The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.T he neuronal K-Cl cotransporter KCC2 transports chloride using the electrochemical gradient of K + ions (1). In mature neurons, this action maintains a low intraneuronal chloride concentration that ensures a hyperpolarizing effect of GABA at chloride-permeable GABA A receptors. KCC2 expression, activity, and membrane traffic are tightly regulated by neuronal activity, particularly through the phosphorylation of its carboxylterminal domain (CTD) (2-4). Activation of postsynaptic glutamate receptors, for instance, reduces KCC2 activity through dephosphorylation and endocytosis within minutes (3, 5). KCC2 expression is also suppressed in pathological conditions associated with enhanced neuronal activity (6), leading to a rise in intraneuronal chloride and an alteration of GABA function (7-9). KCC2 therefore appears to mediate a functional cross-talk between synaptic excitation and inhibition in neurons.Although KCC2 function primarily influences the efficacy of GABAergic signaling, its presence in dendritic spines (10) raises the question of its role in spine morphogenesis and function. Genetic ablation of KCC2 in mice compromises spine maturation and excitatory synapse formation in immature hippocampal neurons (11). This effect appears to be independent of KCC2 function but, instead, involves KCC2 interaction with the neuronal FERM-domain protein 4.1N (12). However, KCC2 expression is up-regulated during postnatal development and is maximal in mature neurons (13), after spine formation, where its role in the maintenance and function of dendritic spines remains unknown. Here, we show that suppression of KCC2 after spine morphogenesis reduces postsynaptic glutamate recept...

Focal adhesion kinase (FAK) is activated following integrin engagement or stimulation of transmembrane receptors. Autophosphorylation of FAK on Tyr-397 is a critical event, allowing binding of Src family kinases and activation of signal transduction pathways. Tissue-specific alternative splicing generates several isoforms of FAK with different autophosphorylation rates. Despite its importance, the mechanisms of FAK autophosphorylation and the basis for differences between isoforms are not known. We addressed these questions using isoforms of FAK expressed in brain. Autophosphorylation of FAK ؉ , which is identical to that of "standard" FAK, was intermolecular in transfected cells, although it did not involve the formation of stable multimeric complexes. Coumermycin-induced dimerization of gyrase B-FAK ؉ chimeras triggered autophosphorylation of Tyr-397. This was independent of cell adhesion but required the C terminus of the protein. In contrast, the elevated autophosphorylation of FAK ؉6,7 , the major neuronal splice isoform, was not accounted for by transphosphorylation. Specifically designed immune precipitate kinase assays confirmed that autophosphorylation of FAK ؉ was intermolecular, whereas autophosphorylation of FAK ؉6,7 or FAK ؉7 was predominantly intramolecular and insensitive to the inhibitory effects of the N-terminal domain. Our results clarify the mechanisms of FAK activation and show how alternative splicing can dramatically alter the mechanism of autophosphorylation of a protein kinase.

Caspr/paranodin, a neuronal transmembrane glycoprotein, is essential for the structure and function of septate-like paranodal axoglial junctions at nodes of Ranvier. A closely related protein, Caspr2, is concentrated in juxtaparanodal regions where it associates indirectly with the shaker-type potassium channels. Although ultrastructural studies indicate that paranodal complexes are linked to the cytoskeleton, the intracellular partners of Caspr/paranodin, as well as those of Caspr2, are poorly characterized. We show that the conserved intracellular juxtamembrane regions (GNP motif) of Caspr/paranodin and Caspr2 bind proteins 4.1R and 4.1B. 4.1B is known to be enriched in paranodal and juxtaparanodal regions. 4.1B immunoreactivity accumulates progressively at paranodes and juxtaparanodes during postnatal development, following the concentration of Caspr/paranodin and Caspr2, respectively, in central and peripheral myelinated axons. These two proteins coimmunoprecipitated with 4.1B in brain homogenates. Our results provide strong evidence for the association of 4.1B with Caspr/paranodin at paranodes and with Caspr2 at juxtaparanodes. We propose that 4.1B anchors these axonal proteins to the actin-based cytoskeleton in these two regions.

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.Focal adhesion kinase (FAK) 1 is a non-receptor cytoplasmic tyrosine kinase of 125 kDa (1, 2) implicated in integrin-mediated signal transduction (reviewed in Refs. 3-5). It is also activated by a variety of extracellular stimuli including G protein-coupled receptors and growth factor receptors (see Refs. 5 and 6). In adherent mammalian cells in culture FAK is located in focal adhesions (1, 2) and appears important for the regulation of their turnover (7). FAK is critical for adhesion-dependent cell survival (8) and integrin-mediated motility (9). Its physiological importance is demonstrated by the embryonic mortality of FAK knockout mice (7), whereas recent work underlines its role in tumor invasiveness (10). Autophosphorylation at Tyr-397 is a crucial event for FAK biological function, because it creates a high affinity binding site for proteins harboring Src homology 2 (SH2) domains, such as Src and Fyn (11), Grb7 (12), and phosphatidylinositide 3Ј-OH-kinase, which activates the anti-apoptotic Akt pathway (13,14). Following their binding to phospho-Tyr-397, Src or Fyn phosphorylate FAK at other tyrosine residues (15, 16) as well as associated proteins, thereby leading to the activation of several signaling pathways (see 5).In contrast to the recent progress in elucidating the signaling pathways downstream from FAK, relatively little is known about the molecular mechanisms regulating FAK activity. The tyrosine kinase ...

Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber.

Axon initial segments (AISs) and nodes of Ranvier (NRs) are essential regions for saltatory conduction of the action potential along the axon. These two domains are enriched in similar multimolecular complexes, which include voltage-gated sodium channels (Na v ), NF186 (neurofascin 186), NrCAM (neuron glia-related cell adhesion molecule), and cytoskeleton linkers ankyrin G (AnkG) and ␤IV-spectrin. Identification of novel members of these complexes is critical to better understand their formation, function, and maintenance. Here we report that IQCJ-SCHIP-1, a recently identified isoform of schwannomin-interacting protein-1 (SCHIP-1), is a novel component of both AISs and NRs in the central and peripheral nervous systems. We show that IQCJ-SCHIP-1 binds calmodulin in the absence of Ca 2ϩ and is highly enriched at AISs and NRs. IQCJ-SCHIP-1 accumulation at AISs and NRs is a late event, suggesting that IQCJ-SCHIP-1 is likely to play a role in mature AISs and NRs rather than during their formation. IQCJ-SCHIP-1 was not detected at

Neuronal activity modulates the membrane diffusion of postsynaptic γ-aminobutyric acid (GABA)(A) receptors (GABA(A)Rs), thereby regulating the efficacy of GABAergic synapses. The K289M mutation in GABA(A)Rs subunit γ2 has been associated with the generalized epilepsy with febrile seizures plus (GEFS+) syndrome. This mutation accelerates receptor deactivation and therefore reduces inhibitory synaptic transmission. Yet, it is not clear why this mutation specifically promotes febrile seizures. We show that upon raising temperature both the number of GABA(A)Rs clusters and the frequency of miniature inhibitory postsynaptic currents decreased in neurons expressing the K289M mutant but not wild-type (WT) recombinant γ2. Single-particle tracking experiments revealed that raising temperature increases the membrane diffusion of synaptic GABA(A)Rs containing the K289M mutant but not WT recombinant γ2. This effect was mediated by enhanced neuronal activity as it was blocked by glutamate receptor antagonists and was mimicked by the convulsant 4-aminopyridine. Our data suggest the K289M mutation in γ2 confers GABA(A)Rs with enhanced sensitivity of their membrane diffusion to neuronal activity. Enhanced activity during hyperthermia may then trigger the escape of receptors from synapses and thereby further reduce the efficacy of GABAergic inhibition. Alteration of the membrane diffusion of neurotransmitter receptors therefore represents a new mechanism in human epilepsy.

Schwannomin/merlin is the product of a tumor suppressor gene mutated in neurofibromatosis type 2 (NF2). Although the consequences of NF2 mutations on Schwann cell proliferation are well established, the physiological role of schwannomin in differentiated cells is not known. To unravel this role, we studied peripheral nerves in mice overexpressing in Schwann cells schwannomin with a deletion occurring in NF2 patients (P0 -SCH-⌬39 -121) or a C-terminal deletion. The myelin sheath and nodes of Ranvier were essentially preserved in both lines. In contrast, the ultrastructural and molecular organization of contacts between Schwann cells and axons in paranodal and juxtaparanodal regions were altered, with irregular juxtaposition of normal and abnormal areas of contact. Similar but more severe alterations were observed in mice with conditional deletion of the Nf2 gene in Schwann cells. The number of SchmidtLanterman incisures, which are cytoplasmic channels interrupting the compact myelin and characterized by distinct autotypic contacts, was increased in the three mutant lines. P0 -SCH-⌬39 -121 and conditionally deleted mice displayed exuberant wrapping of nonmyelinated fibers and short internodes, an abnormality possibly related to altered control of Schwann cell proliferation. In support of this hypothesis, Schwann cell number was increased along fibers before myelination in P0 -SCH-⌬39 -121 mice but not in those with C-terminal deletion. Schwann cell numbers were also more numerous in mice with conditional deletion. Thus, schwannomin plays an important role in the control of Schwann cell number and is necessary for the correct organization and regulation of axoglial heterotypic and glio-glial autotypic contacts.