Brain-derived Neurotrophic Factor Activates ERK5 in Cortical Neurons via a Rap1-MEKK2 Signaling Cascade
2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês
10.1074/jbc.m605503200
ISSN1083-351X
AutoresYupeng Wang, Bing Su, Zhengui Xia,
Tópico(s)RNA regulation and disease
ResumoThe extracellular signal-regulated kinase 5 (ERK5) is activated in neurons of the central nervous system by neurotrophins including brain-derived neurotrophic factor (BDNF). Although MEK5 is known to mediate BDNF stimulation of ERK5 in central nervous system neurons, other upstream signaling components have not been identified. Here, we report that BDNF induces a sustained activation of ERK5 in rat cortical neurons and activates Rap1, a small GTPase, as well as MEKK2, a MEK5 kinase. Our data indicate that activation of Rap1 or MEKK2 is sufficient to stimulate ERK5, whereas inhibition of either Rap1 or MEKK2 attenuates BDNF activation of ERK5. Furthermore, BDNF stimulation of MEKK2 is regulated by Rap1. Our evidence also indicates that Ras and MEKK3, a MEK5 kinase in non-neuronal cells, do not play a significant role in BDNF activation of ERK5. This study identifies Rap1 and MEKK2 as critical upstream signaling molecules mediating BDNF stimulation of ERK5 in central nervous system neurons. The extracellular signal-regulated kinase 5 (ERK5) is activated in neurons of the central nervous system by neurotrophins including brain-derived neurotrophic factor (BDNF). Although MEK5 is known to mediate BDNF stimulation of ERK5 in central nervous system neurons, other upstream signaling components have not been identified. Here, we report that BDNF induces a sustained activation of ERK5 in rat cortical neurons and activates Rap1, a small GTPase, as well as MEKK2, a MEK5 kinase. Our data indicate that activation of Rap1 or MEKK2 is sufficient to stimulate ERK5, whereas inhibition of either Rap1 or MEKK2 attenuates BDNF activation of ERK5. Furthermore, BDNF stimulation of MEKK2 is regulated by Rap1. Our evidence also indicates that Ras and MEKK3, a MEK5 kinase in non-neuronal cells, do not play a significant role in BDNF activation of ERK5. This study identifies Rap1 and MEKK2 as critical upstream signaling molecules mediating BDNF stimulation of ERK5 in central nervous system neurons. The extracellular signal-regulated kinase 5 (ERK5) 2The abbreviations used are: ERK5, extracellular signal-regulated kinase 5; MEKK, MEK kinase; BDNF, brain-derived neurotrophic factor; MAP, mitogen-activated protein; E17, embryonic day 17; DIV, days in vitro; GST, glutathione S-transferase; dn, dominant negative; ca, constitutive active; PAO, phenylarsine oxide; ts, temperature sensitive; MDC, monodansylcadaverine; HA, hemagglutinin. 2The abbreviations used are: ERK5, extracellular signal-regulated kinase 5; MEKK, MEK kinase; BDNF, brain-derived neurotrophic factor; MAP, mitogen-activated protein; E17, embryonic day 17; DIV, days in vitro; GST, glutathione S-transferase; dn, dominant negative; ca, constitutive active; PAO, phenylarsine oxide; ts, temperature sensitive; MDC, monodansylcadaverine; HA, hemagglutinin. or big MAP kinase 1 (BMK1) is a member of the mitogen-activated protein (MAP) kinase family that includes ERK1/2, p38, and N-terminal c-Jun protein kinase (JNK) (1Zhou G. 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Chem. 2003; 278: 18682-18688Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Anthony T.E. Klein C. Fishell G. Heintz N. Neuron. 2004; 41: 881-890Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). ERK5 is expressed in many tissues with the highest levels in the brain (15Yan L. Carr J. Ashby P.R. Murry-Tait V. Thompson C. Arthur J.S. BMC Dev. Biol. 2003; 3: 11Crossref PubMed Scopus (96) Google Scholar). We discovered that ERK5 expression in the brain is maximal during early embryonic development and declines as the brain matures (16Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar). Interestingly, ERK5 and ERK5-regulated MEF2 gene expression contribute to BDNF-promoted survival of developing, but not mature neurons cultured from the cortex and cerebellum (16Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar, 17Shalizi A. Lehtinen M. Gaudilliere B. Donovan N. Han J. Konishi Y. Bonni A. J. Neurosci. 