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• W1978764694 abstract "L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90 rsk in brain. In vitro, ERK2 can phosphorylate L1 at Ser1204 and Ser1248 of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking. L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90 rsk in brain. In vitro, ERK2 can phosphorylate L1 at Ser1204 and Ser1248 of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking. immunoglobulin superfamily cell adhesion molecule AplysiaCAM neural cell adhesion molecule neural-glial CAM neural-glial-related CAM L1 cytoplasmic domain fibroblast growth factor FGF receptor myelin basic protein Raf synthetic peptide mitogen-activated protein kinase Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine high pressure liquid chromatography Immunoglobulin superfamily cell adhesion molecules (IGSF CAMs)1 provide permissive and instructive cues for neuronal migration and neurite outgrowth during the formation of precise connections between neurons and their targets. The functional state and surface expression of IGSF CAMs on extending axons can be altered in response to the complex and changing environments that the axons traverse. For example, axonal expression of L1 is dramatically up-regulated on the contralateral side of commissural axons once they cross the floorplate in the developing mouse spinal cord (1Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (641) Google Scholar). Precise regulation of CAM expression has also been implicated in synapse formation and modification. Adhesion mediated by the invertebrate Aplysia CAM (apCAM) is regulated in culture by internalization during long term facilitation, a cellular model of learning involving structural alteration of synapses (2Bailey C.H. Chen M. Keller F. Kandel E.R. Science. 1992; 256: 645-649Crossref PubMed Scopus (285) Google Scholar, 3Montgomery A.M.P. Becker J.C. Siu C.H. Lemmon V.P. Cheresh D.A. Pancook J.D. Zhao X. Reisfeld R.A. J. Cell Biol. 1996; 132: 475-485Crossref PubMed Scopus (207) Google Scholar). The internalization of apCAM is mediated through activation of the mitogen-activated protein kinase (MAPK) signaling cascade, which results in the phosphorylation of the apCAM cytoplasmic domain by MAPK (4Bailey C.H. Kaang B.-K. Chen M. Martin K.C. Lim C.-S. Casadio A. Kandel E.R. Neuron. 1997; 18: 913-924Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Genetic studies in Drosophila also support the idea that synaptic plasticity is modulated by CAM cell surface expression (5Schuster C.M. Davis G.W. Fetter R.D. Goodman C.S. Neuron. 1996; 17: 655-667Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). L1 is an IGSF CAM that has been implicated in a number of developmentally important processes including neuronal cell migration (6Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Crossref PubMed Scopus (391) Google Scholar), axon outgrowth (7Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Crossref PubMed Scopus (395) Google Scholar), and axon fasciculation (8Landmesser L. Dahm L. Schultz K. Rutishauser U. Dev. Biol. 1988; 130: 645-670Crossref PubMed Scopus (194) Google Scholar, 9Stallcup W.B. Beasley L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1276-1280Crossref PubMed Scopus (121) Google Scholar). Mutations in the human L1 gene cause abnormal brain development, characterized by mental retardation and defects in central nervous system axon tracts such as the corpus callosum and corticospinal tract (Ref. 10Rosenthal A. Jouet M. Kenwrick S. Nat. Genet. 1992; 2: 107-112Crossref PubMed Scopus (255) Google Scholar and for review see Ref. 11Kamiguchi H. Hlavin M.L. Yamasaki M. Lemmon V. Annu. Rev. Neurosci. 1998; 21: 97-125Crossref PubMed Scopus (108) Google Scholar). L1 is also expressed in adult mammals in regions such as the hippocampus and cerebellum, which undergo continual remodeling of synaptic connections, suggesting a possible role for L1 in these processes (12Brümmendorf T. Rathjen F.G. Protein Profile. 