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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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Correlation between PI(3,4)P2 synthesis and circular dorsal ruffle formation. (A) Lipids were extracted from control, SH3YL1-, or SHIP2-depleted NIH3T3 stimulated with 20 ng/ml PDGF for 0, 2, 5, and 15 min. Amounts of PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 were quantified by Qdot-705-Tapp1-2×PH, Qdot-655-PLCδ1-PH, and Qdot-585-Grp1-PH, respectively, in the dot-blot assay. Data are a mean (SD) of three independent experiments. (B) Time course of circular dorsal ruffle formation. Phalloidin staining and quantifications are shown. Results are a mean (SD) of three independent experiments; 200 cells counted per experiment. Bar, 10 µm. (C) Quantification of circular dorsal ruffles in NIH3T3 cells overexpressing lipid-binding domains. Percentages of cells with at least one circular dorsal ruffle are shown. Results are a mean (SD) of three independent experiments; 100 cells counted per experiment. **, P < 0.01.
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fig7: Correlation between PI(3,4)P2 synthesis and circular dorsal ruffle formation. (A) Lipids were extracted from control, SH3YL1-, or SHIP2-depleted NIH3T3 stimulated with 20 ng/ml PDGF for 0, 2, 5, and 15 min. Amounts of PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 were quantified by Qdot-705-Tapp1-2×PH, Qdot-655-PLCδ1-PH, and Qdot-585-Grp1-PH, respectively, in the dot-blot assay. Data are a mean (SD) of three independent experiments. (B) Time course of circular dorsal ruffle formation. Phalloidin staining and quantifications are shown. Results are a mean (SD) of three independent experiments; 200 cells counted per experiment. Bar, 10 µm. (C) Quantification of circular dorsal ruffles in NIH3T3 cells overexpressing lipid-binding domains. Percentages of cells with at least one circular dorsal ruffle are shown. Results are a mean (SD) of three independent experiments; 100 cells counted per experiment. **, P < 0.01.

Mentions: The preferred substrate for SHIP2 is PI(3,4,5)P3, which generates PI(3,4)P2 (Ishihara et al., 1999). To investigate the relationship between PI(3,4)P2 levels and dorsal ruffle formation, we measured cellular levels of phosphoinositides after stimulation with PDGF by dot-blot assay. Production of PI(3,4,5)P3 increased 2 min after stimulation with PDGF, when the dorsal ruffles were scarce (Fig. 7, A and B; and Fig. S5 A). In contrast, the generation of PI(3,4)P2 was delayed compared with PI(3,4,5)P3 and peaked 5 min after stimulation, when the formation of circular dorsal ruffles was nearly complete (Fig. 7, A and B). This result implies a link between PI(3,4)P2 production and circular dorsal ruffle formation.


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)

Correlation between PI(3,4)P2 synthesis and circular dorsal ruffle formation. (A) Lipids were extracted from control, SH3YL1-, or SHIP2-depleted NIH3T3 stimulated with 20 ng/ml PDGF for 0, 2, 5, and 15 min. Amounts of PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 were quantified by Qdot-705-Tapp1-2×PH, Qdot-655-PLCδ1-PH, and Qdot-585-Grp1-PH, respectively, in the dot-blot assay. Data are a mean (SD) of three independent experiments. (B) Time course of circular dorsal ruffle formation. Phalloidin staining and quantifications are shown. Results are a mean (SD) of three independent experiments; 200 cells counted per experiment. Bar, 10 µm. (C) Quantification of circular dorsal ruffles in NIH3T3 cells overexpressing lipid-binding domains. Percentages of cells with at least one circular dorsal ruffle are shown. Results are a mean (SD) of three independent experiments; 100 cells counted per experiment. **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Correlation between PI(3,4)P2 synthesis and circular dorsal ruffle formation. (A) Lipids were extracted from control, SH3YL1-, or SHIP2-depleted NIH3T3 stimulated with 20 ng/ml PDGF for 0, 2, 5, and 15 min. Amounts of PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 were quantified by Qdot-705-Tapp1-2×PH, Qdot-655-PLCδ1-PH, and Qdot-585-Grp1-PH, respectively, in the dot-blot assay. Data are a mean (SD) of three independent experiments. (B) Time course of circular dorsal ruffle formation. Phalloidin staining and quantifications are shown. Results are a mean (SD) of three independent experiments; 200 cells counted per experiment. Bar, 10 µm. (C) Quantification of circular dorsal ruffles in NIH3T3 cells overexpressing lipid-binding domains. Percentages of cells with at least one circular dorsal ruffle are shown. Results are a mean (SD) of three independent experiments; 100 cells counted per experiment. **, P < 0.01.
Mentions: The preferred substrate for SHIP2 is PI(3,4,5)P3, which generates PI(3,4)P2 (Ishihara et al., 1999). To investigate the relationship between PI(3,4)P2 levels and dorsal ruffle formation, we measured cellular levels of phosphoinositides after stimulation with PDGF by dot-blot assay. Production of PI(3,4,5)P3 increased 2 min after stimulation with PDGF, when the dorsal ruffles were scarce (Fig. 7, A and B; and Fig. S5 A). In contrast, the generation of PI(3,4)P2 was delayed compared with PI(3,4,5)P3 and peaked 5 min after stimulation, when the formation of circular dorsal ruffles was nearly complete (Fig. 7, A and B). This result implies a link between PI(3,4)P2 production and circular dorsal ruffle formation.

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