2003; 23: 7326-7336Crossref PubMed Google Scholar). Because ERK5 is highly expressed in developing neurons of the central nervous system and plays a critical role in their survival, it is crucial to define ERK5 signaling mechanisms in central nervous system neurons. The only known signaling molecule mediating ERK5 activation in central nervous system neurons is MEK5 (16Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar, 17Shalizi A. Lehtinen M. Gaudilliere B. Donovan N. Han J. Konishi Y. Bonni A. J. Neurosci. 2003; 23: 7326-7336Crossref PubMed Google Scholar). Here, we used primary cultured cortical neurons from embryonic day (E) 16-17 rodents to investigate signaling pathways that mediate BDNF stimulation of ERK5. Our data strongly implicate Rap1 and MEKK2 in BDNF stimulation of ERK5. Materials—The following plasmids have been described: the Gal4-MEF2C, Gal4-luciferase, and EF-lacZ (9Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (188) Google Scholar); UB6-lacZ (18Poser S. Impey S. Xia Z. Storm D.R. J. Neurosci. 2003; 23: 4420-4427Crossref PubMed Google Scholar); FLAG-tagged wild-type ERK5 (5Kato Y. Kravchenko V.V. Tapping R.I. Han J.H. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (492) Google Scholar); and the HAMEKK2 and HA-MEKK3 (19Jiang K. Coppola D. Crespo N.C. Nicosia S.V. Hamilton A.D. Sebti S.M. Cheng J.Q. Mol. Cell. Biol. 2000; 20: 139-148Crossref PubMed Scopus (229) Google Scholar, 20Adak S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The dynamin constructs were from Dr. Rosalind Segal (Harvard Medical School, Boston, MA) (21Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar, 22Hayashi T. Sakai K. Sasaki C. Zhang W.R. Warita H. Abe K. Neurosci. Lett. 2000; 284: 195-199Crossref PubMed Scopus (69) Google Scholar), the Rap1b constructs from Dr. Philip Stork (Oregon Health Sciences University, Portland, OR) (23York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar), the constitutive active (V12) or dominant negative (N17) H-ras constructs from Dr. Alan Hall (Memorial Sloan-Kettering Cancer Center, NY), the HA-ERK2 construct from Dr. Melanie H. Cobb (The University of Texas Southwestern Medical Center, Dallas, TX), and GST-RalGDSRBD from Drs. Chengbiao Wu and W. C. Mobley (Stanford University) (24Groszer M. Erickson R. Scripture-Adams D.D. Lesche R. Trumpp A. Zack J.A. Kornblum H.I. Liu X. Wu H. Science. 2001; 294: 2186-2189Crossref PubMed Scopus (669) Google Scholar). GST-MEF2C and GST-RalGDSRBD fusion proteins were expressed in Escherichia coli DH5α cells and purified as described (9Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (188) Google Scholar, 25Herrmann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Abstract Full Text PDF PubMed Scopus (301) Google Scholar). The polyclonal anti-ERK5 antibody was generated as described (4Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar). BDNF and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). The anti-Rab5 antibody, Poly-d-lysine and laminin were purchased from BD Biosciences (Bedford, MA). The monoclonal anti-FLAG antibody, polyclonal anti-FLAG antibody, forskolin, monodansylcadaverine (MDC), and phenylarsine oxide (PAO) were purchased from Sigma. The monoclonal anti-HA antibody was purchased from Roche Applied Science. The polyclonal anti-Rap1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against total ERK1/2 or ERK2, and GST-MKK4 fusion protein were purchased from Upstate (Lake Placid, NY). The anti-p-ERK5 antibody was purchased from Cell Signaling (Danvers, MA), the anti-p-ERK1/2 antibody from Promega (Madison, WI). Cell Culture—Primary cortical neurons were prepared from embryonic day 17 (E17) Sprague-Dawley rats as described (4Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar, 16Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar). Briefly, dissociated neurons were plated at a density of 2 × 106 cells per 35-mm dish. Neurons were cultured in basal medium Eagle (BME, Sigma) supplemented with 10% heat-inactivated bovine calf serum, 35 mm glucose, 1 mm l-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin and maintained in a humidified incubator with 5% CO2 at 37 °C. Plates and glass coverslips were coated with