1994; 1: 963-1108Google Scholar). This idea is supported by studies linking L1 to hippocampal long term potentiation (13Lüthl A. Laurent J.P. Figurov A. Muller D. Schachner M. Nature. 1994; 372: 777-779Crossref PubMed Scopus (466) Google Scholar) and spatial learning (14Fransen E. D'Hooge R. Van Camp G. Verhoye M. Sijbers J. Reyniers E. Soriano P. Kamiguchi H. Willemsen R. Koekkoek S.K.E. De Zeeuw C.I. De Deyn P.P. Van Der Linden A. Lemmon V. Kooy F.R. Willems P.J. Hum. Mol. Genet. 1998; 7: 999-1009Crossref PubMed Scopus (211) Google Scholar). Two mechanisms have been proposed for regulating adhesion by L1 family members, both of which can be modulated by phosphorylation events. First, the L1 cytoplasmic domain (L1CD) contains an ankyrin-binding domain that shares homology with other CAMs including vertebrate NrCAM and neurofascin, as well as Drosophilaneuroglian (15Hortsch M. Neuron. 1996; 17: 587-593Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The binding of ankyrin to the L1 subfamily has been shown to stabilize L1-mediated homophilic adhesion (15Hortsch M. Neuron. 1996; 17: 587-593Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) and changes in the phosphorylation state of a critical tyrosine in the ankyrin-binding domain of neurofascin can regulate neurofascin-mediated adhesion (16Tuvia S. Garver T.D. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12957-12962Crossref PubMed Scopus (112) Google Scholar). Second, L1 adhesion may be regulated at the level of cell surface expression (17Itoh K. Stevens B. Schachner M. Fields M. Science. 1995; 270: 1369-11372Crossref PubMed Scopus (156) Google Scholar). The neuronal form of L1 contains an alternatively spliced exon encoding four amino acids (RSLE) within the L1CD (18Miura M. Kobayashi M. Asou H. Uyemura K. FEBS Lett. 1991; 289: 91-95Crossref PubMed Scopus (93) Google Scholar), which contributes to a tyrosine based sorting/endocytosis motif (YRSL) (19Kamiguchi H. Lemmon V. J. Neurosci. 1998; 18: 3749-3756Crossref PubMed Google Scholar). This sequence enables the L1CD to directly bind the μ2 subunit of the adaptin complex AP-2, linking L1 to the clathrin-mediated endocytotic pathway (20Kamiguchi K. Long K. Pendergast M. Schaefer A. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Crossref PubMed Google Scholar). Adaptin proteins are dynamically regulated by phosphorylation (21Ohta Y. Hartwig J.H. J. Biol. Chem. 1996; 271: 11858-11864Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 22Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, 23Slepnev V.I. Ochoa G.-C. Butler M.H. De Camilli P. Science. 1998; 281: 821-824Crossref PubMed Scopus (275) Google Scholar), and examples from G-protein-coupled receptors have demonstrated the importance of phosphorylation of both receptors and intracellular machinery in regulating endocytosis (24Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar, 25Shiratori T. Miyatake O. Nakaseko C. Bonifacino J.S. Saito T. Immunity. 1997; 6: 583-589Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Consequently, it is likely that phosphorylation may also regulate L1 internalization. L1 contains multiple potential phosphorylation sites and is phosphorylated in vivo (26Salton S.R.J. Shelanski M.L. Greene L.A. J. Neurosci. 1983; 3: 2420-2430Crossref PubMed Google Scholar). To understand the role of phosphorylation in L1 function, we have focused on kinases that interact with L1. Previously, we identified two kinases, CKII and p90 rsk, that coimmunoprecipitate with L1 and phosphorylate L1 at Ser1181 and Ser1152, respectively (27Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Crossref PubMed Scopus (72) Google Scholar, 28Wong E.V. Schaefer A. Landreth G. Lemmon V. J. Biol. Chem. 1996; 271: 18217-18223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). p90 rsk is a distal component of the mitogen-activated kinase (MAPK or ERK) signal cascade, which