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Involvement of p90 in Neurite Outgrowth Mediated by the Cell Adhesion Molecule L1

1996; Elsevier BV; Volume: 271; Issue: 30 Linguagem: Inglês

10.1074/jbc.271.30.18217

1083-351X

Eric V. Wong, Andrew W. Schaefer, Gary E. Landreth, Vance Lemmon,

Neurogenesis and neuroplasticity mechanisms

L1 is a neural cell adhesion molecule that has been shown to help guide nascent axons to their targets. This guidance is based on specific interactions of L1 with its binding partners and is likely to involve signaling cascades that alter cytoskeletal elements in response to these binding events. We have examined the phosphorylation of L1 and the role it may have in L1-directed neurite outgrowth. Cytosolic extracts from nerve growth factor-stimulated PC12 cells were fractionated by anion-exchange chromatography, and an activity was found that phosphorylated the cytoplasmic domain of L1. This activity was then assayed using a battery of L1-derived synthetic peptides. Based on these peptide assays and sequencing of radiolabeled L1 proteolytic fragments, the phosphorylation site was determined to be Ser1152. Western blot analysis demonstrated that the L1 kinase activity from PC12 cells that phosphorylated this site was co-eluted with the S6 kinase, p90rsk. Moreover, S6 kinase activity and p90rsk immunoreactivity co-immunoprecipitate with L1 from brain, and metabolic labeling studies have demonstrated that Ser1152 is phosphorylated in vivo in the developing rat brain. The phosphorylation site is located in a region of high conservation between mammalian L1 sequences as well as L1-related molecules in vertebrates from fish to birds. We performed studies to investigate the functional significance of this phosphorylation. Neurons were loaded with peptides that encompass the phosphorylation site, as well as the flanking regions, and their effects on neurite outgrowth were observed. The peptides, which include Ser1152, inhibit neurite outgrowth on L1 but not on a control substrate, laminin. A nonphosphorylatable peptide carrying a Ser to Ala mutation did not affect neurite outgrowth on either substrate. These data demonstrate that the membrane-proximal 15 amino acids of the cytoplasmic domain of L1 are important for neurite outgrowth on L1, and the interactions it mediates may be regulated by phosphorylation of Ser1152. L1 is a neural cell adhesion molecule that has been shown to help guide nascent axons to their targets. This guidance is based on specific interactions of L1 with its binding partners and is likely to involve signaling cascades that alter cytoskeletal elements in response to these binding events. We have examined the phosphorylation of L1 and the role it may have in L1-directed neurite outgrowth. Cytosolic extracts from nerve growth factor-stimulated PC12 cells were fractionated by anion-exchange chromatography, and an activity was found that phosphorylated the cytoplasmic domain of L1. This activity was then assayed using a battery of L1-derived synthetic peptides. Based on these peptide assays and sequencing of radiolabeled L1 proteolytic fragments, the phosphorylation site was determined to be Ser1152. Western blot analysis demonstrated that the L1 kinase activity from PC12 cells that phosphorylated this site was co-eluted with the S6 kinase, p90rsk. Moreover, S6 kinase activity and p90rsk immunoreactivity co-immunoprecipitate with L1 from brain, and metabolic labeling studies have demonstrated that Ser1152 is phosphorylated in vivo in the developing rat brain. The phosphorylation site is located in a region of high conservation between mammalian L1 sequences as well as L1-related molecules in vertebrates from fish to birds. We performed studies to investigate the functional significance of this phosphorylation. Neurons were loaded with peptides that encompass the phosphorylation site, as