poly-d-lysine and laminin. Neurons were cultured for 5 days in vitro (DIV5) before BDNF treatment or harvesting. Cortical Neuron Cultures from MEKK2-deficient Mice—MEKK2-deficient mice have been described (26Guo Z. Clydesdale G. Cheng J. Kim K. Gan L. McConkey D.J. Ullrich S.E. Zhuang Y. Su B. Mol. Cell. Biol. 2002; 22: 5761-5768Crossref PubMed Scopus (49) Google Scholar). The heterozygous MEKK2+/- mice were bred to generate MEKK2-/- and MEKK2+/+ littermates. At E16, cortical neurons were prepared from individual single embryo using the same conditions as for rat E17 cortical neurons described above. The genotype of each embryo was determined afterward by PCR as described (26Guo Z. Clydesdale G. Cheng J. Kim K. Gan L. McConkey D.J. Ullrich S.E. Zhuang Y. Su B. Mol. Cell. Biol. 2002; 22: 5761-5768Crossref PubMed Scopus (49) Google Scholar). The PCR primers used were: P1 (5′-AGTGTTTTGCTTTTATTATGCT-3′), P2 (5′-AGAAAAACCGAAACTTTACACTAGA-3′), and P3 (5′-AAT TCT CTA GAG GTC CAG ATC CC-3′) (P1 + P2 for wild-type, P1 + P3 for knock-out). At DIV 5, neurons were treated with 50 ng/ml BDNF for 1 h and assayed for ERK5 kinase activity or nuclear translocation. Transient Transfection of Primary Cortical Neurons—Cortical neurons (6 × 106 cells/60-mm dish) were transiently transfected at DIV3 using a calcium phosphate co-precipitation protocol as described (27Xia Z. Dudek H. Miranti C.K. Greenberg M.E. J. Neurosci. 1996; 16: 5425-5436Crossref PubMed Google Scholar, 28Hetman M. Kanning K. Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Briefly, the DNA-calcium phosphate precipitates were prepared by mixing 1 volume of DNA in 250 mm CaCl2 with an equal volume of 2× HEPES-buffered saline (HBS; 274 mm NaCl, 10 mm KCl, 1.4 mm Na2HPO4, 15 mm d-glucose, and 42 mm HEPES, pH 7.07). The precipitates were allowed to form for 25-30 min at room temperature before addition to the cultures. The conditioned culture media were removed and saved. Cells were washed three times with BME, and 4.5 ml of transfection media were added to each 60-mm dish. The transfection media consisted of BME supplemented with 1 mm sodium kynurenate, 10 mm MgCl2, and 5 mm HEPES. The pH of the transfection media was kept high by incubating BME in a dish at 37 °C and 0% CO2 for 30 min to de-gas. 180 μl of the DNA-calcium phosphate precipitates were added dropwise to each 60-mm dish and mixed gently. Plates were incubated at room temperature and ambient air for 5 min and then in a humidified incubator with 5% CO2 at 37 °C for 35-45 min. The incubation was stopped 20-25 min after the layer of precipitate formed on the plates by shocking the cells for 2 min with 1× HBS, 1 mm sodium kynurenate, 10 mm MgCl2 in 5 mm HEPES, pH 7.5, and 5% glycerol. Cells were then washed three times with 2 ml of BME. The saved conditioned media were added back to each plate, and cells were returned to the 5% CO2 incubator at 37 °C for 48 h before BDNF treatment or harvesting. Rap1 and Ras Activation Assay—The fusion protein between glutathione S-transferase (GST) and the Rap binding domain of RalGDS (GST-RalGDSRBD) was overexpressed and purified from Escherichia coli DH5α cells as described (29Herrmann C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). The GTP-bound, active Rap1 protein (Rap1GTP) was detected as described (25Herrmann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 30Wu C. Lai C.F. Mobley W.C. J Neurosci. 2001; 21: 5406-5416Crossref PubMed Google Scholar). This assay is based on the specific binding between the GTP-bound, activated form of Rap1 (i.e. Rap1GTP) and the Rap binding domain of the RalGDS protein (RalGDSRBD). Briefly, rat E17 cortical neurons were lysed on ice for 10 min in fishing buffer (FB) which contains10% glycerol, 1% Nonidet P-40, 50 mm Tris-HCl (pH 7.5), 200 mm NaCl, 2.0 mm MgCl2, 10 mm NaF, 1 mm Na3VO4, 250 μm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 10 μg/ml soybean trypsin inhibitor. The samples were centrifuged at 14,000 rpm for 10 min at 4 °C. Ten micrograms of GST-RalGDSRBD fusion protein were prebound to agarose-glutathione conjugates, and then added to 300 μg of cortical neuron lysates. The mixture was incubated at 4 °C for 1-2 h with gentle rotation. The beads were washed four