raises the possibility that L1 may interact with additional components of this pathway. The MAPK cascade is activated by a wide range of extracellular stimuli, and ERK kinases can phosphorylate many proteins, including transcription factors, membrane proteins, cytoskeletal proteins, and other kinases. The ERKs are activated by tyrosine kinase receptors, G-protein-linked receptors, and protein kinase C-dependent pathways, and the best resolved pathway involves the sequential activation of Ras, Raf, MEK, ERK, and p90 rsk (for review see Ref. 29Denhardt D.T. Biochem. J. 1996; 318: 729-747Crossref PubMed Scopus (454) Google Scholar). Activation of the upstream components occurs at the plasma membrane. However, recent evidence suggests that distal components including ERK and p90 rsk require internalization of the receptor tyrosine kinase or G-protein linked receptor to be fully activated (30Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (834) Google Scholar, 31Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 32Ceresa B.P. Kao A.W. Santeler S.R. Pessin J.E. Mol. Cell. Biol. 1998; 18: 3862-3870Crossref PubMed Scopus (200) Google Scholar). Finally, ERK activation has been implicated in the regulation of cell motility. For example, integrin-mediated activation of the MAPK cascade can influence cell motility through the phosphorylation of myosin light chain kinase by ERK (33Macfarlane J. Du J. Pepys M. Ramsden S. Donnai D. Charlton R. Garrett C. Tolmie J. Yates J. Berry C. Goudie D. Moncla A. Lunt P. Hodgson S. Jouet M. Kenwrick S. Hum. Mutat. 1997; 9: 512-518Crossref PubMed Scopus (14) Google Scholar). We present evidence that two additional components of the MAPK cascade, ERK2 and Raf-1, associate with L1. ERK2 phosphorylates L1 and can be activated in L1-expressing 3T3 cells by L1 cross-linking antibodies. The activated ERK colocalizes with endocytosed L1. The activation of ERK by cross-linking cell surface L1 is prevented if endocytosis of L1 is blocked. This suggests that one function of the interaction between ERK and L1 may be in regulating L1 intracellular trafficking because only internalized L1 can be phosphorylated by activated ERK. Protease inhibitors, Pefabloc SC, leupeptin, and aprotinin, as well as horseradish peroxidase-conjugated goat anti-rabbit antibodies were purchased from Roche Molecular Biochemicals. Recombinant bacterially expressed ERK2 was obtained from Upstate Biochemicals, Inc. (Lake Placid, NY). Anti-ERK2 and anti-Ras monoclonal and polyclonal antibodies were purchased from Transduction Laboratories (Lexington, KY). Anti-phospho-specific ERK antibodies were purchased from New England Biolabs (Beverly, MA). Anti-Raf-1, B-Raf, and MEK1 were purchased from Santa Cruz Biotechnology. [32P]H3PO4 was purchased from ICN Biochemicals (Irvine, CA). The anti-NCAM antibody was the gift of Dr. Urs Rutishauser (Sloan-Kettering, New York, NY). The 5G3 anti-human L1 monoclonal antibody was a gift from Dr. R. A. Reisfeld (Scripps Research Institute, La Jolla, CA). The 74–5H7 anti-L1 monoclonal antibody is described in Ref. 34Lemmon V. Farr K. Lagenaur C. Neuron. 1989; 2: 1597-1603Abstract Full Text PDF PubMed Scopus (382) Google Scholar. The rabbit anti-human L1 antibody has been described previously (35Wong E.V. Cheng G. Payne H.R. Lemmon V. Neurosci. Lett. 1995; 200: 155-158Crossref PubMed Scopus (44) Google Scholar). The L1 cytoplasmic domain with a His6 tag was expressed in Escherichia coli (28Wong E.V. Schaefer A. Landreth G. Lemmon V. J. Biol. Chem. 1996; 271: 18217-18223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and used to make a rabbit antibody. Monoclonal and rabbit anti-phosphorylated ERK antibodies were obtained from New England Biolabs. Raf synthetic peptide (RSP) was purchased from Promega (Madison, WI). The epidermal growth factor ERK site peptide, T669, as well as the tyrosine kinase inhibitors, erbstatin analog, and PP1 were purchased from Calbiochem (La Jolla, CA). Immobilon-P polyvinyldifluoridene membrane was from Millipore (Marlborough, MA). RenaissanceTM enhanced chemiluminescent detection reagents were purchased from NEN Life Science Products. Bacterial expression vector pQE13 and Ni-NTA agarose beads were from Qiagen (Valencia, CA). All other chemicals were purchased through Sigma. NIH-3T3 cells (American Type Tissue Culture Collection, Manassas, VA) and dorsal root ganglia from embryonic day 10 chickens were cultured as described previously (20Kamiguchi K. Long K. Pendergast M. Schaefer A. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Crossref PubMed Google Scholar). Briefly, the L1-expressing NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 600 μg/ml G418 (Life Technologies, Inc.) prior to serum starvation. Brains from P7 Harlan Sprague-Dawley rat pups or embryonic day 14 chick embryos were homogenized in 20 mm Tris, pH 7.4, 1 mm EGTA, 1 mmsodium orthovanadate, and 10 mm p-nitrophenyl phosphate (TEV-PNP) containing 0.32 m sucrose, 200 mm Pefabloc SC, and 100 μg/ml aprotinin. The homogenates were separated by ultracentrifugation on a sucrose gradient for 45 min at 58,400 × g at 4 °C. The plasma membrane layer was washed in TEV-PNP and then centrifuged 30 min at 150,000 ×g at 4 °C to pellet the membranes. The plasma membrane pellet was solubilized in TEV-PNP containing 1% Triton X-100 and centrifuged for 45 min at 150,000 × g at 4 °C to remove insoluble material. The solubilized membrane fraction was then incubated for >4 h at 4 °C with Sepharose beads conjugated to a monoclonal anti-L1 antibody, mAb 74–5H7 (34Lemmon V. Farr K. Lagenaur C. Neuron. 1989; 2: 1597-1603Abstract Full Text PDF PubMed Scopus (382) Google Scholar). The beads were washed with TEV-PNP containing 1% Triton X-100 twice followed by four washes with TEV-PNP before use in kinase assays or Western blot analysis. The cytoplasmic domain of human L1, comprising residues 1144–1257, was cloned into the pQE13 bacterial expression vector to produce a recombinant L1CD containing a hexahistidine epitope at the N terminus. This protein was expressed inE. coli, and L1CD was purified from the bacteria by Ni2+ affinity chromatography using nickel-nitrilotriacetic acid agarose beads, using the manufacturer's protocols. Kinase phosphorylation reactions were carried out with L1 immunoprecipitates in TEV-PNP buffer containing 10 mm MgCl2, 2 mm MnCl2, 5 mm [γ-32P]ATP, and myelin basic protein (MBP), ERK substrate peptide (T669), or RSP for 30 min at room temperature. The reactions were stopped by the addition of sample buffer and boiling for 5 min. MBP was separated from other proteins in the reaction by SDS-PAGE, and the radiolabeled MBP was visualized by autoradiography. T669 is a synthetic peptide derived from a potential ERK site on the epidermal growth factor receptor and has the sequence ERELVEPLTPSGEAPNQALLR (36Takihima K. Griswold-Prenner I. Ingebritsen T. Rosner M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2520-2524Crossref PubMed Scopus (108) Google Scholar). RSP is a synthetic peptide with the sequence IVQQFGFQRRSNNGKLTN, which corresponds to a potential autophosphorylation site in the Raf-1 kinase. A tyrosine has been replaced at position seven by a phenylalanine to prevent tyrosine phosphorylation of the substrate (37Hassel B. Rathjen F. Volkmer H. J. Biol. Chem. 1997; 272: 28742-28749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The peptides were separated from other proteins in the reaction on a Tris-Tricine SDS-PAGE system (38Schagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar) modified with a 19–33% linear gradient resolving gel and visualized by autoradiography. L1 immunoprecipitates were mixed with sample buffer and boiled for 5 min. The samples were then separated by SDS-PAGE. The proteins were transferred to Immobilon-P membrane, and the membrane was then blocked with 5% nonfat dry milk in Tris-buffered saline. The commercial primary antibodies were used as