well as the flanking regions, and their effects on neurite outgrowth were observed. The peptides, which include Ser1152, inhibit neurite outgrowth on L1 but not on a control substrate, laminin. A nonphosphorylatable peptide carrying a Ser to Ala mutation did not affect neurite outgrowth on either substrate. These data demonstrate that the membrane-proximal 15 amino acids of the cytoplasmic domain of L1 are important for neurite outgrowth on L1, and the interactions it mediates may be regulated by phosphorylation of Ser1152. INTRODUCTIONThe development of a functional nervous system depends, in part, on the ability of neurons to form the requisite specific connections with their targets. The guidance of axons through varied terrain and over relatively long distances is thought to be influenced by a variety of factors. These include physical channels and chemical signals, either diffusible factors or substrate-bound extracellular matrix molecules or cell surface molecules. Cell adhesion molecules are often involved in providing a suitable substrate upon which neurons can migrate or extend axons. L1 is a cell adhesion molecule that has been implicated in a variety of processes integral to the development of the nervous system, including neuronal migration (Lindner et al., 35Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Google Scholar), neurite outgrowth (Lagenaur and Lemmon, 31Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Google Scholar), and axon fasciculation (Landmesser et al., 32Landmesser L. Dahm L. Schultz K. Rutishauser U. Dev. Biol. 1988; 130: 645-670Google Scholar; Stallcup and Beasley, 52Stallcup W.B. Beasley L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1276-1280Google Scholar). Mutations in the L1 gene are linked to the human mental retardation diseases X-linked hydrocephalus and MASA (ental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome (Rosenthal et al., 46Rosenthal A. Jouet M. Kenwrick S. Nat. Genet. 1992; 2: 107-112Google Scholar; Wong et al., 60Wong E.V. Kenwrick S. Willems P. Lemmon V. Trends Neurosci. 1995; 18: 168-172Google Scholar), in which defects of the cortocospinal tract and corpus callosum are commonly found.Recent evidence suggests a function for L1 beyond adhesion between two cell surfaces. When a growth cone migrating on laminin contacts L1, its morphology changes quickly, broadening and flattening even before the entire growth cone has moved onto the new substrate (Burden-Gulley et al., 9Burden-Gulley S.M. Payne H.R. Lemmon V. J. Neurosci. 1995; 15: 4370-4381Google Scholar). This suggests activation of a signal transduction cascade initiated by L1 contact that eventually affects the cytoskeleton. Further evidence for signal transduction cascades initiated by L1 comes from observations of changes in various intracellular second messenger systems upon activation of L1 by binding with soluble L1 or anti-L1 antibodies (Itoh et al., 27Itoh K. Kawamura H. Asou H. Brain Res. 1992; 580: 233-240Google Scholar; Schuch et al., 50Schuch U. Lohse M.J. Schachner M. Neuron. 1989; 3: 13-20Google Scholar; Von Bohlen und Halbach et al., 56Von Bohlen Halbach F. Taylor J. Schachner M. Eur. J. Neurosci. 1992; 4: 896-909Google Scholar; Williams et al., 57Williams E.J. Doherty P. Turner G. Reid R.A. Hemperly J.J. Walsh F.S. J. Cell Biol. 1992; 119: 883-892Google Scholar).L1 (Moos et al., 38Moos M. Tacke R. Scherer H. Teplow D. Fruh K. Schachner M. Nature. 1988; 334: 701-703Google Scholar) (also termed NILE (Prince et al., 42Prince J.T. Milona N. Stallcup W.B. J. Neurosci. 1989; 9: 1825-1834Google Scholar), 8D9 (Lemmon and McLoon, 33Lemmon V. McLoon S. J. Neurosci. 