Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin.
Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.
Govind
S
S
Department of Neurochemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom.
Kozma
R
R
Monfries
C
C
Lim
L
L
Ahmed
S
S
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002460:Cell Line; D048429:Cell Size; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D003599:Cytoskeleton / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D051379:Mice; D008845:Microinjections; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; C119539:BAIAP2 protein, human; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein