Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth.
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 matrix-bound 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 kinase-active fractions that bind to the cytosolic moiety of N-syndecan. 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.
Kinnunen
T
T
Laboratory of Molecular Neurobiology, Institute of Biotechnology, University of Helsinki, P. O. Box 56, 00014 Helsinki, Finland.
Kaksonen
M
M
Saarinen
J
J
Kalkkinen
N
N
Peng
H B
HB
Rauvala
H
H
D000595:Amino Acid Sequence; D000818:Animals; D051356:Cortactin; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary; D005109:Extracellular Matrix / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016501:Neurites; D010766:Phosphorylation; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D053670:Syndecan-3; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D051356:Cortactin; C495870:Cttn protein, rat; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C508303:Sdc3 protein, rat; D053670:Syndecan-3; D014404:Tubulin; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases