Identification of the binding partners for flightless I, a novel protein bridging the leucine-rich repeat and the gelsolin superfamilies

Yu Tsueng Liu, Helen L. Yin

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63 Citations (Scopus)

Abstract

Flightless-I (fliI) is a novel member of the gelsolin family that is important for actin organization during Drosophila embryogenesis and myogenesis. Drosophila fliI and the human homolog FLI both contain the classic gelsolin 6-fold segmental repeats and an amino-terminal extension of 16 tandem leucine-rich repeats (LRR). LRR repeats form amphipathic β-α structural units that mediate protein-protein interactions. Although there are close to 100 known LRR domain-containing proteins, only a few binding pairs have been identified. In this paper, we used biochemical and genetic approaches to identify proteins that interact with human FLI. In vitro synthesized FLI bound to actin-Sepharose and binding was reduced by competition with excess soluble actin. Actin binding was mediated through the gelsolin-like domain and not the LRR domain. Although the FLI LRR module is most closely related to the LRR domains of Ras-interactive proteins, FLI does not associate with Ras, selected Ras effectors, or other Ras-related small GTPases. Two-hybrid screens using FLI LRR as bait identified a novel LRR binding partner. The 0.65-kilobase pair (kb) clone from the screen survived additional rounds of stringent two-hybrid pairwise assays, establishing a specific interaction. Binding to FLI LRR was corroborated by co- immunoprecipitation with FLI LRR. The translated sequence of the FLI LRR associated protein (FLAP) encodes a novel protein not represented in the data base. Northern blot analyses revealed four FLAP messages of approximately 2.7, 2.9, 3.3, and 5.1 kb, which are differentially expressed in the tissues tested. Skeletal and cardiac muscles are particularly rich in the 3.3-kb FLAP message, and the FLI message as well. Full-length FLAP clones were isolated from a mouse skeletal muscle cDNA library. They have an open reading frame which encodes for a protein containing 626 amino acids. Sequence analyses predict that the FLAP protein is rich in α-helices and contains stretches of dimeric coiled coil in its middle region and COOH terminus. The identification of actin and FLAP as the binding ligands for the gelsolin- like domain and the LRR domain, respectively, suggests that FLI may link the actin cytoskeleton to other modules implicated in intermolecular recognition and structural organization.

Original languageEnglish (US)
Pages (from-to)7920-7927
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number14
DOIs
StatePublished - Apr 3 1998

Fingerprint

Gelsolin
Leucine
Proteins
Actins
Monomeric GTP-Binding Proteins
Drosophila
Skeletal Muscle
leucine-rich repeat proteins
Clone Cells
Muscle
ras Proteins
Two-Hybrid System Techniques
Terminal Repeat Sequences
Muscle Development
Actin Cytoskeleton
Gene Library
Immunoprecipitation
Protein Binding
Northern Blotting
Sepharose

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{74a2ab2c460e4fca99f2402e8b2545ec,
title = "Identification of the binding partners for flightless I, a novel protein bridging the leucine-rich repeat and the gelsolin superfamilies",
abstract = "Flightless-I (fliI) is a novel member of the gelsolin family that is important for actin organization during Drosophila embryogenesis and myogenesis. Drosophila fliI and the human homolog FLI both contain the classic gelsolin 6-fold segmental repeats and an amino-terminal extension of 16 tandem leucine-rich repeats (LRR). LRR repeats form amphipathic β-α structural units that mediate protein-protein interactions. Although there are close to 100 known LRR domain-containing proteins, only a few binding pairs have been identified. In this paper, we used biochemical and genetic approaches to identify proteins that interact with human FLI. In vitro synthesized FLI bound to actin-Sepharose and binding was reduced by competition with excess soluble actin. Actin binding was mediated through the gelsolin-like domain and not the LRR domain. Although the FLI LRR module is most closely related to the LRR domains of Ras-interactive proteins, FLI does not associate with Ras, selected Ras effectors, or other Ras-related small GTPases. Two-hybrid screens using FLI LRR as bait identified a novel LRR binding partner. The 0.65-kilobase pair (kb) clone from the screen survived additional rounds of stringent two-hybrid pairwise assays, establishing a specific interaction. Binding to FLI LRR was corroborated by co- immunoprecipitation with FLI LRR. The translated sequence of the FLI LRR associated protein (FLAP) encodes a novel protein not represented in the data base. Northern blot analyses revealed four FLAP messages of approximately 2.7, 2.9, 3.3, and 5.1 kb, which are differentially expressed in the tissues tested. Skeletal and cardiac muscles are particularly rich in the 3.3-kb FLAP message, and the FLI message as well. Full-length FLAP clones were isolated from a mouse skeletal muscle cDNA library. They have an open reading frame which encodes for a protein containing 626 amino acids. Sequence analyses predict that the FLAP protein is rich in α-helices and contains stretches of dimeric coiled coil in its middle region and COOH terminus. The identification of actin and FLAP as the binding ligands for the gelsolin- like domain and the LRR domain, respectively, suggests that FLI may link the actin cytoskeleton to other modules implicated in intermolecular recognition and structural organization.",
author = "Liu, {Yu Tsueng} and Yin, {Helen L.}",
year = "1998",
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T1 - Identification of the binding partners for flightless I, a novel protein bridging the leucine-rich repeat and the gelsolin superfamilies

AU - Liu, Yu Tsueng

AU - Yin, Helen L.

