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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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Overexpression of IRS-58I267N (1–534) Cdc42Hs binding–defective mutant in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 I267N cDNA as described in the legend to Fig. 4. Cells were double-stained for IRS-58 and F-actin. Compare with Fig. 5 A for IRS-58 effects on Swiss 3T3 cells. F-actin (A), IRS-58 (B), and IRS-58 (green) with F-actin (red) staining with colocalization in blue (C). Colocalization signal of IRS-58 with F-actin alone (D). Bar, 10 μM.
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Figure 7: Overexpression of IRS-58I267N (1–534) Cdc42Hs binding–defective mutant in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 I267N cDNA as described in the legend to Fig. 4. Cells were double-stained for IRS-58 and F-actin. Compare with Fig. 5 A for IRS-58 effects on Swiss 3T3 cells. F-actin (A), IRS-58 (B), and IRS-58 (green) with F-actin (red) staining with colocalization in blue (C). Colocalization signal of IRS-58 with F-actin alone (D). Bar, 10 μM.

Mentions: Next we investigated the effects of deletion of COOH-terminal regions of IRS-58 (IRS-58Δ510–534 and IRS-58Δ143–534) and a Cdc42Hs binding–defective IRS-58I267N. Overexpression of the IRS-58Δ510–534 protein that lacks the area of heterogeneity between the human isoforms gave a more pronounced reorganization of the F-actin cytoskeleton, with most cells possessing numerous well-defined F-actin clusters (Fig. 6A and Fig. B). Stress fibers were present in some cells transfected with this mutant (IRS-58Δ510–534). The reorganization of the F-actin cytoskeleton caused by IRS-58 required amino acid residues 143–534 (Fig. 6C and Fig. D) since expression of IRS-58Δ143–534 did not cause loss of stress fibers or formation of actin clusters. Also, IRS-58Δ143–534 mutant did not induce the formation of peripheral plasma membrane structures; however, cells appeared to be more contracted. It is possible that the contracted morphology seen in cells overexpressing IRS-58Δ143–534 is caused by a thickening of stress fibers. Similar morphologies are seen when RhoA protein is microinjected into cells (Paterson et al. 1990). Overexpression of the mutant IRS-58I267N, which is unable to bind Cdc42Hs, failed to induce filopodia formation or F-actin clusters in most Swiss 3T3 cells (data not shown). However, in some cells expressing higher levels of IRS-58I267N F-actin became disorganized and small F-actin clusters were seen (Fig. 7). Strikingly, IRS-58 I267N did not colocalize with these F-actin clusters or other F-actin structures present within the cell (Fig. 7; also see Fig. 9 B).


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)

Overexpression of IRS-58I267N (1–534) Cdc42Hs binding–defective mutant in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 I267N cDNA as described in the legend to Fig. 4. Cells were double-stained for IRS-58 and F-actin. Compare with Fig. 5 A for IRS-58 effects on Swiss 3T3 cells. F-actin (A), IRS-58 (B), and IRS-58 (green) with F-actin (red) staining with colocalization in blue (C). Colocalization signal of IRS-58 with F-actin alone (D). Bar, 10 μM.
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Related In: Results  -  Collection

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Figure 7: Overexpression of IRS-58I267N (1–534) Cdc42Hs binding–defective mutant in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 I267N cDNA as described in the legend to Fig. 4. Cells were double-stained for IRS-58 and F-actin. Compare with Fig. 5 A for IRS-58 effects on Swiss 3T3 cells. F-actin (A), IRS-58 (B), and IRS-58 (green) with F-actin (red) staining with colocalization in blue (C). Colocalization signal of IRS-58 with F-actin alone (D). Bar, 10 μM.
Mentions: Next we investigated the effects of deletion of COOH-terminal regions of IRS-58 (IRS-58Δ510–534 and IRS-58Δ143–534) and a Cdc42Hs binding–defective IRS-58I267N. Overexpression of the IRS-58Δ510–534 protein that lacks the area of heterogeneity between the human isoforms gave a more pronounced reorganization of the F-actin cytoskeleton, with most cells possessing numerous well-defined F-actin clusters (Fig. 6A and Fig. B). Stress fibers were present in some cells transfected with this mutant (IRS-58Δ510–534). The reorganization of the F-actin cytoskeleton caused by IRS-58 required amino acid residues 143–534 (Fig. 6C and Fig. D) since expression of IRS-58Δ143–534 did not cause loss of stress fibers or formation of actin clusters. Also, IRS-58Δ143–534 mutant did not induce the formation of peripheral plasma membrane structures; however, cells appeared to be more contracted. It is possible that the contracted morphology seen in cells overexpressing IRS-58Δ143–534 is caused by a thickening of stress fibers. Similar morphologies are seen when RhoA protein is microinjected into cells (Paterson et al. 1990). Overexpression of the mutant IRS-58I267N, which is unable to bind Cdc42Hs, failed to induce filopodia formation or F-actin clusters in most Swiss 3T3 cells (data not shown). However, in some cells expressing higher levels of IRS-58I267N F-actin became disorganized and small F-actin clusters were seen (Fig. 7). Strikingly, IRS-58 I267N did not colocalize with these F-actin clusters or other F-actin structures present within the cell (Fig. 7; also see Fig. 9 B).

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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