1986; 6: 2987-2994Google Scholar), Ng-CAM (Grumet et al., 23Grumet M. Hoffman S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 267-271Google Scholar), G4 (Rathjen et al., 45Rathjen F.G. Wolff J.M. Frank R. Bonhoeffer F. Rutishauser U. J. Cell Biol. 1987; 104 (b): 343-353Google Scholar)) is primarily expressed on projection axons of the central nervous system and peripheral nervous system, as well as on a few nonneuronal cell types, including Schwann cells and lymphocytes. It is a member of the immunoglobulin superfamily of adhesion molecules (Burden-Gulley and Lemmon, 7Burden-Gulley S.M. Lemmon V. Semin. Dev. Biol. 1995; 6: 79-87Google Scholar), the extracellular domain of which is characterized by six immunoglobulin-like domains and five fibronectin-type III domains, and highly conserved transmembrane and cytoplasmic domains. The cytoplasmic domain is completely conserved in the known mammalian sequences, and two long stretches are perfectly conserved in the chick, comprising nearly 70% of the cytoplasmic domain (Hlavin and Lemmon, 25Hlavin M.L. Lemmon V. Genomics. 1991; 11: 416-423Google Scholar). Two shorter sequences, one abutting the membrane and one 40 amino acids from the C terminus, are conserved, even in the Drosophila L1 homologue, neuroglian (Bieber et al., 3Bieber A.J. Snow P.M. Hortsch M. Patel N.H. Jacobs J.R. Traquina Z.R. Schilling J. Goodman C.S. Cell. 1989; 59: 447-460Google Scholar). There are also two alternatively spliced exons that are present in neuronal L1 but not in L1 expressed in nonneuronal cells (Miura et al., 37Miura M. Kobayashi M. Asou H. Uyemura K. FEBS Lett. 1991; 289: 91-95Google Scholar). The L1 molecule is both glycosylated and phosphorylated (Faissner et al., 18Faissner A. Kruse J. Nieke J. Schachner M. Dev. Brain Res. 1984; 15: 69-82Google Scholar).One possible mechanism for control of the signal transduction cascades initiated by L1 binding is the regulated phosphorylation of L1. We and others have described a number of kinase activities that coprecipitate with L1 immunoprecipitates (Sadoul et al., 47Sadoul R. Kirchhoff F. Schachner M. J. Neurochem. 1989; 53: 1471-1478Google Scholar; Wong et al., 61Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Google Scholar). We have identified one of these as casein kinase II, which phosphorylates L1 at Ser1181. In this paper, we demonstrate that an S6 family kinase is also associated with L1.The serine/threonine kinase, p90rsk, was initially identified on the basis of its ability to phosphorylate the ribosomal 40 S subunit in vitro. This enzyme has been the focus of much interest due to its ability to be phosphorylated and activated by the mitogen-associated protein kinases and is a component of this growth factor-sensitive signaling cascade (Blenis, 5Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Google Scholar). The role of p90rsk in the nervous system has not been studied directly, but it is reported to be part of an NGF 1The abbreviations used are: NGFnerve growth factorPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatographyFITCfluorescein isothiocyanateCAMcell adhesion moleculeCKIIcasein kinase IIPBSphosphate-buffered saline. -inducible signaling cascade in PC12 pheochromocytoma cells (Scimeca et al., 51Scimeca J.C. Nguyen T.T. Filloux C. Van Obberghen E. J. Biol. Chem. 1992; 267: 17369-17374Google Scholar). This paper describes the phosphorylation of a neural cell adhesion molecule, L1, by p90rsk. p90rsk associates with L1 at the membrane and phosphorylates L1 at Ser1152. This phosphorylation may regulate the interactions of L1 and intracellular signaling cascades or cytoskeletal elements involved in neurite outgrowth on specific substrates.DISCUSSIONL1 is a cell adhesion molecule of the immunoglobulin superfamily that binds to L1 molecules on opposing surfaces and to several other molecules as well (Brummendorf et al., 6Brummendorf T. Hubert M. Treubert U. Leuschner R. Tarnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Google Scholar; Felsenfeld et al., 19Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Google Scholar; Kuhn et al., 29Kuhn T.B. Stoeckli E.T. Condrau M.A. Rathjen F.G. Sonderegger P. J. Cell Biol. 1991; 115: 1113-1126Google Scholar; Milev et al., 36Milev P. Friedlander D.R. Sakuri T. Karthikeyan L. Flad M. Margolis R.K. Grumet M. Margolis R.U. J. Cell Biol. 1994; 127: 1703-1715Google Scholar). Accumulating evidence suggests that L1 not only mediates adhesion but also acts as a receptor, transducing extracellular interactions into an intracellular second messenger cascade (Doherty and Walsh, 15Doherty P. Walsh F.S. Curr. Opin. Neurobiol. 