Biomedical subjects

Lionel Arnaud

Publications and source records attributed to Lionel Arnaud.

9 recordsLinked to original sources

PubMed2026

Large-scale functional annotation establishes a reference framework for human LRRK2 variants.

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2)1are among the most frequent monogenic causes of Parkinson's disease (PD)2 and act through a gain-of-function mechanism of increased kinase activity. LRRK2-targeted therapies are in clinical development, but interpretation of the rapidly expanding catalogue of rare LRRK2 variants remains a barrier to translation. Here, we present functionally annotated data on >350 LRRK2 coding variants using a standardized cellular assay with Rab10 phosphorylation as a readout of kinase activity and integrated these data with curated genetic and clinical annotations from the Movement Disorders Society Genetic Mutation Database (MDSGene). Variants differed in activation magnitude, ranging from modest increases (e.g., p.G2019S) to strongly activating substitutions such as p.Y1699C or p.L1795F. Activating variants occurred across the full length of LRRK2, although the largest effects clustered within the ROC-COR regulatory hub, where structural analysis identified subdomains forming an allosteric scaffold controlling kinase output. All known/established pathogenic variants showed increased activity, whereas benign and likely benign variants remained within the wild-type range. Functional effect sizes correlated with pathway activation in patient-derived immune cells, altogether providing a framework for ACMG-based variant interpretation in which kinase activation can support PS3 functional evidence for reclassification of variants.

Protein phosphorylation↗

PubMed2005

Both the phosphoinositide and receptor binding activities of Dab1 are required for Reelin-stimulated Dab1 tyrosine phosphorylation.

Reelin-stimulated tyrosine phosphorylation of the Dab1 adaptor protein is required during brain development for Reelin-dependent neuron positioning in the cerebral cortex and various other laminated regions. Dab1 contains an amino-terminal PTB/PI domain through which it can bind to Reelin receptors and membrane phosphoinositides. The relative contributions of these binding activities were unknown. Here, we identify a mutation in the PTB domain of Dab1 that inhibits membrane localization without inhibiting receptor binding. In neurons, this mutation reduces both basal and Reelin-stimulated Dab1 tyrosine phosphorylation. In contrast, a mutation that inhibits receptor binding reduces Reelin-stimulated but not basal tyrosine phosphorylation. These results support a model in which phospholipids recruit Dab1 to membranes but do not play a direct role in relaying the Reelin signal, while direct Dab1-receptor interaction is responsible for relaying the Reelin signal but not for membrane recruitment.

Animals↗

PubMed2005

Absence of Fyn and Src causes a reeler-like phenotype.

Nonreceptor protein tyrosine kinases of the Src family regulate the survival, proliferation, differentiation, and motility of many cell types, but their roles in brain development are unclear. Biochemical and in vitro experiments implicate Src and Fyn in the Reelin-dependent tyrosine phosphorylation of Dab1, which controls the positioning of radially migrating neurons in many brain regions. However, genetic evidence that either Src or Fyn mediates Reelin-dependent migrations in vivo has been lacking. Here, we report that, although Src is dispensable and although the absence of Fyn causes an intermediate phenotype, the combined absence of Src and Fyn almost abolishes tyrosine phosphorylation of Dab1 and causes defects in the fetal cortex and cerebellum very similar to those of dab1 mutants of the same age. Neurogenesis is not detectably affected, but the layering of neurons in the cortex is inverted, and the formation of the Purkinje plate is impaired. This implies that Src and Fyn are needed for Reelin-dependent events during brain development.

Animals↗

PubMed2004

Activation of a Dab1/CrkL/C3G/Rap1 pathway in Reelin-stimulated neurons.

During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Tyrosine phosphorylation of Disabled-1 is essential for Reelin-stimulated activation of Akt and Src family kinases.