recommended by the manufacturer. The membrane was incubated with primary antibodies for 1 h at room temperature with shaking and washed with 0.1% Tween-20 in Tris-buffered saline. The membrane was then probed with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1000 in 5% milk/0.05% Tween-20/phosphate-buffered saline) for 1 h, washed, and then visualized by chemiluminescence. The Western blots were scanned onto a Macintosh power PC using a AGFA duoscanner, and images were analyzed with NIH Image. In the experiments designed to detect ERK activation, NIH-3T3 cells stably transfected with full-length human L1 (20Kamiguchi K. Long K. Pendergast M. Schaefer A. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Crossref PubMed Google Scholar) were plated at a density of 2 × 105 cells/60-mm dish. Prior to stimulation, the cells were maintained in low serum, 0.5% fetal calf serum in DMEM for 48 h followed by 2 h in serum-free DMEM. At all times the tissue culture medium was maintained at 37 °C and equilibrated with CO2. The cells were then treated with rabbit polyclonal anti-L1 antibody for various periods. After the treatments, cells were directly extracted from the tissue culture dishes with 300 μl of sample buffer supplemented with 1 mm sodium orthovanadate. The sample was boiled for 5 min followed by sonication with a vibrating probe sonicator to shear DNA. The samples were separated by SDS-PAGE and analyzed by Western blot as above. Blots were first probed with the anti-phosphorylation-specific ERK antibodies and then stripped and reprobed with other antibodies recognizing both phosphorylated and unphosphorylated forms of ERK (total ERK) to compare loading between lanes and relative ERK activation levels. Recombinant L1CD (10 μg) was phosphorylated with recombinant ERK2 in TEV-PNP containing 10 mm MgCl2, 2 mm MnCl2, 5 mm ATP, and 5 μCi of [γ-32P]ATP. The samples were then digested with endoproteinase Asp-N for 18 h at 37 °C, and the resulting peptides were separated by HPLC on a C-18 reverse phase column. Fractions were collected and analyzed for protein concentration and radioactivity. The fractions containing significant protein and radioactivity were then sequenced on an ABI protein sequencer. L1-expressing NIH-3T3 cells (20Kamiguchi K. Long K. Pendergast M. Schaefer A. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Crossref PubMed Google Scholar) cultured on two-chamber plastic slides (Lab-Tek, Naperville, IL) coated with fibronectin (5 μg/cm2; Roche Molecular Biochemicals) were maintained in 0.5% serum in DMEM for 48 h followed by 2 h in serum-free DMEM. Then the cells were treated with either rabbit polyclonal anti-L1 antisera or preimmune sera for 20 min and processed for immunocytochemistry to examine the subcellular distribution of phosphorylated ERK. Following fixation with 4% formaldehyde and permeabilization with 0.02% Triton X-100, the cells were incubated with mouse monoclonal anti-phospho ERK (1:500; New England Biolabs) at 4 °C for 16 h. Phosphorylated ERK was then visualized with Texas Red-conjugated anti-mouse IgG (1:100; Molecular Probes, Eugene, OR). In some experiments, the cells were double-labeled for L1 and phosphorylated ERK to analyze colocalization. Differential labeling of cell surface and internalized L1 was performed as described previously (20Kamiguchi K. Long K. Pendergast M. Schaefer A. Rapoport I. Kirchhausen T. Lemmon V. J. Neurosci. 