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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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Endogenous IRS-58 colocalizes with F-actin in filopodia. (A) N1E-115 cells were transfected with IRS-58 cDNA or empty vector control, left for 18–24 h, and then stained with anti–IRS-58–specific monoclonal antibodies and phalloidin. (B) Serum-starved cells were double-stained with anti–IRS-58–specific monoclonal and phalloidin. Staining procedures were as described in Materials and Methods. Inserts in B shows a magnified region of the cell (position of magnification shown by dashed line box). Bars, 20 μM.
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Figure 11: Endogenous IRS-58 colocalizes with F-actin in filopodia. (A) N1E-115 cells were transfected with IRS-58 cDNA or empty vector control, left for 18–24 h, and then stained with anti–IRS-58–specific monoclonal antibodies and phalloidin. (B) Serum-starved cells were double-stained with anti–IRS-58–specific monoclonal and phalloidin. Staining procedures were as described in Materials and Methods. Inserts in B shows a magnified region of the cell (position of magnification shown by dashed line box). Bars, 20 μM.

Mentions: Finally, the effect of overexpression of the Cdc42Hs binding–defective mutation, IRS-58I267N, on N1E-115 cells was compared with wild-type IRS-58 (Fig. 9 B). IRS-58 induced neurite outgrowth, as described above, and localized throughout the neurite and with F-actin in filopodia. In contrast, IRS-58I267N did not induce neurite outgrowth, although in some cases cells were seen to elongate (Fig. 9 B, panel A, cell on right). IRS-58I267N was distributed throughout the cytoplasm in small aggregates, but was never found at the cell periphery and did not colocalize with F-actin structures (Fig. 9 B). Using monoclonal antibodies specific to IRS-58, we have found endogenous protein to colocalize with F-actin in filopodia in neuroblastoma cells (Fig. 11).


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)

Endogenous IRS-58 colocalizes with F-actin in filopodia. (A) N1E-115 cells were transfected with IRS-58 cDNA or empty vector control, left for 18–24 h, and then stained with anti–IRS-58–specific monoclonal antibodies and phalloidin. (B) Serum-starved cells were double-stained with anti–IRS-58–specific monoclonal and phalloidin. Staining procedures were as described in Materials and Methods. Inserts in B shows a magnified region of the cell (position of magnification shown by dashed line box). Bars, 20 μM.
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Related In: Results  -  Collection

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Figure 11: Endogenous IRS-58 colocalizes with F-actin in filopodia. (A) N1E-115 cells were transfected with IRS-58 cDNA or empty vector control, left for 18–24 h, and then stained with anti–IRS-58–specific monoclonal antibodies and phalloidin. (B) Serum-starved cells were double-stained with anti–IRS-58–specific monoclonal and phalloidin. Staining procedures were as described in Materials and Methods. Inserts in B shows a magnified region of the cell (position of magnification shown by dashed line box). Bars, 20 μM.
Mentions: Finally, the effect of overexpression of the Cdc42Hs binding–defective mutation, IRS-58I267N, on N1E-115 cells was compared with wild-type IRS-58 (Fig. 9 B). IRS-58 induced neurite outgrowth, as described above, and localized throughout the neurite and with F-actin in filopodia. In contrast, IRS-58I267N did not induce neurite outgrowth, although in some cases cells were seen to elongate (Fig. 9 B, panel A, cell on right). IRS-58I267N was distributed throughout the cytoplasm in small aggregates, but was never found at the cell periphery and did not colocalize with F-actin structures (Fig. 9 B). Using monoclonal antibodies specific to IRS-58, we have found endogenous protein to colocalize with F-actin in filopodia in neuroblastoma cells (Fig. 11).

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