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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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Localization of the Cdc42Hs-binding site of IRS-58. Different portions of IRS-58 were expressed as GST fusion proteins and either dot-blotted or Western-transferred after SDS-PAGE onto nitrocellulose and then probed with Cdc42HsL61 as described in the legend to Fig. 2. (A) Deletion mutants coming in from the 3′ end using restriction sites. Lane 1, amino acid residues 1–534; lane 2, 1–432; lane 3, 1–358; lane 4, 1–142; lane 5, 142–358; lane 6, GST. Rows a–c, ∼30, 15, and 1 μg protein, respectively. (B–D) Shown in parallel with the overlay blot and corresponding Coomassie-stained protein gel. (B) PCR was used to generate minimal binding fragments (amino acid residues 202–305 and 266–305). Lanes 1 and 1′, GST 25 μg; lanes 2 and 2′, IRS-58 (202–305) 45 μg; lanes 3 and 3′, IRS-58 (266–305) 35 μg. (C) Point mutations were made into the minimal IRS-58 (267–305) Cdc42Hs-binding fragment: NSDA, NSDP, and IPDP. Lanes 1 and 1′, IRS-58 (267–305) 60 μg; lanes 2 and 2′, GST 2 μg; lanes 3 and 3′, IRS-58I267N/P270A 55 μg; lanes 4 and 4′, IRS-58I267N 45 μg; lanes 5 and 5′, IRS-58 S268P 45 μg. (D) Lanes 1 and 1′, GST 30 μg; lanes 2 and 2′, IRS-58 (1–534) 60 μg; lanes 3 and 3′, IRS-58I267N (1–534) 45 μg; lanes 4 and 4′, IRS-58 (1–266) 40 μg; lanes 5 and 5′, IRS-58 (306–534) 20 μg. (E) A summary of binding analysis, showing the fragments of IRS-58 examined and the domain positions. Point mutations are shown on a expanded scale of 40–amino acid residues. The polyproline sequence is underlined. 1 indicates that Cdc42H5 binds much more weakly to the 40–amino acid residue fragment than to the larger polypeptides.
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Figure 3: Localization of the Cdc42Hs-binding site of IRS-58. Different portions of IRS-58 were expressed as GST fusion proteins and either dot-blotted or Western-transferred after SDS-PAGE onto nitrocellulose and then probed with Cdc42HsL61 as described in the legend to Fig. 2. (A) Deletion mutants coming in from the 3′ end using restriction sites. Lane 1, amino acid residues 1–534; lane 2, 1–432; lane 3, 1–358; lane 4, 1–142; lane 5, 142–358; lane 6, GST. Rows a–c, ∼30, 15, and 1 μg protein, respectively. (B–D) Shown in parallel with the overlay blot and corresponding Coomassie-stained protein gel. (B) PCR was used to generate minimal binding fragments (amino acid residues 202–305 and 266–305). Lanes 1 and 1′, GST 25 μg; lanes 2 and 2′, IRS-58 (202–305) 45 μg; lanes 3 and 3′, IRS-58 (266–305) 35 μg. (C) Point mutations were made into the minimal IRS-58 (267–305) Cdc42Hs-binding fragment: NSDA, NSDP, and IPDP. Lanes 1 and 1′, IRS-58 (267–305) 60 μg; lanes 2 and 2′, GST 2 μg; lanes 3 and 3′, IRS-58I267N/P270A 55 μg; lanes 4 and 4′, IRS-58I267N 45 μg; lanes 5 and 5′, IRS-58 S268P 45 μg. (D) Lanes 1 and 1′, GST 30 μg; lanes 2 and 2′, IRS-58 (1–534) 60 μg; lanes 3 and 3′, IRS-58I267N (1–534) 45 μg; lanes 4 and 4′, IRS-58 (1–266) 40 μg; lanes 5 and 5′, IRS-58 (306–534) 20 μg. (E) A summary of binding analysis, showing the fragments of IRS-58 examined and the domain positions. Point mutations are shown on a expanded scale of 40–amino acid residues. The polyproline sequence is underlined. 1 indicates that Cdc42H5 binds much more weakly to the 40–amino acid residue fragment than to the larger polypeptides.

Mentions: To localize the Cdc42Hs-binding site, COOH-terminal deletions were made using unique restriction sites present in the full-length pGEX–IRS-58 construct. The proteins encoded by these constructs were used in binding experiments with Cdc42HsQ61L [γ-32P]GTP as probe. Cdc42Hs binding was lost between deletions three and four, suggesting that amino acid residues 142–358 were essential for this activity (Fig. 3A and Fig. e). Using PCR cloning a 40–amino acid residue fragment was generated that could bind Cdc42Hs (amino acid residues 266–305; Fig. 3B and Fig. C). However, Cdc42Hs binding to this fragment was significantly weaker than to amino acid residues 202–305 (∼10–20-fold). Cdc42Hs did not bind to amino acid residues 304–534 showing that there were no COOH-terminal Cdc42Hs-binding sites. The sequence isoleucine, serine, andy amino acid residue, proline at the beginning of this 40–amino acid residue fragment has similarity to the Cdc42/Rac interactive binding (CRIB) motif. (The CRIB motif [Burbelo et al. 1995], a sequence of 16 amino acid residues, a smaller version of a G protein–binding domain first identified in the proteins PAK and ACK, has been suggested as a consensus for Cdc42Hs/Rac1 binding.) To determine whether the conserved I, P, and/or S were required for binding, mutations were made in these residues (N267, P268, and N267/A270). Binding assays showed that these amino acid residues were required, as Cdc42Hs binding was lost in these mutant proteins (Fig. 3 C). Introduction of the N267 mutation into a full-length IRS-58 construct also resulted in a loss of Cdc42 binding (Fig. 3 D). These experiments localize the Cdc42Hs-binding site to a position overlapping with the polyproline-rich sequence, which may be an SH3 domain–binding site (Fig. 1). A summary of these binding experiments is presented in Fig. 3 E.


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)

Localization of the Cdc42Hs-binding site of IRS-58. Different portions of IRS-58 were expressed as GST fusion proteins and either dot-blotted or Western-transferred after SDS-PAGE onto nitrocellulose and then probed with Cdc42HsL61 as described in the legend to Fig. 2. (A) Deletion mutants coming in from the 3′ end using restriction sites. Lane 1, amino acid residues 1–534; lane 2, 1–432; lane 3, 1–358; lane 4, 1–142; lane 5, 142–358; lane 6, GST. Rows a–c, ∼30, 15, and 1 μg protein, respectively. (B–D) Shown in parallel with the overlay blot and corresponding Coomassie-stained protein gel. (B) PCR was used to generate minimal binding fragments (amino acid residues 202–305 and 266–305). Lanes 1 and 1′, GST 25 μg; lanes 2 and 2′, IRS-58 (202–305) 45 μg; lanes 3 and 3′, IRS-58 (266–305) 35 μg. (C) Point mutations were made into the minimal IRS-58 (267–305) Cdc42Hs-binding fragment: NSDA, NSDP, and IPDP. Lanes 1 and 1′, IRS-58 (267–305) 60 μg; lanes 2 and 2′, GST 2 μg; lanes 3 and 3′, IRS-58I267N/P270A 55 μg; lanes 4 and 4′, IRS-58I267N 45 μg; lanes 5 and 5′, IRS-58 S268P 45 μg. (D) Lanes 1 and 1′, GST 30 μg; lanes 2 and 2′, IRS-58 (1–534) 60 μg; lanes 3 and 3′, IRS-58I267N (1–534) 45 μg; lanes 4 and 4′, IRS-58 (1–266) 40 μg; lanes 5 and 5′, IRS-58 (306–534) 20 μg. (E) A summary of binding analysis, showing the fragments of IRS-58 examined and the domain positions. Point mutations are shown on a expanded scale of 40–amino acid residues. The polyproline sequence is underlined. 1 indicates that Cdc42H5 binds much more weakly to the 40–amino acid residue fragment than to the larger polypeptides.
