Cortactin-Src Kinase Signaling Pathway Is Involved in N-syndecan-dependent Neurite Outgrowth

Abstract

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrixbound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinaseactive fractions that bind to the cytosolic moiety of Nsyndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway. HB-GAM1 was initially isolated from neonatal rat brain as a neurite outgrowth-promoting protein, the expression of which in brain corresponds to the stage of rapid axonal growth (1). Molecular cloning of full-length cDNA identified a novel secretory sequence (2). The same cDNA sequence was reported for pleiotrophin, a protein suggested to be mitogen for fibroblastic cells (3, 4). The HB-GAM/pleiotrophin sequence shares approximately 50% homology with the midkine protein involved in retinoic acid-induced cell differentiation (5-7).The expression of HB-GAM in the axon pathways of the brain and in the basement membranes outside of brain (8 -9) and the neurite outgrowth-promoting property of HB-GAM in vitro (see Refs. 1 and 4) have suggested interaction of matrixassociated HB-GAM with a cell surface receptor. Furthermore, HB-GAM is expressed at the surface of developing muscle cells and is suggested to play a role in the development of nerve/ muscle contacts (10 -12). N-syndecan (syndecan-3) has recently been isolated from detergent extracts of perinatal rat brain as a receptor or coreceptor for HB-GAM using recombinant HB-GAM as an affinity matrix (13). N-syndecan is localized at the surface of neurites and their growth cones in rat primary neurons growing on HB-GAM-coated matrix in vitro (13). Furthermore, HB-GAM and N-syndecan are spatiotemporally co-expressed in developing rat brain (14).The cell surface N-syndecan interacts with HB-GAM through its heparan sulfate chains (15). This interaction is enhanced by assembly of the heparan sulfat...

Haptotactic Migration Induced by Midkine

“…Consistent with our data, Souttou et al reported that Erk1 and 2 and PI3-kinase are involved in PTN/HB-GAM-mediated cell proliferation (57). In addition, Src, JAK1, and 2 and ␤-catenin have been reported to be involved in PTN/HB-GAM signaling (58,59,60). A study on the possible involvement of these molecules in MK-induced cell migration is underway in our laboratory.…”

Molecular Mediators for Raft-dependent Endocytosis of Syndecan-1, a Highly Conserved, Multifunctional Receptor

“…The syndecan-1 cytoplasmic tail contains four absolutely conserved tyrosyl residues (Fig. 1) (14, 19, 20), and as just noted, the C1 domain of syndecan-3 can bind Src family kinases (32). Here, we found that ligand binding, but only with clustering, provoked tyrosine phosphorylation of the syndecan transmembrane and cytoplasmic domains of the FcR-Synd1 chimera (Fig.…”

“…A previous report indicated that an affinity column made from a Sepharose-linked peptide corresponding to the cytoplasmic tail of syndecan-3 (neural syndecan) was able to bind ␣-tubulin, Src family kinases, and cortactin from crude brain homogenates (32). Binding was attributed to the C1 domain of the syndecan-3 peptide (32). Here, to examine a possible association between ␣-tubulin and the syndecan-1 cytoplasmic tail in our cultured cells, we used co-immunoprecipitations.…”

Cortactin: coupling membrane dynamics to cortical actin assembly

“…In NIH3T3 ®broblasts, a 5 b 1 integrin-mediated cell adhesion and spreading on ®bronectin results in elevated cortactin tyrosine phosphorylation (Vuori and Ruoslahti, 1995), a condition where Src is activated following binding to activated focal adhesion kinase (FAK) (Schaller et al, 1994). In N18 neuroblastoma cells, cortactin is tyrosine phosphorylated following binding of N-syndecan (syndecan 3) with heparin-binding growth-associated molecule (HB-GAM), and both cortactin and Src copurify following a nity chromotography using the N-syndecan cytoplasmic domain (Kinnunen et al, 1998). Similarly, binding of CD44 to hyaluronic acid (HA) in ovarian tumor cells leads to activation and association of Src with the CD44 cytoplasmic domain and subsequent cortactin tyrosine phosphorylation (Bourguignon et al, 2001).…”