An ankyrin-binding motif regulates nuclear levels of L1-type neuroglian and expression of the oncogene Myc in Drosophila neurons

Abstract

Edited by Eric R. Fearon L1 cell adhesion molecule (L1CAM) is well-known for its importance in nervous system development and cancer progression. In addition to its role as a plasma membrane protein in cytoskeletal organization, recent in vitro studies have revealed that both transmembrane and cytosolic fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus. In vitro studies indicate that nuclear L1CAM affects genes with functions in DNA post-replication repair, cell cycle control, and cell migration and differentiation, but its in vivo role and how its nuclear levels are regulated is less well-understood. Here, we report that mutations in the conserved ankyrin-binding domain affect nuclear levels of the sole Drosophila homolog neuroglian (Nrg) and that it also has a noncanonical role in regulating transcript levels of the oncogene Myc in the adult nervous system. We further show that altered nuclear levels of Nrg correlate with altered transcript levels of Myc in neurons, similar to what has been reported for human glioblastoma stem cells. However, whereas previous in vitro studies suggest that increased nuclear levels of L1CAM promote tumor cell survival, we found here that elevated levels of nuclear Nrg in neurons are associated with increased sensitivity to oxidative stress and reduced life span of adult animals. We therefore conclude that these findings are of potential relevance to the management of neurodegenerative diseases associated with oxidative stress and cancer.L1-type cell adhesion molecules (CAMs) 4 are single-pass transmembrane glycoproteins belonging to the immunoglobu-lin family of receptors that are highly conserved from invertebrates to vertebrates (1). The structure of L1CAM consists of extracellular immunoglobulin and fibronectin type III domains, a transmembrane domain, and a cytoplasmic tail harboring an ezrin-binding FERM domain as well as an ankyrinbinding FIGQY domain (Fig. 1A). In its unphosphorylated state, the highly conserved FIGQY domain reversibly binds to ankyrin (Fig. 1A), which couples L1-type CAMs to actin. This interaction is known to mediate neuritogenesis, synapse growth, and stability (2-4). In contrast, phosphorylation of the tyrosine in the FIGQY domain inhibits ankyrin binding (5,6).In addition to its function as a cytoskeleton-organizing protein at the plasma membrane, vertebrate L1CAM can be proteolytically cleaved with fragments translocating to the nucleus (7-11). The 200-kDa full-length L1CAM is cleaved proximal to the plasma membrane by metalloproteases to a 32-kDa fragment (12,13). The 32-kDa fragment is further cleaved by ␥-secretase/presenilin, releasing a 28-kDa cytosolic fragment containing the intracellular domain (ICD), which translocates to the nucleus (10, 11). Similar to full-length L1CAM, recombinant expression of L1-ICD in nonneuronal cell lines affects gene expression of CRABPII and ␤3-integrin (11). In addition, it was shown that nuclear L1-ICD also led to up-regulation of NBS1 (Nijmegen breakage syndrome gene) via c-Myc, w...

Neuronal‐derived extracellular vesicles are enriched in the brain and serum of HIV‐1 transgenic rats

“…We also confirmed the presence of L1CAM on EVs using antibodies against L1CAM tagged with biotin by western blotting (Figure 2(e)). Similar to previous studies, we observed cleaved form (~70, ~55 and ~30 kDa) of L1CAM in L1CAM + EVs [46,47]. Further, we confirmed the presence of another neuronal marker βIII Tubulin as well as exosomal markers TSG101 and CD9 on the L1CAM + EVs (Figure 2(e)).…”

“…To detect the biotinylated fragment after its release in EVs, biotinylated proteins were isolated from these fractions using streptavidin beads and analysed for the presence of the biotinylated fragments by western blot analysis using c-terminal antibody. Interestingly, we observed the L1CAM cleaved form (~70,~55 and~30 kDa) in L1CAM + EVs derived from both brain and sera, suggesting the ectodomain cleavage of full-length L1CAM, as previously reported [46,47]. Similar to our findings, L1CAM fragment of 55 kDa was also observed in EVs isolated from cell culture supernatants after cell surface biotinylation and L1 stimulation of neuroblastoma cells [56].…”

“…Neuronal enrichment of EVs (L1CAM + EVs) has been suggested to improve the signal to noise ratio and lower the detection threshold when assessing brain‐specific serum EV expression in several neurodegenerative diseases [29,43,55]. The 220‐kDa full‐length molecule L1CAM is proteolytically processed at different sites by different enzymes, leading to generation of differently sized cleaved products of ~140, ~80, ~70, ~55 and ~30 kDa, and serves as a substrate for regulating intramembrane proteolysis [46,56,57]. Ectodomain shedding of L1CAM yields a C‐terminal stub (L1‐32), which is retained in the plasma membrane [57], with the ectodomain cleavage of membrane molecules accelerated under pathological conditions involving neurodegeneration [58].…”

“…Lutz et al showed that the 70 kDa fragment generated from serine protease‐dependent cleavage of full‐length L1CAM at the plasma membrane was transported from the plasma membrane to a late endosomal compartment cleavage with alterations in the levels of the fragment that were found to be associated with neurodegeneration [56]. The 70 kDa L1CAM fragment can also be cleaved into a sumoylated ~30 kDa cytosolic fragment [46]. To detect the biotinylated fragment after its release in EVs, biotinylated proteins were isolated from these fractions using streptavidin beads and analysed for the presence of the biotinylated fragments by western blot analysis using c‐terminal antibody.…”

Neuroglian regulatesDrosophilaintestinal stem cell proliferation through enhanced signaling via the Epidermal Growth Factor Receptor

“…3A, C). In previous studies in the Drosophila nervous system, the human homolog of Nrg, hL1CAM, was found to rescue neurodevelopmental defects in Nrg mutants (Godenschwege et al, 2006;Kakad et al, 2018;Kudumala et al, 2013).…”