times in cold FB and boiled in SDS-PAGE sample buffer. The amount of active Rap1GTP bound to GSTRalGDSRBD fusion protein that is coupled to glutathione-agarose beads was analyzed by SDS-PAGE followed by immunoblotting using an anti-Rap1 antibody. Ras activation was analyzed using Ras Activation Assay kit (Upstate) per the manufacturer's instruction. Kinase Assays—Kinase assays were performed as described (4Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar, 31Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5028) Google Scholar). Cell lysates were prepared and protein concentrations assayed by the Bradford method. Equal amounts of protein extracts (300 or 600 μg) were used for each kinase assay. To measure ERK5 activity, cell lysates were incubated at 4 °C for 2.5 h with either 6 μl of a polyclonal anti-ERK5 antibody (for endogenous ERK5) or 2.5 μg of a polyclonal anti-FLAG antibody (for transfected FLAG-ERK5). Protein A-Sepharose beads (60 μl) were then added, and the mixture was incubated at 4 °C for an additional hour. The activity of ERK5 in the immune precipitates was quantitated by a kinase assay using recombinant GSTMEF2C (5 μg) and [32P]ATP as the substrates (5Kato Y. Kravchenko V.V. Tapping R.I. Han J.H. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (492) Google Scholar). Activated MEKK2 or MEKK3 can phosphorylate multiple MEKs in vitro including MKK4 and MEK5 (32Deacon K. Blank J.L. J. Biol. Chem. 1997; 272: 14489-14496Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Because GSTMKK4 is commercially available, we used it as the substrate in the in vitro kinase assay to monitor the biochemical activities of MEKK2 and MEKK3. To measure the activities of transfected MEKK2 or MEKK3, cell lysates were incubated at 4 °C for 2.5 h with 2.5 μg of anti-HA antibody. Protein G-Sepharose beads (60 μl) were then added, and the mixture was incubated at 4 °C for an additional hour. The activity of MEKK2 and MEKK3 in the immune precipitates was quantitated by a kinase assay using recombinant GST-MKK4 (2 μg) and [32P]ATP as the substrates. Quantitation of kinase activity was achieved by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA) or by using the ImageQuant program after scanning the autoradiographic images, and normalized by protein expression based on Western analysis. Reporter Gene Assays—The Gal4-MEF2C-driven, Gal4-luciferase reporter gene expression was assayed as described (4Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar). Cortical neurons were transfected using Lipofectamine 2000 (33Impey S. Mark M. Villacres E.C. Poser S. Chavkin C. Storm D.R. Neuron. 1996; 16: 973-982Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Briefly, 0.5 × 106 cells were plated onto each well of a 24-well plate coated with poly-d-lysine and laminin. At DIV3, cells were co-transfected with the Gal4-luciferase reporter construct (1.2 μg DNA/4 wells), Gal4-MEF2C fusion protein construct (0.9 μg DNA/4 wells), and EF1a.LacZ DNA (0.55 μg DNA/4 wells). Where indicated, cortical neurons were also cotransfected with various expression vectors for the ERK5 signaling pathways. Cells were treated 40 h after transfection with 50 ng/ml BDNF for 6 h when indicated. Cell lysates were prepared, and the activities of luciferase and β-galactosidase were measured as described (33Impey S. Mark M. Villacres E.C. Poser S. Chavkin C. Storm D.R. Neuron. 1996; 16: 973-982Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). The reporter gene luciferase activity was normalized to β-galactosidase activity and expressed as the fold induction relative to control. Assay of Apoptosis—Apoptosis was determined by nuclear condensation and/or fragmentation after Hoechst staining (28Hetman M. Kanning K. Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Healthy cells have evenly and uniformly stained nuclei. To facilitate detection of apoptosis in transfected cells, cortical cultures were cotransfected with an expression vector encoding β-galactosidase (UB6-Lacz) as a marker for transfected cells, To obtain unbiased counting, slides were coded, and cells were scored blindly without prior knowledge of treatment. Data Analysis—Data were either the averages or representatives of at least three independent experiments. Statistical analysis of data were performed using one-way analysis of variance. Error bars represent S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not statistically significant (p > 0.05). BDNF Induces Sustained Activation of ERK5 in E17 Cortical Neurons—To investigate signaling pathways responsible for BDNF stimulation of ERK5, we prepared primary neurons from embryonic day (E) 17 rat cortex. In initial experiments, we examined the kinetics for ERK5 activation after BDNF treatment and compared it to ERK1/2 activation. The kinase activities of ERK1/2 and ERK5 were directly measured by in vitro kinase assays using MBP or GST-MEF2C fusion protein as substrates, respectively (Fig. 1). BDNF activated both ERK1/2 and ERK5 in E17 cortical neurons. Like ERK1/2 activation, ERK5 activation was prolonged and sustained for at least 12 h after BDNF treatment. However, the peak of ERK5 activity occurred later than ERK1/2 and did not reach a maximum until 1-2 h after BDNF treatment. In contrast, ERK1/2 was maximally activated by BDNF 10 min after BDNF treatment. The kinetics for activation of ERK1/2 and ERK5 in E17 cortical neurons is similar to that in postnatal cortical neurons (4Cavanaugh J.E. Ham J. Hetman M. Poser S. Yan C. Xia Z. J. Neurosci. 2001; 21: 434-443Crossref PubMed Google Scholar). BDNF Activation of ERK5 Is Mediated by Receptor-mediated Endocytosis—Receptor-mediated endocytosis has been implicated in sustained neurotrophin signaling (34Grimes M.L. Zhou J. Beattie E.C. Yuen E.C. Hall D.E. Valletta J.S. Topp K.S. LaVail J.H. Bunnett N.W. Mobley W.C. J. Neurosci. 1996; 16: 7950-7964Crossref PubMed Google Scholar, 35Howe 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, 36York R.D. Molliver D.C. Grewal S.S. Stenberg P.E. McCleskey E.W. Stork P.J. Mol. Cell. Biol. 2000; 20: 8069-8083Crossref PubMed Scopus (203) Google Scholar, 37Yano H. Chao M.V. J. Neurobiol. 2004; 58: 244-257Crossref PubMed Scopus (58) Google Scholar). Upon BDNF treatment for 20 min, we detected co-immunostaining of phospho-ERK5 and Rab5 (supplemental Fig. S1), a marker for endosomes (38Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2688) Google Scholar). These data are consistent with the notion that ERK5 is found in endosomes following neurotrophin stimulation (39Shao Y. Akmentin W. Toledo-Aral J.J. Rosenbaum J. Valdez G. Cabot J.B. Hilbush B.S. Halegoua S. J. Cell Biol. 2002; 157: 679-691Crossref PubMed Scopus (141) Google Scholar). Dynamin, a large G protein, is involved in clathrin-dependent and -independent receptor-mediated endocytosis (40Song B.D. Schmid S.L. Biochemistry. 2003; 42: 1369-1376Crossref PubMed Scopus (102) Google Scholar). Moreover, mutation of dynamin inhibits internalization of neurotrophin receptors (10Watson F.L. Heerssen H.M. Bhattacharyya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (383) Google Scholar, 21Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). To determine if BDNF activation of ERK5 depends on receptor-mediated endocytosis, cortical neurons were transiently transfected with a dominant negative (dn) dynamin mutant (K44A) to block receptor-mediated endocytosis (10Watson F.L. Heerssen H.M. Bhattacharyya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (383) Google Scholar, 21Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). The wild-type dynamin and cloning vector were used as controls. Cortical neurons were also co-transfected with FLAG-tagged, wild-type ERK5 to facilitate detection of ERK5 in transfected cells. In contrast to the wild-type dynamin, expression of the dnDynamin completely abolished BDNF stimulation of ERK5 (Fig. 2A). As a control for specificity, neurons were co-transfected with the dynamin constructs and wild-type, HA-tagged ERK2. Although dnDynamin attenuated BDNF stimulation of ERK2 (Fig. 2B), it had no effect on ERK2 activation by forskolin (Fig. 2C). Forskolin activates adenylyl cyclases which increase intracellular cAMP, thereby activating ERK2 (41Xia Z. Storm D.R. The Mammalian Adenylyl Cyclases. R. G. Landes Company, Mol. Biol. Intelligence Unit, Austin, TX1996Google Scholar). Our results are consistent with the reports that neurotrophin but not cAMP regulation of ERK1/2 signaling in neurons is regulated by receptor-mediated endocytosis (36York R.D. Molliver D.C. Grewal S.S. Stenberg P.E. McCleskey E.W. Stork P.J. Mol. Cell. Biol. 2000; 20: 8069-8083Crossref PubMed Scopus (203) Google Scholar). These results also demonstrate that expression of the dnDynamin specifically inhibits receptor-mediated endocytosis, rather than nonspecifically interfering with MAP kinase signaling. Cortical neurons were also co-transfected with ERK5 and a temperature-sensitive (ts) dynamin mutant (G273D) (10Watson F.L. Heerssen H.M. Bhattacharyya A. Klesse L. Lin M.Z. Segal R.A. Nat. Neurosci. 2001; 4: 981-988Crossref PubMed Scopus (383) Google Scholar, 21Zhang Y. Moheban D.B. Conway B.R. Bhattacharyya A. Segal R.A. J. Neurosci. 2000; 20: 5671-5678Crossref PubMed Google Scholar). The G273D Dynamin protein functions as a wild-type protein at the permissive temperature (33 °C), but as a dominant-negative protein at the non-permissive temperature (39 °C). Cells were incubated at 37 °C for 48 h after transfection to allow expression of the transgenes, at which time the expression of tsDynamin can be detected by Western analysis. The cells were then incubated at 33 °C or 39 °C for 30 min, followed by BDNF treatment for 1 h. BDNF stimulated ERK5 in the presence of co-transfected tsDynamin at the permissive temperature (33 °C), but not at the non-permissive temperature (39 °C) (Fig. 2D). The fact that transfection of tsDynamin did not affect the ability of BDNF to stimulate ERK5 at the permissive temperature suggests that overexpression of tsDynamin per se does not interfere with endocytosis. In the absence of co-transfected tsDynamin, BDNF activated ERK5 at 39 °C, excluding the possibility that BDNF activation of ERK5 is inhibited at 39 °C. Moreover, we did not observe any morphological changes or cell death of cortical neurons during the 1.5-h incubation at 33 or 39 °C. Thus, expression of the tsDynamin selectively blocks BDNF activation of ERK5, presumably because it blocks receptor-mediated endocytosis. To further investigate the role of receptor-mediated endocytosis in BDNF signaling to ERK5, we utilized phenylarsine oxide (PAO), an inhibitor widely used to inhibit receptor-mediated endocytosis (42Boudin H. Sarret P. Mazella J. Schonbrunn A. Beaudet A. J. Neurosci. 2000; 20: 5932-5939Crossref PubMed Google Scholar, 43Zwaagstra J.C. El-Alfy M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 27237-27245Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 44Piper M. Salih S. Weinl C. Holt C.E. Harris W.A. Nat. Neurosci. 2005; 8: 179-186Crossref PubMed Scopus (137) Google Scholar). We pretreated cortical neurons with 5 nm PAO, a concentration specific for inhibition of endocytosis (45Gibson A.E. Noel R.J. Herlihy J.T. Ward W.F. Am. J. Physiol. 1989; 257: C182-C184Crossref PubMed Google Scholar). The endogenous ERK5 is normally a cytoplasmic protein but translocates to the nucleus upon BDNF stimulation (Fig. 3, A and B). PAO inhibited BDNF-induced nuclear translocation of endogenous ERK5, suggesting PAO suppression of ERK5 activation. Furthermore, treatment with PAO greatly attenuated BDNF stimulation of endogenous ERK5 kinase activity (Fig. 3C). Similar results were obtained using monodansylcadaverine (MDC) (Fig. 4A), another endocytosis blocker (35Howe 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). Treatment with PAO or MDC attenuated BDNF stimulation of ERK1/2 (Fig. 3D and Fig. 4B) but had no effect on forskolin stimulation of ERK1/2 (Fig. 3E and Fig. 4C), demonstrating the specificity of PAO and MDC for endocytosis. Together, data in Figs. 2, 3, and 4 suggest that BDNF activation of ERK5 in cortical neurons depends on receptor-mediated endocytosis.FIGURE 4BDNF activation of ERK5 in cortical neurons is inhibited by MDC, an endocytosis blocker. A, MDC blocks BDNF activation of endogenous ERK5. Cortical neurons (DIV5) were treated with 100 μm MDC or vehicle control (Veh) 10 min before BDNF treatment (50 ng/ml, 1 h). B, MDC blocks ERK1/2 phosphorylation induced by BDNF (50 ng/ml, 0-120 min). C, MDC does not inhibit ERK1/2 phosphorylation induced by forskolin treatment (10 μm, 10 min).View Large
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