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Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin.

Govind S, Kozma R, Monfries C, Lim L, Ahmed S - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom.

ABSTRACT
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.

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Primary structure and expression of IRS-58. (A) The Bgl2 fragment of IRS-58 isolated from the two-hybrid screen was cloned into the Bluescript vector and sequenced on both strands with an automated ABI sequencer. The derived protein sequence is shown with three putative protein–protein interaction domains as reported previously (Yeh et al. 1996, Yeh et al. 1998). SH3-binding domain is in bold type and underlined; SH3 domain is underlined; and the WW-binding domain is in bold. (B) BLAST searches of three protein domains: an SH3-binding site, an SH3-binding domain, and a WW domain–binding site. (C) A multiple tissue mRNA blot was probed with IRS-58 cDNA and processed, as described in Materials and Methods, and exposed to film for 48 h.
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Figure 1: Primary structure and expression of IRS-58. (A) The Bgl2 fragment of IRS-58 isolated from the two-hybrid screen was cloned into the Bluescript vector and sequenced on both strands with an automated ABI sequencer. The derived protein sequence is shown with three putative protein–protein interaction domains as reported previously (Yeh et al. 1996, Yeh et al. 1998). SH3-binding domain is in bold type and underlined; SH3 domain is underlined; and the WW-binding domain is in bold. (B) BLAST searches of three protein domains: an SH3-binding site, an SH3-binding domain, and a WW domain–binding site. (C) A multiple tissue mRNA blot was probed with IRS-58 cDNA and processed, as described in Materials and Methods, and exposed to film for 48 h.

Mentions: BLAST searches with IRS-58 show the presence of three putative domains, a polyproline-rich sequence (a potential SH3-binding site), a PPXY motif (a WW domain–binding site), and an SH3 domain (amino acid residues 400–460; Fig. 1a and Fig. b). The polyproline stretch (amino acid residues 267–285) also aligned with channel proteins (data not shown). IRS-58 did not have clear homology with Cdc42Hs-binding sites of ACK, PAK, WASP, myotonic dystrophy kinase–related Cdc42-binding kinase (MRCK), IQGAP, or CIP4, suggesting the presence of a novel binding site. Northern analysis, using a cDNA probe of IRS-58 encoding amino acid residues 1–137 of IRS-58, was used to examine its expression pattern in human tissues. Two signals were detected at 2.4 and 3.5 kb (Fig. 1 C). The smaller message was ubiquitously expressed, but present at higher levels in placenta, liver, and kidney. The 3.5-kb message was brain enriched and possibly brain specific (Fig. 1 C).


Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin.

Govind S, Kozma R, Monfries C, Lim L, Ahmed S - J. Cell Biol. (2001)

Primary structure and expression of IRS-58. (A) The Bgl2 fragment of IRS-58 isolated from the two-hybrid screen was cloned into the Bluescript vector and sequenced on both strands with an automated ABI sequencer. The derived protein sequence is shown with three putative protein–protein interaction domains as reported previously (Yeh et al. 1996, Yeh et al. 1998). SH3-binding domain is in bold type and underlined; SH3 domain is underlined; and the WW-binding domain is in bold. (B) BLAST searches of three protein domains: an SH3-binding site, an SH3-binding domain, and a WW domain–binding site. (C) A multiple tissue mRNA blot was probed with IRS-58 cDNA and processed, as described in Materials and Methods, and exposed to film for 48 h.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195994&req=5

Figure 1: Primary structure and expression of IRS-58. (A) The Bgl2 fragment of IRS-58 isolated from the two-hybrid screen was cloned into the Bluescript vector and sequenced on both strands with an automated ABI sequencer. The derived protein sequence is shown with three putative protein–protein interaction domains as reported previously (Yeh et al. 1996, Yeh et al. 1998). SH3-binding domain is in bold type and underlined; SH3 domain is underlined; and the WW-binding domain is in bold. (B) BLAST searches of three protein domains: an SH3-binding site, an SH3-binding domain, and a WW domain–binding site. (C) A multiple tissue mRNA blot was probed with IRS-58 cDNA and processed, as described in Materials and Methods, and exposed to film for 48 h.
Mentions: BLAST searches with IRS-58 show the presence of three putative domains, a polyproline-rich sequence (a potential SH3-binding site), a PPXY motif (a WW domain–binding site), and an SH3 domain (amino acid residues 400–460; Fig. 1a and Fig. b). The polyproline stretch (amino acid residues 267–285) also aligned with channel proteins (data not shown). IRS-58 did not have clear homology with Cdc42Hs-binding sites of ACK, PAK, WASP, myotonic dystrophy kinase–related Cdc42-binding kinase (MRCK), IQGAP, or CIP4, suggesting the presence of a novel binding site. Northern analysis, using a cDNA probe of IRS-58 encoding amino acid residues 1–137 of IRS-58, was used to examine its expression pattern in human tissues. Two signals were detected at 2.4 and 3.5 kb (Fig. 1 C). The smaller message was ubiquitously expressed, but present at higher levels in placenta, liver, and kidney. The 3.5-kb message was brain enriched and possibly brain specific (Fig. 1 C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom.

ABSTRACT
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.

Show MeSH