1994; 4: 49-56Google Scholar), leading ultimately to changes in the behavior of the neuron, influencing migration (Lindner et al., 35Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Google Scholar), fasciculation (Stallcup and Beasley, 52Stallcup W.B. Beasley L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1276-1280Google Scholar; Landmesser et al., 32Landmesser L. Dahm L. Schultz K. Rutishauser U. Dev. Biol. 1988; 130: 645-670Google Scholar; Cervello et al., 11Cervello M. Lemmon V. Landreth G. Rutishauser U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10548-10552Google Scholar), or axonal outgrowth (Lagenaur and Lemmon, 31Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Google Scholar).The morphology of growth cones from chick retinal ganglion cell neurons differs radically depending upon the substrate on which they are growing (Payne et al., 41Payne H.R. Burden S.M. Lemmon V. Cell Motil. Cytoskeleton. 1992; 21: 65-73Google Scholar). When growth cones migrating on laminin first encounter L1, there are significant morphological changes in the growth cone within 1 min (Burden-Gulley et al., 9Burden-Gulley S.M. Payne H.R. Lemmon V. J. Neurosci. 1995; 15: 4370-4381Google Scholar). This change is reflected in the redistribution of cytoskeletal components (Burden-Gulley and Lemmon, 8Burden-Gulley S.M. Lemmon V. Cell Motil. Cytoskeleton. 1996; (in press)Google Scholar) and is consistent with the idea that L1 binding triggers an intracellular signal that leads to cytoskeletal rearrangement (Atashi et al., 2Atashi J.R. Klinz S.G. Ingraham C.A. Matten W.T. Schachner M. Maness P.F. Neuron. 1992; 8: 831-842Google Scholar). In addition to generating signals via L1-L1 binding, L1 may also act as a signal transducing receptor for other adhesion molecules. Although axonin-1 homophilic interactions are sufficient for adhesion (Rader et al., 43Rader C. Stoeckli E.T. Ziegler U. Osterwalder T. Kunz B. Sonderegger P. Eur. J. Biochem. 1993; 215: 133-141Google Scholar), neurite outgrowth involving axonin-1 requires an interaction with the chick L1 homologue, Ng-CAM (Kuhn et al., 29Kuhn T.B. Stoeckli E.T. Condrau M.A. Rathjen F.G. Sonderegger P. J. Cell Biol. 1991; 115: 1113-1126Google Scholar). Similarly, TAG-1, the mammalian homologue of axonin-1, interacts with L1 to produce neurite outgrowth (Felsenfeld et al., 19Felsenfeld D.P. Hynes M.A. Skoler K.M. Furley A.J. Jessell T.M. Neuron. 1994; 12: 675-690Google Scholar). In these situations, L1/Ng-CAM could act as a signal transducing receptor for TAG-1/axonin-1 in TAG-1/axonin-1 directed neurite outgrowth, since these glycosylphosphatidylinositol-linked molecules do not have direct communication inside the cell.Several different second messenger systems may be involved in L1-mediated signaling, as evidenced by reports of changes in intracellular Ca2+, pH, and inositol phosphates upon activation of L1 in a variety of cell types (Schuch et al., 50Schuch U. Lohse M.J. Schachner M. Neuron. 1989; 3: 13-20Google Scholar; Von Bohlen und Halbach et al., 1992). Recently, Ca2+ signaling was linked to Ng-CAM expression during neuronal migration in bird forebrain (Goldman et al., 21Goldman S.A. Williams S. Barami K. Lemmon V. Nedergaard M. Mol. Cell. Neurosci. 1996; 7: 29-45Google Scholar). Doherty and Walsh (15Doherty P. Walsh F.S. Curr. Opin. Neurobiol. 