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SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain.

Hasegawa J, Tokuda E, Tenno T, Tsujita K, Sawai H, Hiroaki H, Takenawa T, Itoh T - J. Cell Biol. (2011)

Bottom Line: Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation.Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure.These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Membrane Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan.

ABSTRACT
Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P(3)) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

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An amphipathic α helix at the N terminus of the SYLF domain is necessary for lipid binding. (A) Multiple sequence alignment of N-terminal regions of the SYLF domain from various species. The predicted α helix is indicated by a red line (residues 9–23), and mutated, positively charged residues in M1 and M2 are shown by asterisks. Identical amino acids are shaded in black, and similar residues are in gray. (B) A helical wheel representation of the SH3YL1 N-terminal region. Yellow, hydrophobic residues; purple, serine; blue, basic; pink, asparagine; red, glutamic acid; green, proline; and gray, other residues. Mutated residues are shown by asterisks. (C) Liposome cosedimentation assay using full-length or Δ1–29 SH3YL1 mutant and brain liposomes. Proteins are visualized by immunoblotting with anti-SH3YL1 antibodies. (D) PE/PC-based liposomes supplemented with 10% of PI(4,5)P2 or PI(3,4,5)P3 were used in the cosedimentation assay and quantitative representation. WT, wild type. Results are a mean (SD) of three independent experiments. (E) Liposome cosedimentation assay using 1–30 or ΔSH3 SH3YL1-GFP mutant and brain liposomes. Proteins are visualized by CBB staining. (F) Gel filtration chromatography of SH3YL1 (full length and SYLF). Eluted fractions were measured at 280 nm and then visualized by CBB staining. Abs, absorbance.
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fig2: An amphipathic α helix at the N terminus of the SYLF domain is necessary for lipid binding. (A) Multiple sequence alignment of N-terminal regions of the SYLF domain from various species. The predicted α helix is indicated by a red line (residues 9–23), and mutated, positively charged residues in M1 and M2 are shown by asterisks. Identical amino acids are shaded in black, and similar residues are in gray. (B) A helical wheel representation of the SH3YL1 N-terminal region. Yellow, hydrophobic residues; purple, serine; blue, basic; pink, asparagine; red, glutamic acid; green, proline; and gray, other residues. Mutated residues are shown by asterisks. (C) Liposome cosedimentation assay using full-length or Δ1–29 SH3YL1 mutant and brain liposomes. Proteins are visualized by immunoblotting with anti-SH3YL1 antibodies. (D) PE/PC-based liposomes supplemented with 10% of PI(4,5)P2 or PI(3,4,5)P3 were used in the cosedimentation assay and quantitative representation. WT, wild type. Results are a mean (SD) of three independent experiments. (E) Liposome cosedimentation assay using 1–30 or ΔSH3 SH3YL1-GFP mutant and brain liposomes. Proteins are visualized by CBB staining. (F) Gel filtration chromatography of SH3YL1 (full length and SYLF). Eluted fractions were measured at 280 nm and then visualized by CBB staining. Abs, absorbance.

Mentions: We observed that the predicted α-helical sequence at the N terminus of the SYLF domain (residues 9–23) contained many positively charged residues (Fig. 2 A). Moreover, this region was predicted to possess amphipathic properties (Fig. 2 B), suggesting that it plays an important role in lipid binding. To test this hypothesis, a deletion mutant (Δ1–29) was constructed and subjected to lipid-binding assay. The deletion mutant (Δ1–29) showed much lower affinity than the full-length protein (Fig. 2 C), indicating the importance of this region. The contribution of the positively charged residues on the hydrophilic surface of this region (Lys14 + Lys15 [M1] or Lys18 + Arg21 [M2]) was determined by replacing them with alanine (Fig. 2, A and B). In comparison with wild type, the binding affinity of these mutants for PI(3,4,5)P3, especially that of the M1 mutant, was significantly low (Fig. 2 D).


SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain.

Hasegawa J, Tokuda E, Tenno T, Tsujita K, Sawai H, Hiroaki H, Takenawa T, Itoh T - J. Cell Biol. (2011)

An amphipathic α helix at the N terminus of the SYLF domain is necessary for lipid binding. (A) Multiple sequence alignment of N-terminal regions of the SYLF domain from various species. The predicted α helix is indicated by a red line (residues 9–23), and mutated, positively charged residues in M1 and M2 are shown by asterisks. Identical amino acids are shaded in black, and similar residues are in gray. (B) A helical wheel representation of the SH3YL1 N-terminal region. Yellow, hydrophobic residues; purple, serine; blue, basic; pink, asparagine; red, glutamic acid; green, proline; and gray, other residues. Mutated residues are shown by asterisks. (C) Liposome cosedimentation assay using full-length or Δ1–29 SH3YL1 mutant and brain liposomes. Proteins are visualized by immunoblotting with anti-SH3YL1 antibodies. (D) PE/PC-based liposomes supplemented with 10% of PI(4,5)P2 or PI(3,4,5)P3 were used in the cosedimentation assay and quantitative representation. WT, wild type. Results are a mean (SD) of three independent experiments. (E) Liposome cosedimentation assay using 1–30 or ΔSH3 SH3YL1-GFP mutant and brain liposomes. Proteins are visualized by CBB staining. (F) Gel filtration chromatography of SH3YL1 (full length and SYLF). Eluted fractions were measured at 280 nm and then visualized by CBB staining. Abs, absorbance.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig2: An amphipathic α helix at the N terminus of the SYLF domain is necessary for lipid binding. (A) Multiple sequence alignment of N-terminal regions of the SYLF domain from various species. The predicted α helix is indicated by a red line (residues 9–23), and mutated, positively charged residues in M1 and M2 are shown by asterisks. Identical amino acids are shaded in black, and similar residues are in gray. (B) A helical wheel representation of the SH3YL1 N-terminal region. Yellow, hydrophobic residues; purple, serine; blue, basic; pink, asparagine; red, glutamic acid; green, proline; and gray, other residues. Mutated residues are shown by asterisks. (C) Liposome cosedimentation assay using full-length or Δ1–29 SH3YL1 mutant and brain liposomes. Proteins are visualized by immunoblotting with anti-SH3YL1 antibodies. (D) PE/PC-based liposomes supplemented with 10% of PI(4,5)P2 or PI(3,4,5)P3 were used in the cosedimentation assay and quantitative representation. WT, wild type. Results are a mean (SD) of three independent experiments. (E) Liposome cosedimentation assay using 1–30 or ΔSH3 SH3YL1-GFP mutant and brain liposomes. Proteins are visualized by CBB staining. (F) Gel filtration chromatography of SH3YL1 (full length and SYLF). Eluted fractions were measured at 280 nm and then visualized by CBB staining. Abs, absorbance.
Mentions: We observed that the predicted α-helical sequence at the N terminus of the SYLF domain (residues 9–23) contained many positively charged residues (Fig. 2 A). Moreover, this region was predicted to possess amphipathic properties (Fig. 2 B), suggesting that it plays an important role in lipid binding. To test this hypothesis, a deletion mutant (Δ1–29) was constructed and subjected to lipid-binding assay. The deletion mutant (Δ1–29) showed much lower affinity than the full-length protein (Fig. 2 C), indicating the importance of this region. The contribution of the positively charged residues on the hydrophilic surface of this region (Lys14 + Lys15 [M1] or Lys18 + Arg21 [M2]) was determined by replacing them with alanine (Fig. 2, A and B). In comparison with wild type, the binding affinity of these mutants for PI(3,4,5)P3, especially that of the M1 mutant, was significantly low (Fig. 2 D).

Bottom Line: Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation.Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure.These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Membrane Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan.

ABSTRACT
Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P(3)) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

Show MeSH