RPTPalpha is essential for NCAM-mediated p59fyn activation and neurite elongation

The neural cell adhesion molecule (NCAM) forms a complex with p59fyn kinase and activates it via a mechanism that has remained unknown. We show that the NCAM140 isoform directly interacts with the intracellular domain of the receptor-like protein tyrosine phosphatase RPTPalpha, a known activator of p59fyn. Whereas this direct interaction is Ca2+ independent, formation of the complex is enhanced by Ca2+-dependent spectrin cytoskeleton-mediated cross-linking of NCAM and RPTPalpha in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTPalpha-deficient brains and is disrupted by dominant-negative RPTPalpha mutants, demonstrating that RPTPalpha is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTPalpha-deficient neurons, and disruption of the NCAM-p59fyn complex in RPTPalpha-deficient neurons or with dominant-negative RPTPalpha mutants blocks NCAM-dependent neurite outgrowth, implicating RPTPalpha as a major phosphatase involved in NCAM-mediated signaling.     

Alternative Splicing of the Cytoplasmic Domain of Neural Cell Adhesion Molecule Alters Its Ability to Act as a Substrate for Neurite Outgrowth

A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.     

神经细胞粘附分子通过P21活化激酶1促进神经突生长

目的探索神经细胞粘附分子(NCAM)促进神经突生长的分子机制。方法对新生小鼠脑组织行免疫共沉淀以筛选NCAM的结合伴侣。向体外培养的海马神经元中加入免疫共沉淀的阳性筛选分子的抑制剂,观察其对NCAM促进神经突生长作用的影响。提取新生小鼠脑内生长锥以及脂筏,检测NCAM、NCAM的结合伴侣及其上、下游分子在小鼠脑内的空间分布。结果免疫共沉淀发现P21活化激酶1(Pak1)为NCAM的结合伴侣,Pak1抑制剂可以阻断NCAM促进神经突生长的作用。对小鼠脑内脂筏的研究发现NCAM和Pak1上游激活物Pak相互作用交换因子(PIX)、细胞分裂周期蛋白42(Cdc42)在生长锥脂筏上富集,提示NCAM与Pak1的结合以及Pak1的活化可能在脂筏上完成。结论 NCAM通过Pak1途径促进神经突生长,且这一作用的实现可能依赖于脂筏。     

GAP-43 regulates NCAM-180-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.     

Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis

The neural cell adhesion molecule (NCAM) has been reported to stimulate neuritogenesis either via nonreceptor tyrosine kinases or fibroblast growth factor (FGF) receptor. Here we show that lipid raft association of NCAM is crucial for activation of the nonreceptor tyrosine kinase pathway and induction of neurite outgrowth. Transfection of hippocampal neurons of NCAM-deficient mice revealed that of the three major NCAM isoforms only NCAM140 can act as a homophilic receptor that induces neurite outgrowth. Disruption of NCAM140 raft association either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the tyrosine focal adhesion kinase and extracellular signal-regulated kinase 1/2, completely blocking neurite outgrowth. Likewise, NCAM-triggered neurite outgrowth is also completely blocked by a specific FGF receptor inhibitor, indicating that cosignaling via raft-associated kinases and FGF receptor is essential for neuritogenesis.     

Identification of an amino acid sequence motif in the cytoplasmic domain of the NCAM-140 kDa isoform essential for its neuritogenic activity

The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated. We investigated the importance of the cytoplasmic domain of the 140-kDa NCAM isoform (cytNCAM-140) and of the 180-kDa NCAM isoform (cytNCAM-180) in NCAM-induced neurite extension by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding cytNCAM-140, cytNCAM-180, the constitutively active form of the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP). EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM-180 had no effect on NCAM-stimulated neuritogenesis, whereas expression of cytNCAM-140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM-140, neurite outgrowth was rescued, indicating that cytNCAM-140 is involved in signaling via the Ras-MAP kinase pathway. PC12-E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM-140 and various full-length cytNCAM-140 mutants, and the residues Thr-Glu-Val-Lys-Thr (839-843) were identified as essential in NCAM-stimulated neuritogenesis. The combined substitution of Glu(840) and Lys(842) with Ala abrogated the effect of the construct, assigning a critical role to these two residues.     

Expression of NCAM containing VASE in neurons can account for a developmental loss in their neurite outgrowth response to NCAM in a cellular substratum

Binding of the neural cell adhesion molecule (NCAM) in neurons to NCAM on non-neuronal cells can stimulate axonal growth. A developmentally regulated loss of this response is associated with the insertion of 10 amino acids (called VASE) into the fourth Ig domain in up to 50% of the NCAM receptors in neurons. In the present study we have transfected PC12 cells with the major neuronal isoforms of human NCAM and tested cells expressing these isoforms for their ability to respond to NCAM in a cellular substratum. Whereas both the 140- and 180-kD isoforms of NCAM can act as functional receptors for neurite outgrowth, the presence of the VASE sequence in a minority of the receptors specifically inhibited this response. A synthetic peptide containing the VASE sequence inhibits neurite outgrowth from PC12 cells and primary neurons stimulated by NCAM. The same peptide has no effect on integrin dependent neurite outgrowth or neurite outgrowth stimulated by N-cadherin or L1. We discuss the possibility that the VASE peptide inhibits the NCAM response by preventing NCAM from binding to the FGF receptor in the plasma membrane.     

Polysialyltransferase ST8Sia II (STX) polysialylates all of the major isoforms of NCAM and facilitates neurite outgrowth

The neural cell adhesion molecule (NCAM) has different isoforms due to different sizes in its polypeptide and plays a significant role in neural development. In neural development, the function of NCAM is modified by polysialylation catalyzed by two polysialyltransferases, ST8Sia II and ST8Sia IV. Previously, it was reported by others that ST8Sia II polysialylates only transmembrane isoforms of the NCAM, such as NCAM-140 and NCAM-180, but not NCAM-120 and NCAM-125 anchored by a glycosylphosphotidylinositol. In the present study, we first discovered that ST8Sia II polysialylates all isoforms of the NCAM examined, and we demonstrated that polysialylation of NCAM expressed on 3T3 cells facilitates neurite outgrowth regardless of isoforms of NCAM, where polysialic acid is attached. We then show that neurite outgrowth is significantly facilitated only when polysialylated NCAM is present in cell membranes. Moreover, the soluble NCAM coated on plates did not have an effect on neurite outgrowth exerted by soluble L1 adhesion molecule coated on plates. These results, taken together, indicate that ST8Sia II plays critical roles in modulating the function of all major isoforms of NCAM. The results also support previous studies showing that a signal cascade initiated by NCAM differs from that initiated by L1 molecule.     

Metalloprotease-induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180(1)) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM-transfected L-fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate-induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM-dependent neurite branching and outgrowth. Moreover, NCAM-dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease-induced ectodomain shedding of NCAM down-regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity.     

Tyrosine 734 of NCAM180 interferes with FGF receptor-dependent signaling implicated in neurite growth

The cytoplasmic domain of the neural cell adhesion molecule (NCAM) contains multiple phosphorylation sites. We report here that in addition to serine and threonine residues a tyrosine of the NCAM180 isoform is phosphorylated as shown by phosphoamino acid analysis. Exchange of the only cytoplasmic tyrosine at position 734 of human NCAM180 (NCAM180-Y734F) to phenylalanine resulted in increased neurite outgrowth of NCAM180-Y734F transfected B35 neuroblastoma cells compared to NCAM180-wt transfectants on poly-L-lysine as substrate. As demonstrated by inhibitor studies the increased neurite outgrowth was due to higher FGF receptor 1 and ERK1 activity in NCAM180-Y734F cells, indicating that tyrosine residue 734 plays a role in signal transduction mediated by the FGF receptor. On an NCAM expressing monolayer of COS-7 cells the Y734F mutation also influences FGF receptor 1 dependent neurite outgrowth, but under these conditions additional mechanisms seem to be responsible for the increased neurite length observed for NCAM180-Y734F transfected cells.     

Metalloprotease‐induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180¹) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM‐transfected L‐fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate‐induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM‐dependent neurite branching and outgrowth. Moreover, NCAM‐dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease‐induced ectodomain shedding of NCAM down‐regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Clustering of the neural cell adhesion molecule (NCAM) at the neuronal cell surface induces caspase-8- and -3-dependent changes of the spectrin meshwork required for NCAM-mediated neurite outgrowth

Changes in neuronal morphology underlying neuronal differentiation depend on rapid and sustained cytoskeleton rearrangements in the growing neurites. Whereas cell adhesion molecules are well established as regulators of neuronal differentiation, less is known about the signaling mechanisms by which they influence the cytoskeleton. Here we show that the neural cell adhesion molecule (NCAM) associates with the active form of caspase-8 and that clustering of NCAM at the neuronal cell surface leads to activation of caspase-8 and -3 followed by the cleavage of the sub-membranous brain spectrin meshwork, but not of the actin or tubulin cytoskeleton. Inhibitors of caspase-8 and -3 specifically block the NCAM-dependent spectrin cleavage and abolish NCAM-dependent neurite outgrowth. NCAM-dependent rearrangements of the membrane associated spectrin meshwork via caspase-8 dependent caspase-3 activation are thus indispensable for NCAM-mediated neurite outgrowth.     

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca²⁺ channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca²⁺ channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca²⁺-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.     