1994; 4: 49-56Google Scholar) have advanced the idea that activation of a variety of cell adhesion molecules, including L1, leads to activation of the fibroblast growth factor receptor and subsequently to an arachidonic acid second messenger cascade (Doherty and Walsh, 15Doherty P. Walsh F.S. Curr. Opin. Neurobiol. 1994; 4: 49-56Google Scholar). This cascade involves generation of diacylglycerol by phospholipase Cγ, conversion to arachidonic acid by diacylglycerol lipase, and calcium influx through L- and N-type channels (Doherty et al., 16Doherty P. Furness J. Williams E.J. Walsh F.S. J. Neurochem. 1994; 62: 2124-2131Google Scholar; Williams et al., 58Williams E.J. Furness J. Walsh F.S. Doherty P. Neuron. 1994; 13 (a): 583-594Google Scholar, 59Williams E.J. Walsh F.S. Doherty P. J. Neurochem. 1994; 62 (b): 1231-1234Google Scholar). The nonreceptor tyrosine kinase Src has also been implicated in neurite outgrowth on L1: neurons from src-knockout mice have a diminished capacity to extend neurites on an L1 substrate (Ignelzi et al., 26Ignelzi Jr., M.A. Miller D.R. Soriano P. Maness P.F. Neuron. 1994; 12: 873-884Google Scholar).On the other hand, relatively little is known about the factors that may regulate the functions of L1 in activating such signaling systems. L1 is both alternatively spliced (Miura et al., 37Miura M. Kobayashi M. Asou H. Uyemura K. FEBS Lett. 1991; 289: 91-95Google Scholar) and phosphorylated (Faissner et al., 18Faissner A. Kruse J. Nieke J. Schachner M. Dev. Brain Res. 1984; 15: 69-82Google Scholar) in the cytoplasmic domain. The phosphorylation suggested a potential mechanism by which L1 activity could be modulated. L1 has been found to be associated with a number of kinases (Sadoul et al., 47Sadoul R. Kirchhoff F. Schachner M. J. Neurochem. 1989; 53: 1471-1478Google Scholar), including casein kinase II (Wong et al., 61Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Google Scholar). We have previously shown that CKII can phosphorylate L1 on Ser1181 and that it is associated with L1. Continuation of the search for L1 kinases revealed p90rsk, which also coprecipitates with L1 from rat brain membrane preparations.p90rsk is well described in several systems (Blenis, 5Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Google Scholar; Erikson, 17Erikson R. J. Biol. Chem. 1991; 266: 6007-6010Google Scholar) and can be activated when it is phosphorylated by mitogen-associated protein kinase kinases. The p90rsk kinase is composed of two kinase domains, an N-terminal cGMP-dependent kinase-like domain and a C-terminal domain bearing resemblance to the catalytic domains of phosphorylase b and Ca2+/calmodulin kinases (Alcorta et al., 1Alcorta D.A. Crews C.M. Sweet L.J. Bankston L. Jones S.W. Erikson R.L. Mol. Cell. Biol. 1989; 9: 3850-3859Google Scholar). Recent data suggest that the N-terminal catalytic domain mediates substrate phosphorylation, whereas the C-terminal domain is involved in autophosphorylation (Bjorbaek et al., 4Bjorbaek C. Zhao Y. Moller D.E. J. Biol. Chem. 1995; 270: 18848-18852Google Scholar). There is no clear consensus recognition site for p90rsk, but like the cGMP- and cAMP-dependent kinases, there is a general requirement for basic residues in the vicinity of the target serine or threonine. The residues around Ser1152 do not fit the RXXS consensus found in several p90rsk target sites, but the serine is bracketed by arginine residues in the form RXSXR and may represent a novel site for p90rsk phosphorylation.The discovery of p90rsk began with a search for protein kinases that inducibly phosphorylate the S6 protein (Erikson, 17Erikson R. J. Biol. Chem. 1991; 266: 6007-6010Google Scholar; Novak-Hofer and Thomas, 39Novak-Hofer I. Thomas G. J. Biol. Chem. 1984; 259: 5995-6000Google Scholar; Sturgill and Wu, 53Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Google Scholar). Thus, it was expected to be involved in mitogen-stimulated pathways. However, several other functions have now been linked to this kinase. Among these are insulin-regulated glycogen metabolism, platelet (Papkoff et al., 40Papkoff J. Chen R.H. Blenis J. Forsman J. Mol. Cell. Biol. 1994; 14: 463-472Google Scholar) and T-cell activation (Calvo et al., 10Calvo V. Bierer B.E. Vik T.A. Eur. J. Immunol. 