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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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Cdc42Hs, Rac1, and RhoA interaction with IRS-58. Cdc42HsL61, Rac1L61, and RhoAL63 were labeled with [γ-32P]GTP by exchange in the presence of EDTA and used to probe IRS-58–GST fusion protein (142–363 amino acid residues) immobilized on nitrocellulose filter. (A) Lanes 1–3, IRS-58, 30, 10, and 1 μg total protein; lane 4, GST 25 μg; lane 5, βPAK (37–137 amino acid residues) 27.5 μg (for Cdc42Hs and Rac1) and p190 GAP domain 20 μg (for RhoA). (B) Cdc42Hs wild-type was labeled as described above and either used immediately or incubated for 60 min before use. Due to the high intrinsic GTPase activity of Cdc42Hs the α-labeled GTP probe becomes GDP. LANES 1 and 2, IRS-58–GST fusion protein (202–305 amino acid residues) 1 μg and 0.1 μg. (C) Lanes 1–3, IRS-58 fusion protein (202–305) 10, 1, and 0.1 μg, was probed with L61, L61C40, and L61A37; lane 4, GST 10 μg; lanes 5 and 6, purified PAK fraction from neutrophil cytosol extract 50 μg and 10 μg total protein.
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Figure 2: Cdc42Hs, Rac1, and RhoA interaction with IRS-58. Cdc42HsL61, Rac1L61, and RhoAL63 were labeled with [γ-32P]GTP by exchange in the presence of EDTA and used to probe IRS-58–GST fusion protein (142–363 amino acid residues) immobilized on nitrocellulose filter. (A) Lanes 1–3, IRS-58, 30, 10, and 1 μg total protein; lane 4, GST 25 μg; lane 5, βPAK (37–137 amino acid residues) 27.5 μg (for Cdc42Hs and Rac1) and p190 GAP domain 20 μg (for RhoA). (B) Cdc42Hs wild-type was labeled as described above and either used immediately or incubated for 60 min before use. Due to the high intrinsic GTPase activity of Cdc42Hs the α-labeled GTP probe becomes GDP. LANES 1 and 2, IRS-58–GST fusion protein (202–305 amino acid residues) 1 μg and 0.1 μg. (C) Lanes 1–3, IRS-58 fusion protein (202–305) 10, 1, and 0.1 μg, was probed with L61, L61C40, and L61A37; lane 4, GST 10 μg; lanes 5 and 6, purified PAK fraction from neutrophil cytosol extract 50 μg and 10 μg total protein.

Mentions: To determine the Rho family (Cdc42Hs, Rac1, and RhoA) binding specificity of IRS-58, purified GST fusion protein was dot-blotted onto nitrocellulose and probed with Cdc42HsQ61L, Rac1Q61L, and RhoAG14V labeled with [γ-32P]GTP. PAK and p190 GAP domain were used as positive controls. This analysis showed that IRS-58 is a Cdc42Hs-specific binding protein, as it did not bind Rac1 or RhoA (Fig. 2 A). Cdc42Hs needs to be in a GTP-bound form to interact with IRS-58, as we could not detect binding to Cdc42Hs-GDP (Fig. 2 B). The binding strength (cpm/μg protein) of Cdc42Hs to IRS-58 and PAK is similar (data not shown). Unlike PAK and WASP, IRS-58 binds the Cdc42Hs C40 mutation, but does not bind A37 (Fig. 2 C). However, the C40 mutation did reduce the strength of interaction between Cdc42Hs and IRS-58.


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)

Cdc42Hs, Rac1, and RhoA interaction with IRS-58. Cdc42HsL61, Rac1L61, and RhoAL63 were labeled with [γ-32P]GTP by exchange in the presence of EDTA and used to probe IRS-58–GST fusion protein (142–363 amino acid residues) immobilized on nitrocellulose filter. (A) Lanes 1–3, IRS-58, 30, 10, and 1 μg total protein; lane 4, GST 25 μg; lane 5, βPAK (37–137 amino acid residues) 27.5 μg (for Cdc42Hs and Rac1) and p190 GAP domain 20 μg (for RhoA). (B) Cdc42Hs wild-type was labeled as described above and either used immediately or incubated for 60 min before use. Due to the high intrinsic GTPase activity of Cdc42Hs the α-labeled GTP probe becomes GDP. LANES 1 and 2, IRS-58–GST fusion protein (202–305 amino acid residues) 1 μg and 0.1 μg. (C) Lanes 1–3, IRS-58 fusion protein (202–305) 10, 1, and 0.1 μg, was probed with L61, L61C40, and L61A37; lane 4, GST 10 μg; lanes 5 and 6, purified PAK fraction from neutrophil cytosol extract 50 μg and 10 μg total protein.
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Figure 2: Cdc42Hs, Rac1, and RhoA interaction with IRS-58. Cdc42HsL61, Rac1L61, and RhoAL63 were labeled with [γ-32P]GTP by exchange in the presence of EDTA and used to probe IRS-58–GST fusion protein (142–363 amino acid residues) immobilized on nitrocellulose filter. (A) Lanes 1–3, IRS-58, 30, 10, and 1 μg total protein; lane 4, GST 25 μg; lane 5, βPAK (37–137 amino acid residues) 27.5 μg (for Cdc42Hs and Rac1) and p190 GAP domain 20 μg (for RhoA). (B) Cdc42Hs wild-type was labeled as described above and either used immediately or incubated for 60 min before use. Due to the high intrinsic GTPase activity of Cdc42Hs the α-labeled GTP probe becomes GDP. LANES 1 and 2, IRS-58–GST fusion protein (202–305 amino acid residues) 1 μg and 0.1 μg. (C) Lanes 1–3, IRS-58 fusion protein (202–305) 10, 1, and 0.1 μg, was probed with L61, L61C40, and L61A37; lane 4, GST 10 μg; lanes 5 and 6, purified PAK fraction from neutrophil cytosol extract 50 μg and 10 μg total protein.
Mentions: To determine the Rho family (Cdc42Hs, Rac1, and RhoA) binding specificity of IRS-58, purified GST fusion protein was dot-blotted onto nitrocellulose and probed with Cdc42HsQ61L, Rac1Q61L, and RhoAG14V labeled with [γ-32P]GTP. PAK and p190 GAP domain were used as positive controls. This analysis showed that IRS-58 is a Cdc42Hs-specific binding protein, as it did not bind Rac1 or RhoA (Fig. 2 A). Cdc42Hs needs to be in a GTP-bound form to interact with IRS-58, as we could not detect binding to Cdc42Hs-GDP (Fig. 2 B). The binding strength (cpm/μg protein) of Cdc42Hs to IRS-58 and PAK is similar (data not shown). Unlike PAK and WASP, IRS-58 binds the Cdc42Hs C40 mutation, but does not bind A37 (Fig. 2 C). However, the C40 mutation did reduce the strength of interaction between Cdc42Hs and IRS-58.

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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