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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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The N-terminal region of SH3YL1 with high affinity for phosphoinositides. (A) A schematic presentation of human SH3YL1 and domain linker prediction by the SVM long protocol. Smoothed SVM output values were plotted against the amino acid sequence. Larger values indicate higher probabilities of domain linkers. (B) Limiting proteolytic digestion. Purified FLAG-SH3YL1 (full length and SYLF) proteins were treated with trypsin for the indicated times and then immunoblotted (IB) with anti-FLAG antibodies. The asterisk shows a minor digestion product. (C) Liposome cosedimentation assay using brain liposomes and SH3YL1 (full length, SYLF, and ΔSYLF). Proteins in the supernatant (S) and pellet (P) are visualized by CBB staining. (D) Liposome cosedimentation assay using brain liposomes and Ysc84p and Lsb3p (1–218). (E) PE/PC-based liposomes supplemented with 10% of the indicated lipids were used in the cosedimentation assay and quantitative representation. Results are a mean (SD) of three independent experiments.
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fig1: The N-terminal region of SH3YL1 with high affinity for phosphoinositides. (A) A schematic presentation of human SH3YL1 and domain linker prediction by the SVM long protocol. Smoothed SVM output values were plotted against the amino acid sequence. Larger values indicate higher probabilities of domain linkers. (B) Limiting proteolytic digestion. Purified FLAG-SH3YL1 (full length and SYLF) proteins were treated with trypsin for the indicated times and then immunoblotted (IB) with anti-FLAG antibodies. The asterisk shows a minor digestion product. (C) Liposome cosedimentation assay using brain liposomes and SH3YL1 (full length, SYLF, and ΔSYLF). Proteins in the supernatant (S) and pellet (P) are visualized by CBB staining. (D) Liposome cosedimentation assay using brain liposomes and Ysc84p and Lsb3p (1–218). (E) PE/PC-based liposomes supplemented with 10% of the indicated lipids were used in the cosedimentation assay and quantitative representation. Results are a mean (SD) of three independent experiments.

Mentions: To determine whether this entire region forms an independent structure that extends from the DUF500 domain to the N terminus, we predicted the domain boundary by using the support vector machine (SVM) long method (Ebina et al., 2009). In the primary sequence of human SH3YL1 (Aoki et al., 2000), a putative linker region separating two structural domains was predicted to be Gln230–Glu265 with a peak probability at Pro242 (Fig. 1 A), suggesting that ∼230 N-terminal residues form a structurally independent unit. A similar result was obtained using the DomCut server (Suyama and Ohara, 2003), which predicted Pro246 as the most probable linker position (unpublished data). These predictions were supported by the results of proteolytic digestion experiments, wherein purified FLAG-SH3YL1 (full length) protein was incubated with limiting amounts of trypsin. The N-terminal region of SH3YL1, which was recognized by anti-FLAG, was cleaved to an ∼25-kD fragment, slightly larger than FLAG-SH3YL1 SYLF (1–216) (Fig. 1 B). A faint band was also observed around ∼16 kD (Fig. 1 B, asterisk), suggesting another very minor cleavage site. These results indicate that the C-terminal region extending from Glu217 acts as the primary domain linker that is accessible to trypsin. Based on these data and our findings from this study, we define this ∼220-residue-long N-terminal region as a single domain module named “SYLF” domain on the basis of its representative members (SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE protein; Fig. S1 B).


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)

The N-terminal region of SH3YL1 with high affinity for phosphoinositides. (A) A schematic presentation of human SH3YL1 and domain linker prediction by the SVM long protocol. Smoothed SVM output values were plotted against the amino acid sequence. Larger values indicate higher probabilities of domain linkers. (B) Limiting proteolytic digestion. Purified FLAG-SH3YL1 (full length and SYLF) proteins were treated with trypsin for the indicated times and then immunoblotted (IB) with anti-FLAG antibodies. The asterisk shows a minor digestion product. (C) Liposome cosedimentation assay using brain liposomes and SH3YL1 (full length, SYLF, and ΔSYLF). Proteins in the supernatant (S) and pellet (P) are visualized by CBB staining. (D) Liposome cosedimentation assay using brain liposomes and Ysc84p and Lsb3p (1–218). (E) PE/PC-based liposomes supplemented with 10% of the indicated lipids were used in the cosedimentation assay and quantitative representation. Results are a mean (SD) of three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105542&req=5

fig1: The N-terminal region of SH3YL1 with high affinity for phosphoinositides. (A) A schematic presentation of human SH3YL1 and domain linker prediction by the SVM long protocol. Smoothed SVM output values were plotted against the amino acid sequence. Larger values indicate higher probabilities of domain linkers. (B) Limiting proteolytic digestion. Purified FLAG-SH3YL1 (full length and SYLF) proteins were treated with trypsin for the indicated times and then immunoblotted (IB) with anti-FLAG antibodies. The asterisk shows a minor digestion product. (C) Liposome cosedimentation assay using brain liposomes and SH3YL1 (full length, SYLF, and ΔSYLF). Proteins in the supernatant (S) and pellet (P) are visualized by CBB staining. (D) Liposome cosedimentation assay using brain liposomes and Ysc84p and Lsb3p (1–218). (E) PE/PC-based liposomes supplemented with 10% of the indicated lipids were used in the cosedimentation assay and quantitative representation. Results are a mean (SD) of three independent experiments.
Mentions: To determine whether this entire region forms an independent structure that extends from the DUF500 domain to the N terminus, we predicted the domain boundary by using the support vector machine (SVM) long method (Ebina et al., 2009). In the primary sequence of human SH3YL1 (Aoki et al., 2000), a putative linker region separating two structural domains was predicted to be Gln230–Glu265 with a peak probability at Pro242 (Fig. 1 A), suggesting that ∼230 N-terminal residues form a structurally independent unit. A similar result was obtained using the DomCut server (Suyama and Ohara, 2003), which predicted Pro246 as the most probable linker position (unpublished data). These predictions were supported by the results of proteolytic digestion experiments, wherein purified FLAG-SH3YL1 (full length) protein was incubated with limiting amounts of trypsin. The N-terminal region of SH3YL1, which was recognized by anti-FLAG, was cleaved to an ∼25-kD fragment, slightly larger than FLAG-SH3YL1 SYLF (1–216) (Fig. 1 B). A faint band was also observed around ∼16 kD (Fig. 1 B, asterisk), suggesting another very minor cleavage site. These results indicate that the C-terminal region extending from Glu217 acts as the primary domain linker that is accessible to trypsin. Based on these data and our findings from this study, we define this ∼220-residue-long N-terminal region as a single domain module named “SYLF” domain on the basis of its representative members (SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE protein; Fig. S1 B).

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
Related in: MedlinePlus