1992; 22: 457-462Google Scholar), stress responses (Jurivich et al., 28Jurivich D.A. Chung J. Blenis J. J. Cell. Physiol. 1991; 148: 252-259Google Scholar), and neuronal differentiation of PC12 cells (Scimeca et al., 51Scimeca J.C. Nguyen T.T. Filloux C. Van Obberghen E. J. Biol. Chem. 1992; 267: 17369-17374Google Scholar). The mitogen-activated pathways leading to p90rsk stimulation involve activation of a receptor tyrosine kinase, followed by sequential activation of Raf, MEK (mitogen-associated protein kinase kinase), erk-1 and erk-2 mitogen-associated protein kinases, and p90rsk (Blenis, 5Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Google Scholar). Activation of such a cascade has been described upon binding of IgM on B lymphocytes (Tordai et al., 55Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Google Scholar). Although p90rsk is considered a cytosolic protein, which when activated can translocate to the nucleus, it is also found in membrane fractions (Chen et al., 12Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Google Scholar), and we have recently found p90rsk in growth cone particle preparations purified from rat brain (data not shown).Ser1152, the site of p90rsk action, is located nine amino acids from the membrane, within one of the most highly conserved regions of the molecule. Of note, this serine is conserved only in the closest homologues of L1 and is not in related members of the L1 group of immunoglobulin superfamily adhesion molecules, including the chick proteins Nr-CAM (Grumet et al., 24Grumet M. Mauro V. Burgoon M.P. Edelman G.M. Cunningham B.A. J. Cell Biol. 1991; 113: 1399-1412Google Scholar) and neurofascin (Rathjen et al., 44Rathjen F.G. Wolff J.M. Chang S. Bonhoeffer F. Raper J. Cell. 1987; 51 (a): 841-849Google Scholar) or the rat ankyrin-binding glycoprotein (Davis et al., 14Davis J.Q. McLaughlin T. Bennett V. J. Cell Biol. 1993; 121: 121-133Google Scholar). The 10 membrane-proximal intracellular residues of Nr-CAM, neurofascin, and ankyrin-binding glycoprotein are identical to L1 except Ser1152, which is changed to a proline residue, implying an important function for this region. The presence of the serine at residue 1152 only in L1 may allow its functional regulation by phosphorylation. The KRSK peptide inhibition studies described here show that perturbation of interactions with this region of the L1 cytoplasmic domain disturbs L1-mediated neurite outgrowth. There is a 33% decrease in the percentage of cells bearing neurites when loaded with the KRSK peptide compared to tracer dye alone or scrambled sequence peptide. Interestingly, the S/A1152 peptide, which is a nonphosphorylatable KRSK peptide, has no significant effect by this measure, indicating that phosphorylation of KRSK is involved in this inhibition of neurite outgrowth. One mechanism by which the inhibition may take place is by competitive inhibition of p90rsk phosphorylation of L1, preventing it from undergoing phosphorylation-dependent conformational changes or protein-protein interactions. Another possible mechanism is that phosphorylation of the KRSK peptide allows it to interact with some other protein, which normally interacts with L1 only when Ser1152 is phosphorylated. Although the putative protein interaction is not known, it is unlikely to be the recently described ankyrin-L1 interaction (Davis and Bennett, 13Davis J.Q. Bennett V. J. Biol. Chem. 1994; 269: 27163-27166Google Scholar), which has been mapped to a region between residues 1200-1230.The data presented here indicate that