1998; 18: 5311-5321Crossref PubMed Google Scholar). In the experiment designed to double-label cell surface L1 and phosphorylated ERK, live cells were incubated with rabbit polyclonal anti-L1 antibody for 1 h at 37 °C, followed by incubation with Oregon Green-conjugated anti-rabbit IgG (1:200; Molecular Probes) for 1 h at 4 °C. Subsequently, the cells were fixed with 4% formaldehyde for 30 min, permeabilized, and blocked with a mixture of 10% horse serum and 0.02% Triton X-100 in phosphate-buffered saline. The cells were then incubated with mouse monoclonal anti-phospho ERK overnight at 4 °C followed by Texas Red-conjugated anti-mouse IgG (1:100). In the experiment designed to double-label internalized L1 and phosphorylated ERK, live cells were incubated with rabbit polyclonal anti-L1 antibody for 1 h at 37 °C, followed by incubation with unconjugated anti-rabbit IgG (200 μg/ml; Molecular Probes) for 1 h at 4 °C. The cells were fixed, permeabilized, and incubated with mouse monoclonal anti-phospho ERK overnight at 4 °C. Then the cells were incubated with a mixture of Texas Red-conjugated anti-mouse IgG (1:100) and Oregon Green-conjugated anti-rabbit IgG (1:200). The labeled cells were mounted with SlowFade (Molecular Probes), and images taken with a Zeiss LSM 410 confocal laser microscope (Zeiss, Göttingen, Germany) using an argon/krypton laser (excitation lines, 488 and 568 nm) and a 100× Plan-Neofluor, numerical aperture 1.3, oil objective. Transfection of cDNA encoding for K44A dynamin or β-galactosidase was done using recombinant adenovirus vectors (kind gift of Dr. Jeffrey E. Pessin, The University of Iowa, Iowa City, IA). Production of concentrated adenovirus and infection of NIH-3T3 cells were done as described previously (32Ceresa B.P. Kao A.W. Santeler S.R. Pessin J.E. Mol. Cell. Biol. 1998; 18: 3862-3870Crossref PubMed Scopus (200) Google Scholar). Briefly, 85–90% confluent 293 cells (American Type Culture Collection) were infected with adenovirus and incubated for 36–48 h. The cells were collected and lysed by repeated freezing and thawing, and concentrated adenovirus (1 ml of cell lysate/10-cm culture dish of 293 cells) was prepared. Then, L1-expressing 3T3 cells (50–60% confluent) plated on fibronectin-coated 35-mm dishes were infected with 50 μl/dish of concentrated adenovirus medium. After 48 h incubation, the cells were serum-starved, treated with anti-L1 antibody, and processed for Western blot analysis to detect phosphorylated ERK. Immunocytochemistry of infected 3T3 cells showed that approximately 95% of the cells expressed the transgene products (data not shown). At least two distinct kinase activities have previously been shown to coimmunoprecipitate with L1 (27Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Crossref PubMed Scopus (72) Google Scholar, 39Schuch U. Lohse M.J. Schachner M. Neuron. 1989; 3: 13-20Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 40Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar). These have been identified as CKII (27Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Crossref PubMed Scopus (72) Google Scholar) and p90 rsk (28Wong E.V. Schaefer A. Landreth G. Lemmon V. J. Biol. Chem. 1996; 271: 18217-18223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), which phosphorylate L1 at Ser1181 and Ser1152, respectively. p90 rsk is a distal component of the MAPK signaling cascade and has been found to associate with ERK in PC12 cells, inXenopus oocytes, and in COS cells transfected with p90 rsk isoforms (41Scimeca J. Nguyen T. Filloux C. Van Obberghen E. J. Biol. Chem. 1992; 267: 17369-17374Abstract Full Text PDF PubMed Google Scholar, 42Hsiao K.-M. Chou S.-Y. Shih S.-J. Ferrell J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5480-5484Crossref PubMed Scopus (97) Google Scholar, 43Zhao Y. Bjørbæk C. Moller D.E. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These findings raise the possibility that L1 may associate with other kinases involved in the activation of p90 rsk, such as ERK. To determine whether any other kinases in the MAPK pathway associate with L1, Western blots of L1 immunoprecipitates from rat brain membrane preparations were probed for Ras, Raf-1, B-Raf, MEK-1, ERK, and p90 rsk (Fig. 1 A). The results demonstrate that Raf-1 and ERK2, in addition to the previously identified p90 rsk, are associated with L1. As a control for the stringency of the wash conditions, the abundant IGSF CAM, NCAM, was shown not to coimmunoprecipitate with L1. The MAPK