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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.

Show MeSH
IRS-58 phenotypes in N1E-115 cells produced by deletion and mutation. A schematic of IRS-58 is shown in which protein–protein interactions are summarized. The insulin receptor (IR) phosphorylates the NH2 terminus at three potential sites (Okamura-Oho et al. 1999). A polyproline sequence lies within the Cdc42Hs-binding site. IRS-58 possesses at least two other protein–protein interaction sites, an SH3 domain, and a WW-binding domain. Atrophin-1 and BA1I have been shown to bind to the SH3 domain. IRS-58 induces neurite outgrowth with high complexity and hyperactivity which is increased in *IRS-58 (1–510) mutant. IRS-58 (1–390) induces neurite extension with little complexity, suggesting that the region between amino acid residues 391–526 is responsible for the complexity. 1IRS-58I267N and 2IRS-58 (1–142) do not induce neurite outgrowth. The 1IRS-58I267N mutant induces cell elongation in some cells but no longer colocalizes with F-actin, suggesting that interaction with Cdc42Hs is required for F-actin association. Similar results were observed in Swiss 3T3 cells in Fig. 7.
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Figure 12: IRS-58 phenotypes in N1E-115 cells produced by deletion and mutation. A schematic of IRS-58 is shown in which protein–protein interactions are summarized. The insulin receptor (IR) phosphorylates the NH2 terminus at three potential sites (Okamura-Oho et al. 1999). A polyproline sequence lies within the Cdc42Hs-binding site. IRS-58 possesses at least two other protein–protein interaction sites, an SH3 domain, and a WW-binding domain. Atrophin-1 and BA1I have been shown to bind to the SH3 domain. IRS-58 induces neurite outgrowth with high complexity and hyperactivity which is increased in *IRS-58 (1–510) mutant. IRS-58 (1–390) induces neurite extension with little complexity, suggesting that the region between amino acid residues 391–526 is responsible for the complexity. 1IRS-58I267N and 2IRS-58 (1–142) do not induce neurite outgrowth. The 1IRS-58I267N mutant induces cell elongation in some cells but no longer colocalizes with F-actin, suggesting that interaction with Cdc42Hs is required for F-actin association. Similar results were observed in Swiss 3T3 cells in Fig. 7.

Mentions: A summary of the IRS-58 and mutant phenotypes obtained by cell transfection in N1E-115 cells is presented in Fig. 12.


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)

IRS-58 phenotypes in N1E-115 cells produced by deletion and mutation. A schematic of IRS-58 is shown in which protein–protein interactions are summarized. The insulin receptor (IR) phosphorylates the NH2 terminus at three potential sites (Okamura-Oho et al. 1999). A polyproline sequence lies within the Cdc42Hs-binding site. IRS-58 possesses at least two other protein–protein interaction sites, an SH3 domain, and a WW-binding domain. Atrophin-1 and BA1I have been shown to bind to the SH3 domain. IRS-58 induces neurite outgrowth with high complexity and hyperactivity which is increased in *IRS-58 (1–510) mutant. IRS-58 (1–390) induces neurite extension with little complexity, suggesting that the region between amino acid residues 391–526 is responsible for the complexity. 1IRS-58I267N and 2IRS-58 (1–142) do not induce neurite outgrowth. The 1IRS-58I267N mutant induces cell elongation in some cells but no longer colocalizes with F-actin, suggesting that interaction with Cdc42Hs is required for F-actin association. Similar results were observed in Swiss 3T3 cells in Fig. 7.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195994&req=5

Figure 12: IRS-58 phenotypes in N1E-115 cells produced by deletion and mutation. A schematic of IRS-58 is shown in which protein–protein interactions are summarized. The insulin receptor (IR) phosphorylates the NH2 terminus at three potential sites (Okamura-Oho et al. 1999). A polyproline sequence lies within the Cdc42Hs-binding site. IRS-58 possesses at least two other protein–protein interaction sites, an SH3 domain, and a WW-binding domain. Atrophin-1 and BA1I have been shown to bind to the SH3 domain. IRS-58 induces neurite outgrowth with high complexity and hyperactivity which is increased in *IRS-58 (1–510) mutant. IRS-58 (1–390) induces neurite extension with little complexity, suggesting that the region between amino acid residues 391–526 is responsible for the complexity. 1IRS-58I267N and 2IRS-58 (1–142) do not induce neurite outgrowth. The 1IRS-58I267N mutant induces cell elongation in some cells but no longer colocalizes with F-actin, suggesting that interaction with Cdc42Hs is required for F-actin association. Similar results were observed in Swiss 3T3 cells in Fig. 7.
Mentions: A summary of the IRS-58 and mutant phenotypes obtained by cell transfection in N1E-115 cells is presented in Fig. 12.

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