No abstract

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885-17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N-and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80 -110 nM range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80 -90% by heparin (2 g/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N-or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.In previous studies we biochemically characterized a major heparan sulfate proteoglycan of nervous tissue (1, 2) that we later cloned and identified as the rat homologue of glypican-1 (3), the initial member of a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that is currently composed of six vertebrate proteins. High levels of glypican-1 mRNA are present in brain and skeletal muscle, and in situ hybridization histochemistry demonstrated that glypican-1 mRNA is especially prominent in cerebellar granule cells, large motor neurons in the brain stem, and CA3 pyramidal cells of the hippocampus (4). These results and parallel immunocytochemical studies indicate that glypican-1 is predominantly a neuronal product in the late embryonic and postnatal rat nervous system. Based on genetic studies, it would appear that the glypicans play a significant role in cell growth and development. Dally, the Drosophila homologue of glypican-1, is required for the control of cell division in the developing visual system and for morphogenesis of other tissues (5), and the human homologue of glypican-3/OCI-5 (GPC3) is found to be mutated in pat...

Apoptosis is an evolutionarily conserved process that is critical for tissue homeostasis and development including sex determination in essentially all multicellular organisms. Here, we report the cloning of an ankyrin repeat-containing protein, termed F1A␣, in a yeast twohybrid screen using the cytoplasmic domain of Fas (CD95/APO-1) as bait. Amino acid sequence analysis indicates that F1A␣ has extensive homology to the sexdetermining protein FEM-1 of the Caenorhabditis elegans, which is required for the development of all aspects of the male phenotype. F1A␣ associates with the cytoplasmic domains of Fas and tumor necrosis factor receptor 1, two prototype members of the "death receptor" family. The F1A␣ protein also oligomerizes. Overexpression of F1A␣ induces apoptosis in mammalian cells, and co-expression of Bcl-XL or the dominant negative mutants of either FADD or caspase-9 blocks this effect. Deletion analysis revealed the center region of F1A␣, including a cluster of five ankyrin repeats to be necessary and sufficient for maximum apoptotic activity, and the N-terminal region appears to regulate negatively this activity. Furthermore, F1A␣ is cleaved by a caspase-3-like protease at Asp 342 , and the cleavage-resistant mutant is unable to induce apoptosis upon overexpression.

We have studied the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, on the regulation of apoptosis in the human neuroblastoma cell line, SH-SY5Y. H-7 (20 -100 M) induced apoptosis in these cells characterized by DNA fragmentation and chromatin condensation. Immunoblot analyses were performed with specific antibody against BCL-2, BCL-X S/L , BAX, JUNB, c-JUN, ICH-1 L , c-FOS, RB, CDK-2, and p53. H-7 treatment did not significantly alter the level of these proteins with the exception of p53. H-7, but not staurosporine, caused a dramatic nuclear accumulation of p53. The kinetics of nuclear accumulation of p53 correlates well with the kinetics of induction of apoptosis. The effect of H-7 was further assessed in a group of human cell lines. Only cell lines harboring the wild-type p53 gene were responsive to the stimulatory effect of H-7 on nuclear accumulation of p53. Furthermore, cell lines carrying a mutated p53 gene were resistant to the cytotoxic effect of H-7. The ability of H-7 in mediating apoptosis in the SH-SY5Y line expressing a dominant negative mutant of p53 was significantly diminished. Taken together, these data strongly suggest that a p53-dependent mechanism contributes to the cytotoxicity of H-7 in human neuroblastoma cells.