Prion protein recruits its neuronal receptor NCAM to lipid rafts to activate p59fyn and to enhance neurite outgrowth

In spite of advances in understanding the role of the cellular prion protein (PrP) in neural cell interactions, the mechanisms of PrP function remain poorly characterized. We show that PrP interacts directly with the neural cell adhesion molecule (NCAM) and associates with NCAM at the neuronal cell surface. Both cis and trans interactions between NCAM at the neuronal surface and PrP promote recruitment of NCAM to lipid rafts and thereby regulate activation of fyn kinase, an enzyme involved in NCAM-mediated signaling. Cis and trans interactions between NCAM and PrP promote neurite outgrowth. When these interactions are disrupted in NCAM-deficient and PrP-deficient neurons or by PrP antibodies, NCAM/PrP-dependent neurite outgrowth is arrested, indicating that PrP is involved in nervous system development cooperating with NCAM as a signaling receptor.     

NCAM induces CaMKIIalpha-mediated RPTPalpha phosphorylation to enhance its catalytic activity and neurite outgrowth

Receptor protein tyrosine phosphatase α (RPTPα) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPα activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPα. NCAM associates with T- and L-type voltage-dependent Ca²⁺ channels, and NCAM clustering at the cell surface results in Ca²⁺ influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIα (CaMKIIα). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM–RPTPα–CaMKIIα complex, resulting in serine phosphorylation of RPTPα by CaMKIIα. Overexpression of RPTPα with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPα activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity.     

Phosphorylation of Serine 774 of the Neural Cell Adhesion Molecule (NCAM) Is Involved in the Interaction with Collapsin Response Mediator Protein-2

The neural cell adhesion molecule NCAM is a major adhesion receptor involved in the development and regeneration of the nervous system. It is expressed in three major isoforms of which two have large intracellular domains of different lengths (NCAM140 and NCAM180). Several intracellular ligands of NCAM have been described. One of them is the collapsin response mediator protein-2 (CRMP-2), which is known to be involved in cell differentiation and axonal growth. The cytoplasmic domains of NCAM contain up to 49 phosphorylation sites and it has been demonstrated recently that the phosphorylation of serine 774 is crucial for NCAM-mediated signal transduction and neurite outgrowth. Here we analyzed the interaction of NCAM with CRMP-2 in more detail using a biochemical approach. We found that CRMP-2 binds specifically to NCAM180 in a sequence between amino acid 788 and 819. In addition we could demonstrate that serine 774, which has been shown previously to be phosphorylated and involved in neurite outgrowth, is also important for the interaction of CRMP-2 with NCAM.     

The neural cell adhesion molecule is associated with major components of the cytoskeleton

The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily. Two of the three major isoforms (NCAM 140 and NCAM 180) are transmembrane glycoproteins, which differ in their intracellular domains. The present study is concerned with the identification of novel intracellular binding partners of NCAM. We expressed and purified both cytoplasmic domains of NCAM. Using ligand affinity chromatography followed by peptide mass fingerprinting, we could identify several novel binding partners of the cytoplasmic domains of NCAM 140 and 180. We present data that alpha- and beta-tubulin as well as alpha-actinin 1 are associated with both NCAM 140 and 180. In contrast, beta-actin, tropomyosin, microtubuli-associated protein MAP 1A, and rhoA-binding kinase-alpha preferentially bind to NCAM 180. Furthermore, we demonstrate that inhibition of rhoA-binding kinase-alpha stimulates neurite outgrowth independently from NCAM.     

Distinct roles of PKC isoforms in NCAM-mediated neurite outgrowth

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)-mediated neurite outgrowth was tested using a co-culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12-E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM-stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM-mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase-deficient forms of PKCalpha, betaI and betaII blocked NCAM-mediated neurite extension, but had no effect on nerve growth factor (NGF)-mediated neurite outgrowth. Expression of two PKCepsilon constructs: (i) a fragment from PKCepsilon encompassing the pseudosubstrate, the C1a domain (including the actin-binding site, ABS), and parts of the V3 region, or (ii) the PKCepsilon-specific ABS blocked NCAM-mediated neurite extension in both cases. These two constructs also partially inhibited NGF-stimulated neuritogenesis indicating that PKCepsilon is a positive regulator of both NCAM- and NGF-mediated differentiation. We suggest that PKCepsilon is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM-stimulated neurite outgrowth.     

Neurite Outgrowth is Dependent on the Association of c-Src and Lipid Rafts

Regulation of neurite outgrowth is an important aspect not only for proper development of the nervous system but also for tissue regeneration after nerve injury and the treatment of neuropathological conditions. Here, we report that neurite outgrowth in cortical neuron and neuro 2A (N2A) cell was dependent on intact lipid rafts, as well as the enhanced localization of c-Src in the lipid rafts. Src inhibition or lipid rafts disruption could specifically block c-Src phosphorylation profile, pY416 Src increase and pY529 Src decrease, they also resulted in pY529 Src and c-terminal Src kinase (Csk) partition out of lipid rafts. Thus, we concluded that c-Src signal cascades within the lipid rafts is crucial for efficient neurite outgrowth.     

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.