cascade components, Ras, B-Raf, and MEK-1, were not detected in the L1 immunoprecipitates (data not shown). The predominant bands in silver-stained L1 immunoprecipitates correspond to L1 products of 220, 135, and 80 kDa, indicating that associated kinases are present well below stoichiometric levels and may associate with a specific subset of L1 (Fig. 1 B). To demonstrate that the ERK association with L1 in brain was specific, anti-L1-coated beads, anti-NCAM-coated beads, and uncoated beads were incubated with detergent extracts from P7 rat brains and embryonic day 14 chick brains. ERK was found in the L1 immunoprecipitations but not in the NCAM immunoprecipitations or bead controls (Fig. 1 C). Similar results were found for Raf-1 (data not shown). To determine the activity of the kinases identified by Western blot analysis, L1 immunoprecipitates from rat brain were incubated with [γ-32P]ATP and either Raf substrate (RSP) or the ERK substrates (MBP or T669, an ERK substrate peptide derived from the epidermal growth factor receptor). The L1 immunoprecipitates were able to phosphorylate all three substrates consistent with the Western blot results (Fig. 2). Previous work has demonstrated that L1 is phosphorylated on at least two serines (27Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Crossref PubMed Scopus (72) Google Scholar, 28Wong E.V. Schaefer A. Landreth G. Lemmon V. J. Biol. Chem. 1996; 271: 18217-18223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Our earlier studies showed that endoproteinase Asp-N digested L1 from L1 immunoprecipitation kinase reactions and from in vivometabolically labeled L1 both contained at least three radiolabeled peptide fragments. The major peaks of radioactivity ran at 30–32, 48–54, and 58–64 min on reverse phase HPLC. The 30–32-min peak contains a peptide fragment containing Ser1152 that can be phosphorylated by p90 rsk (28Wong E.V. Schaefer A. Landreth G. Lemmon V. J. Biol. Chem. 1996; 271: 18217-18223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and the 58–64-min peptide fragment peak contains peptides with Ser1181 that can be phosphorylated by CKII (27Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Crossref PubMed Scopus (72) Google Scholar). We reasoned that the newly characterized Raf-1 and/or ERK2 kinase activities associated with L1 may account for the phosphorylated peptide peak of 48–54 min. ERK2 was the most likely candidate because the L1CD contains a potential proline directed phosphorylation site at Ser1248. In addition, Sonderegger and colleagues (40Kunz S. Ziegler U. Kunz B. Sonderegger P. J. Cell Biol. 1996; 135: 253-267Crossref PubMed Scopus (63) Google Scholar) found that the synthetic substrate peptide, syntide-2, which can act as a competitive substrate for Raf-1, did not affect the phosphorylation of chicken L1 (NgCAM) by L1-associated kinase activities. We tested the ability of recombinant ERK2 to phosphorylate recombinant L1CD in vitro to determine whether L1 could be a substrate for ERK2 phosphorylation. The phosphorylated L1CD was digested with endoproteinase Asp-N, and the resulting fragments were separated by reverse phase HPLC. Two peaks of radioactivity were detected, a mi" @default.
• W1978764694 created "2016-06-24" @default.
• W1978764694 creator A5003437461 @default.
• W1978764694 creator A5049959111 @default.
• W1978764694 creator A5062194249 @default.
• W1978764694 creator A5065482109 @default.
• W1978764694 creator A5073496526 @default.
• W1978764694 creator A5086812132 @default.
• W1978764694 date "1999-12-01" @default.
• W1978764694 modified "2023-10-13" @default.
• W1978764694 title "Activation of the MAPK Signal Cascade by the Neural Cell Adhesion Molecule L1 Requires L1 Internalization" @default.
• W1978764694 cites W1480030874 @default.
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• W1978764694 doi "https://doi.org/10.1074/jbc.274.53.37965" @default.
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