GSK3β/Axin-1/β-Catenin Complex Is Involved in Semaphorin3A Signaling

Abstract

Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3␤ (GSK3␤). To delineate how GSK3␤ mediates Sema3A signaling, we here determined the substrates of GSK3␤ involved. Introduction of either GSK3␤ mutants, GSK3␤-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3␤. The phosphorylation of Axin-1 by GSK3␤ accelerates the association of Axin-1 with ␤-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of ␤-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3␤, Axin-1, and ␤-catenin in the growth cones. The increase of ␤-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or ␤-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3␤/Axin-1/␤-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.

A functional coupling between CRMP1 and Nav1.7 for retrograde propagation of Semaphorin3A signaling

“…The colocalization of PlexA4 and TrkA in both the growth cones and distal axons was suppressed by nimodipine or MDC (Figs 1 and 2). This finding suggests that the colocalization of these two molecules occurred downstream of Sema3A-induced Ca 2+ -dependent endocytosis (Castellani et al, 2004;Fournier et al, 2000;Hida et al, 2012;Tojima et al, 2010). Notably, at 5 min after stimulation, the increased colocalization in the growth cones returned to the basal level and was followed by an increase in colocalization in distal axons (Figs 1 and 2).…”

“…The initial signaling event of Sema3A involves endocytosis of Sema3A and the formation of the GSK3β-Axin-1-β-catenin complex (Hida et al, 2012). Furthermore, we have recently provided evidence that an interaction between PlexA4 and TrkA occurs in response to Sema3A, which is essential for retrograde Sema3A signaling (Yamashita et al, 2016b).…”

“…The sense strands for the two 19-oligonucleotide Na v 1.7 siRNAs were synthesized with the following sequences: siRNAa: 5′-GUUCUUAUCU-GCCUCAAUA-3′; siRNAb: 5′-CUCACUCGUGUUAAUAAGA-3′; siRNAc: 5′-CACCAGGUGUUUAGUAUGA-3′ (#SC149784A, Santa Cruz, CA). The siRNAs against Na v 1.6 and Na v 1.7, mixed to use the same amount of the three siRNAs for each of the corresponding target genes, were introduced into cultured dissociated mouse DRG neurons using DharmaFECT-3 (Thermo Scientific, Hudson, NH, USA) according to the manufacturer's protocol (Hida et al, 2012). Cultured dissociated mouse DRG neurons were transfected with each siRNA (final concentration, 50 nM) 10-12 h after the start of cultures.…”

Amino- and carboxyl-terminal domains of Filamin-A interact with CRMP1 to mediate Sema3A signalling

“…After 4 h incubation at 37°C, the medium was replaced with the fresh mouse DRG medium. The DRG explants were further incubated for 2 to 3 days 49 .…”

Regulation of dendritic development by semaphorin 3A through novel intracellular remote signaling

“…60 Sema3A-induced growth cone collapse is suppressed by introduction of GSK3b mutants, GSK3b-R96A, L128A, or K85M, into DRG neurons, indicating that unprimed and primed substrates are involved in stimulating endocytosis of the growth cone plasma membrane. 63 Either Axin-1 or b-catenin RNAi knockdown suppresses the internalization of venus-tagged Sema3A. In addition, local treatment by monodancylcadaverin, an inhibitor of clathrin-dependent endocytosis, at the distal axon suppressed growth cone and distal axon distribution of venus-tagged Sema3A.…”