RESUMEN
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 Ser(1204) and Ser(1248) 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.
Asunto(s)
Endocitosis , Sistema de Señalización de MAP Quinasas , Glicoproteínas de Membrana/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Activación Enzimática , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/química , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMEN
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, p90(rsk). Moreover, S6 kinase activity and p90(rsk) 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.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/farmacología , Membrana Celular/metabolismo , Pollos , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Complejo de Antígeno L1 de Leucocito , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/farmacología , Neuritas/efectos de los fármacos , Células PC12 , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Fosforilación , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144-1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173-1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177-1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Quinasa de la Caseína II , Humanos , Complejo de Antígeno L1 de Leucocito , Datos de Secuencia Molecular , Células PC12 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Pruebas de Precipitina , Ratas , Ratas Sprague-DawleyRESUMEN
L1 is a highly conserved cell adhesion molecule with complete homology of the cytoplasmic domain between the known mammalian protein sequences. Since the cytoplasmic domains of other adhesion molecules have been shown to influence adhesion, we have investigated the effects of deletion of the cytoplasmic domain on the ability of L1 to mediate homophilic adhesion. Full length L1 and a truncated L1, lacking 95% of the cytoplasmic domain, were expressed in myeloma cells. Independent stable transfectants were assayed for the ability to form aggregates. Myelomas expressing L1 lacking the cytoplasmic domain were able to form cell aggregates as well as the myelomas expressing full length L1. Cell aggregate formation was correlated with the level of L1 expression, and the aggregation could be blocked by anti-L1 Fabs. Similar results were obtained in adhesion assays of the myeloma cells to substrate-bound L1. These results indicate that the cytoplasmic domain of L1 is not required for homophilic interactions.
Asunto(s)
Citoplasma/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Secuencia de Aminoácidos , Animales , Adhesión Celular/fisiología , Pollos , Secuencia Conservada , ADN Complementario/biosíntesis , Humanos , Complejo de Antígeno L1 de Leucocito , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Unión Proteica , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
Recently, studies in the usually disparate fields of human genetics and developmental neurobiology have converged to reveal that some types of human mental retardation and brain malformations are due to mutations that affect the neural cell adhesion molecule L1. L1 has a very complex biology, interacting with a variety of ligands, and functioning in migration of neurons and growth of axons. Over the past few years, it has also become clear that L1 is able to influence intracellular second messengers. The identification of a number of different mutations in L1, some of which alter the extracellular portion of the molecule, and others that change only the cytoplasmic tail, confirm that L1 is a crucial player in normal brain development. The information gained from genetic analysis of human L1 is giving new insights into how L1 functions in the formation of major axon pathways, but it also raises unanticipated questions about how L1 participates in the development of cortical and ventricular systems.