PY - 1998/4/3

Y1 - 1998/4/3

N2 - Flightless-I (fliI) is a novel member of the gelsolin family that is important for actin organization during Drosophila embryogenesis and myogenesis. Drosophila fliI and the human homolog FLI both contain the classic gelsolin 6-fold segmental repeats and an amino-terminal extension of 16 tandem leucine-rich repeats (LRR). LRR repeats form amphipathic β-α structural units that mediate protein-protein interactions. Although there are close to 100 known LRR domain-containing proteins, only a few binding pairs have been identified. In this paper, we used biochemical and genetic approaches to identify proteins that interact with human FLI. In vitro synthesized FLI bound to actin-Sepharose and binding was reduced by competition with excess soluble actin. Actin binding was mediated through the gelsolin-like domain and not the LRR domain. Although the FLI LRR module is most closely related to the LRR domains of Ras-interactive proteins, FLI does not associate with Ras, selected Ras effectors, or other Ras-related small GTPases. Two-hybrid screens using FLI LRR as bait identified a novel LRR binding partner. The 0.65-kilobase pair (kb) clone from the screen survived additional rounds of stringent two-hybrid pairwise assays, establishing a specific interaction. Binding to FLI LRR was corroborated by co- immunoprecipitation with FLI LRR. The translated sequence of the FLI LRR associated protein (FLAP) encodes a novel protein not represented in the data base. Northern blot analyses revealed four FLAP messages of approximately 2.7, 2.9, 3.3, and 5.1 kb, which are differentially expressed in the tissues tested. Skeletal and cardiac muscles are particularly rich in the 3.3-kb FLAP message, and the FLI message as well. Full-length FLAP clones were isolated from a mouse skeletal muscle cDNA library. They have an open reading frame which encodes for a protein containing 626 amino acids. Sequence analyses predict that the FLAP protein is rich in α-helices and contains stretches of dimeric coiled coil in its middle region and COOH terminus. The identification of actin and FLAP as the binding ligands for the gelsolin- like domain and the LRR domain, respectively, suggests that FLI may link the actin cytoskeleton to other modules implicated in intermolecular recognition and structural organization.

AB - Flightless-I (fliI) is a novel member of the gelsolin family that is important for actin organization during Drosophila embryogenesis and myogenesis. Drosophila fliI and the human homolog FLI both contain the classic gelsolin 6-fold segmental repeats and an amino-terminal extension of 16 tandem leucine-rich repeats (LRR). LRR repeats form amphipathic β-α structural units that mediate protein-protein interactions. Although there are close to 100 known LRR domain-containing proteins, only a few binding pairs have been identified. In this paper, we used biochemical and genetic approaches to identify proteins that interact with human FLI. In vitro synthesized FLI bound to actin-Sepharose and binding was reduced by competition with excess soluble actin. Actin binding was mediated through the gelsolin-like domain and not the LRR domain. Although the FLI LRR module is most closely related to the LRR domains of Ras-interactive proteins, FLI does not associate with Ras, selected Ras effectors, or other Ras-related small GTPases. Two-hybrid screens using FLI LRR as bait identified a novel LRR binding partner. The 0.65-kilobase pair (kb) clone from the screen survived additional rounds of stringent two-hybrid pairwise assays, establishing a specific interaction. Binding to FLI LRR was corroborated by co- immunoprecipitation with FLI LRR. The translated sequence of the FLI LRR associated protein (FLAP) encodes a novel protein not represented in the data base. Northern blot analyses revealed four FLAP messages of approximately 2.7, 2.9, 3.3, and 5.1 kb, which are differentially expressed in the tissues tested. Skeletal and cardiac muscles are particularly rich in the 3.3-kb FLAP message, and the FLI message as well. Full-length FLAP clones were isolated from a mouse skeletal muscle cDNA library. They have an open reading frame which encodes for a protein containing 626 amino acids. Sequence analyses predict that the FLAP protein is rich in α-helices and contains stretches of dimeric coiled coil in its middle region and COOH terminus. The identification of actin and FLAP as the binding ligands for the gelsolin- like domain and the LRR domain, respectively, suggests that FLI may link the actin cytoskeleton to other modules implicated in intermolecular recognition and structural organization.

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