 38Thomas D. Bradshaw R.A. J. Biol. Chem. 1997; 272: 22293-22299Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The failure of Shc to compete for with p62 for binding to TrkA reveals that p62 activates the κB 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 κB signal is attenuated (37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); inhibition of TrkA by K252a enhances survival signaling, impairs TrkA internalization, and promotes activation of NF-κB. Alternatively, overexpression of p62 can enhance survival signaling and activation of NF-κB (37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, our data support a model whereby survival signaling occurs predominantly from NGF bound at the cell surface (11Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). 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-κB (12Wooten M.W. Seibenhener M.L. Mamidipudi V. Diaz-Meco M.T. Barker P.A. Moscat J. J. Biol. Chem. 2001; 276: 7709-7712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 37Mamidipudi V. Li X. Wooten M.W. J. Biol. Chem. 2002; 277: 28010-28018Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) 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-κB activation in neurons. 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.