L1 is associated with and phosphorylated by the S6 kinase p90rsk, the substrate site of which is Ser1152. Disruption of interactions between L1 and p90rsk or other proteins in the vicinity of Ser1152 has a significant deleterious effect on neurite outgrowth. One of the initial hypotheses in searching for L1 kinases was that they may transiently alter L1 function. The first L1 kinase we found, CKII, is generally in a constitutively active state and unlikely to be acutely regulated. However, p90rsk has previously been well studied as part of an extracellularly initiated signal transduction cascade. Therefore, in contrast to CKII, p90rsk could be involved in a transient change in the phosphorylation state of L1 and consequently lead to changes in the conformational and functional state of L1 that determine the distinct morphological and motile characteristics of neurite outgrowth on L1. INTRODUCTIONThe development of a functional nervous system depends, in part, on the ability of neurons to form the requisite specific connections with their targets. The guidance of axons through varied terrain and over relatively long distances is thought to be influenced by a variety of factors. These include physical channels and chemical signals, either diffusible factors or substrate-bound extracellular matrix molecules or cell surface molecules. Cell adhesion molecules are often involved in providing a suitable substrate upon which neurons can migrate or extend axons. L1 is a cell adhesion molecule that has been implicated in a variety of processes integral to the development of the nervous system, including neuronal migration (Lindner et al., 35Lindner J. Rathjen F.G. Schachner M. Nature. 1983; 305: 427-430Google Scholar), neurite outgrowth (Lagenaur and Lemmon, 31Lagenaur C. Lemmon V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7753-7757Google Scholar), and axon fasciculation (Landmesser et al., 32Landmesser L. Dahm L. Schultz K. Rutishauser U. Dev. Biol. 1988; 130: 645-670Google Scholar; Stallcup and Beasley, 52Stallcup W.B. Beasley L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1276-1280Google Scholar). Mutations in the L1 gene are linked to the human mental retardation diseases X-linked hydrocephalus and MASA (ental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome (Rosenthal et al., 46Rosenthal A. Jouet M. Kenwrick S. Nat. Genet. 1992; 2: 107-112Google Scholar; Wong et al., 60Wong E.V. Kenwrick S. Willems P. Lemmon V. Trends Neurosci. 1995; 18: 168-172Google Scholar), in which defects of the cortocospinal tract and corpus callosum are commonly found.Recent evidence suggests a function for L1 beyond adhesion between two cell surfaces. When a growth cone migrating on laminin contacts L1, its morphology changes quickly, broadening and flattening even before the entire growth cone has moved onto the new substrate (Burden-Gulley et al., 9Burden-Gulley S.M. Payne H.R. Lemmon V. J. Neurosci. 1995; 15: 4370-4381Google Scholar). This suggests activation of a signal transduction cascade initiated by L1 contact that eventually affects the cytoskeleton. Further evidence for signal transduction cascades initiated by L1 comes from observations of changes in various intracellular second messenger systems upon activation of L1 by binding with soluble L1 or anti-L1 antibodies (Itoh et al., 27Itoh K. Kawamura H. Asou H. Brain Res. 1992; 580: 233-240Google Scholar; Schuch et al., 50Schuch U. Lohse M.J. Schachner M. Neuron. 1989; 3: 13-20Google Scholar; Von Bohlen und Halbach et al., 56Von Bohlen Halbach F. Taylor J. Schachner M. Eur. J. Neurosci. 1992; 4: 896-909Google Scholar; Williams et al., 57Williams E.J. Doherty P. Turner G. Reid R.A. Hemperly J.J. Walsh F.S. J. Cell Biol. 1992; 119: 883-892Google Scholar).L1 (Moos et al., 38Moos M. Tacke R. Scherer H. Teplow D. Fruh K. Schachner M. Nature. 