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Figure 3: Localization of the Cdc42Hs-binding site of IRS-58. Different portions of IRS-58 were expressed as GST fusion proteins and either dot-blotted or Western-transferred after SDS-PAGE onto nitrocellulose and then probed with Cdc42HsL61 as described in the legend to Fig. 2. (A) Deletion mutants coming in from the 3′ end using restriction sites. Lane 1, amino acid residues 1–534; lane 2, 1–432; lane 3, 1–358; lane 4, 1–142; lane 5, 142–358; lane 6, GST. Rows a–c, ∼30, 15, and 1 μg protein, respectively. (B–D) Shown in parallel with the overlay blot and corresponding Coomassie-stained protein gel. (B) PCR was used to generate minimal binding fragments (amino acid residues 202–305 and 266–305). Lanes 1 and 1′, GST 25 μg; lanes 2 and 2′, IRS-58 (202–305) 45 μg; lanes 3 and 3′, IRS-58 (266–305) 35 μg. (C) Point mutations were made into the minimal IRS-58 (267–305) Cdc42Hs-binding fragment: NSDA, NSDP, and IPDP. Lanes 1 and 1′, IRS-58 (267–305) 60 μg; lanes 2 and 2′, GST 2 μg; lanes 3 and 3′, IRS-58I267N/P270A 55 μg; lanes 4 and 4′, IRS-58I267N 45 μg; lanes 5 and 5′, IRS-58 S268P 45 μg. (D) Lanes 1 and 1′, GST 30 μg; lanes 2 and 2′, IRS-58 (1–534) 60 μg; lanes 3 and 3′, IRS-58I267N (1–534) 45 μg; lanes 4 and 4′, IRS-58 (1–266) 40 μg; lanes 5 and 5′, IRS-58 (306–534) 20 μg. (E) A summary of binding analysis, showing the fragments of IRS-58 examined and the domain positions. Point mutations are shown on a expanded scale of 40–amino acid residues. The polyproline sequence is underlined. 1 indicates that Cdc42H5 binds much more weakly to the 40–amino acid residue fragment than to the larger polypeptides.
Mentions: To localize the Cdc42Hs-binding site, COOH-terminal deletions were made using unique restriction sites present in the full-length pGEX–IRS-58 construct. The proteins encoded by these constructs were used in binding experiments with Cdc42HsQ61L [γ-32P]GTP as probe. Cdc42Hs binding was lost between deletions three and four, suggesting that amino acid residues 142–358 were essential for this activity (Fig. 3A and Fig. e). Using PCR cloning a 40–amino acid residue fragment was generated that could bind Cdc42Hs (amino acid residues 266–305; Fig. 3B and Fig. C). However, Cdc42Hs binding to this fragment was significantly weaker than to amino acid residues 202–305 (∼10–20-fold). Cdc42Hs did not bind to amino acid residues 304–534 showing that there were no COOH-terminal Cdc42Hs-binding sites. The sequence isoleucine, serine, andy amino acid residue, proline at the beginning of this 40–amino acid residue fragment has similarity to the Cdc42/Rac interactive binding (CRIB) motif. (The CRIB motif [Burbelo et al. 1995], a sequence of 16 amino acid residues, a smaller version of a G protein–binding domain first identified in the proteins PAK and ACK, has been suggested as a consensus for Cdc42Hs/Rac1 binding.) To determine whether the conserved I, P, and/or S were required for binding, mutations were made in these residues (N267, P268, and N267/A270). Binding assays showed that these amino acid residues were required, as Cdc42Hs binding was lost in these mutant proteins (Fig. 3 C). Introduction of the N267 mutation into a full-length IRS-58 construct also resulted in a loss of Cdc42 binding (Fig. 3 D). These experiments localize the Cdc42Hs-binding site to a position overlapping with the polyproline-rich sequence, which may be an SH3 domain–binding site (Fig. 1). A summary of these binding experiments is presented in Fig. 3 E.

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