Reelin is a large secreted signaling protein that is essential for proper positioning of migratory neurons during mammalian brain development. The Reelin signal is transduced into the cell by the lipoprotein receptors VLDLR and ApoER2, leading to tyrosine phosphorylation of the associated intracellular adaptor protein Disabled-1 (Dab1). Tyrosine phosphorylation of Dab1 is essential for responding to Reelin, as knock-in mice expressing a form of Dab1 that cannot be phosphorylated on tyrosine are indistinguishable from mice lacking Reelin, Reelin-receptors or Dab1. Molecular events dependent on Dab1 tyrosine phosphorylation are unknown. However, Reelin has recently been shown to activate the phosphoinositide-3-kinase (PI 3-K)-dependent kinase, Akt, as well as Src family kinases in wild type but not Dab1-/- primary embryonic neuronal cultures. Using pharmacological inhibitors and mice harboring mutant alleles of Dab1, we show here that tyrosine phosphorylation, but not the carboxyl-terminal region, of Dab1 is required for Reelin-induced activation of Akt and Src family kinases. Additionally, although Fyn is an important regulator of Dab1, Fyn deficiency does not prevent acute Reelin-induced Akt activation. Finally, whereas a number of growth factors propagate signals simultaneously through PI 3-K and mitogen-activated protein kinase (MAPK) cascades, we find Reelin does not engage the canonical MAPK cascade. These results define the first molecular events strictly dependent on Reelin-induced Dab1 tyrosine phosphorylation, and suggest that propagation of the Reelin signal is mediated by Akt, substrates of Src family kinases and/or unidentified molecules that share with these a common molecular link to phosphorylated Dab1.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2003

Lipid-dependent recruitment of neuronal Src to lipid rafts in the brain.

Although most Src family tyrosine kinases are modified by palmitoylation as well as myristoylation, Src itself is only myristoylated. Dual acylation is important for attachment to liquid-ordered microdomains or lipid rafts. Accordingly, Src is excluded from lipid rafts in fibroblasts. Evidence of partial genetic redundancy between Src and Fyn for brain-specific targets suggests that these two kinases may occupy overlapping subcellular locations. Neuronal Src (NSrc), an alternative isoform of Src with a 6-amino acid insert in the Src homology 3 domain, is highly expressed in neurons. We investigated whether this structural difference in NSrc allows it to associate with lipid rafts. We found that perinatal mouse brains express predominantly NSrc, which is partly (10-20%) in a lipid raft fraction from brain but not fibroblasts. The association of Src with brain lipid rafts does not depend on the NSrc insert but depends on the amino-terminal myristoylation signal. A crude lipid fraction from brain promotes NSrc entry into rafts in vitro. Moreover, lipid raft-localized NSrc is more catalytically active than NSrc from the soluble fraction, possibly because raft localization alters access to other tyrosine kinases and phosphatases. These findings suggest that NSrc may be involved in signaling from lipid rafts in mouse brain.

Animals↗

PubMed2003

Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development.

BACKGROUND: Disabled-1 (Dab1) is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood. RESULTS: We found that Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. We analyzed the Dab1 protein level and tyrosine phosphorylation stoichiometry by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. We found that fyn is required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src. CONCLUSIONS: Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway.

Animals↗

PubMed2003

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development.

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.

Animals↗

PubMed2002

Human Mps1 kinase is required for the spindle assembly checkpoint but not for centrosome duplication.

Budding yeast Mps1p kinase has been implicated in both the duplication of microtubule-organizing centers and the spindle assembly checkpoint. Here we show that hMps1, the human homolog of yeast Mps1p, is a cell cycle-regulated kinase with maximal activity during M phase. hMps1 localizes to kinetochores and its activity and phosphorylation state increase upon activation of the mitotic checkpoint. By antibody microinjection and siRNA, we demonstrate that hMps1 is required for human cells to undergo checkpoint arrest in response to microtubule depolymerization. In contrast, centrosome (re-)duplication as well as cell division occur in the absence of hMps1. We conclude that hMps1 is required for the spindle assembly checkpoint but not for centrosome duplication.

Animals↗