1988; 334: 701-703Google Scholar) (also termed NILE (Prince et al., 42Prince J.T. Milona N. Stallcup W.B. J. Neurosci. 1989; 9: 1825-1834Google Scholar), 8D9 (Lemmon and McLoon, 33Lemmon V. McLoon S. J. Neurosci. 1986; 6: 2987-2994Google Scholar), Ng-CAM (Grumet et al., 23Grumet M. Hoffman S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 267-271Google Scholar), G4 (Rathjen et al., 45Rathjen F.G. Wolff J.M. Frank R. Bonhoeffer F. Rutishauser U. J. Cell Biol. 1987; 104 (b): 343-353Google Scholar)) is primarily expressed on projection axons of the central nervous system and peripheral nervous system, as well as on a few nonneuronal cell types, including Schwann cells and lymphocytes. It is a member of the immunoglobulin superfamily of adhesion molecules (Burden-Gulley and Lemmon, 7Burden-Gulley S.M. Lemmon V. Semin. Dev. Biol. 1995; 6: 79-87Google Scholar), the extracellular domain of which is characterized by six immunoglobulin-like domains and five fibronectin-type III domains, and highly conserved transmembrane and cytoplasmic domains. The cytoplasmic domain is completely conserved in the known mammalian sequences, and two long stretches are perfectly conserved in the chick, comprising nearly 70% of the cytoplasmic domain (Hlavin and Lemmon, 25Hlavin M.L. Lemmon V. Genomics. 1991; 11: 416-423Google Scholar). Two shorter sequences, one abutting the membrane and one 40 amino acids from the C terminus, are conserved, even in the Drosophila L1 homologue, neuroglian (Bieber et al., 3Bieber A.J. Snow P.M. Hortsch M. Patel N.H. Jacobs J.R. Traquina Z.R. Schilling J. Goodman C.S. Cell. 1989; 59: 447-460Google Scholar). There are also two alternatively spliced exons that are present in neuronal L1 but not in L1 expressed in nonneuronal cells (Miura et al., 37Miura M. Kobayashi M. Asou H. Uyemura K. FEBS Lett. 1991; 289: 91-95Google Scholar). The L1 molecule is both glycosylated and phosphorylated (Faissner et al., 18Faissner A. Kruse J. Nieke J. Schachner M. Dev. Brain Res. 1984; 15: 69-82Google Scholar).One possible mechanism for control of the signal transduction cascades initiated by L1 binding is the regulated phosphorylation of L1. We and others have described a number of kinase activities that coprecipitate with L1 immunoprecipitates (Sadoul et al., 47Sadoul R. Kirchhoff F. Schachner M. J. Neurochem. 1989; 53: 1471-1478Google Scholar; Wong et al., 61Wong E.V. Schaefer A.W. Landreth G. Lemmon V. J. Neurochem. 1996; 66: 779-786Google Scholar). We have identified one of these as casein kinase II, which phosphorylates L1 at Ser1181. In this paper, we demonstrate that an S6 family kinase is also associated with L1.The serine/threonine kinase, p90rsk, was initially identified on the basis of its ability to phosphorylate the ribosomal 40 S subunit in vitro. This enzyme has been the focus of much interest due to its ability to be phosphorylated and activated by the mitogen-associated protein kinases and is a component of this growth factor-sensitive signaling cascade (Blenis, 5Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Google Scholar). The role of p90rsk in the nervous system has not been studied directly, but it is reported to be part of an NGF 1The abbreviations used are: NGFnerve growth factorPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatographyFITCfluorescein isothiocyanateCAMcell adhesion moleculeCKIIcasein kinase IIPBSphosphate-buffered saline. -inducible signaling cascade in PC12 pheochromocytoma cells (Scimeca et al., 51Scimeca J.C. Nguyen T.T. Filloux C. Van Obberghen E. J. Biol. Chem. 1992; 267: 17369-17374Google Scholar). This paper describes the phosphorylation of a neural cell adhesion molecule, L1, by p90rsk. p90rsk associates with L1 at the membrane and phosphorylates L1 at Ser1152. This phosphorylation may regulate the interactions of L1 and intracellular signaling cascades or cytoskeletal elements involved in neurite outgrowth on specific substrates.