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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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SH3YL1 forms a complex with SHIP2. (A) The GST or GST-SH3 domain of SH3YL1 was incubated with (+) or without (−) HeLa cell lysates. Bound fractions were visualized by CBB staining. Proteins identified by mass spectrometry are shown. (B) Lysates of COS-1 cells expressing Myc-SHIP2 were pulled down by GST, GST fusions with the SH3 domain, or full-length SH3YL1 and then immunoblotted with anti-Myc or anti-GST antibodies. (C) FLAG-SH3YL1 (full length, ΔSH3, or W322A) expressed in HeLa cells was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-SHIP2 antibodies. (D) Endogenous SH3YL1 was immunoprecipitated with anti-SH3YL1 antibodies using lysates of NIH3T3 cells and immunoblotted with anti-SH3YL1 or anti-SHIP2 antibodies. (E) Purified FLAG-SHIP2 was pulled down by GST and GST fusions with the SH3 domain and then visualized by CBB staining. (F) A schematic presentation of SHIP2 deletion and point mutants used in this study. The red text indicates introduced alanines instead of intact amino acids (proline and leucine). PRD, proline-rich domain; SAM, sterile α motif. (G) FLAG-SH3YL1 (full length) and the indicated Myc-SHIP2 mutants expressed in COS-1 cells were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies. (H) NIH3T3 cells transiently transfected with FLAG-SH3YL1 and Myc-SHIP2 were serum starved and then stimulated with 20 ng/ml PDGF for 0 and 5 min. Cells were harvested, and the lysate was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies.
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fig5: SH3YL1 forms a complex with SHIP2. (A) The GST or GST-SH3 domain of SH3YL1 was incubated with (+) or without (−) HeLa cell lysates. Bound fractions were visualized by CBB staining. Proteins identified by mass spectrometry are shown. (B) Lysates of COS-1 cells expressing Myc-SHIP2 were pulled down by GST, GST fusions with the SH3 domain, or full-length SH3YL1 and then immunoblotted with anti-Myc or anti-GST antibodies. (C) FLAG-SH3YL1 (full length, ΔSH3, or W322A) expressed in HeLa cells was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-SHIP2 antibodies. (D) Endogenous SH3YL1 was immunoprecipitated with anti-SH3YL1 antibodies using lysates of NIH3T3 cells and immunoblotted with anti-SH3YL1 or anti-SHIP2 antibodies. (E) Purified FLAG-SHIP2 was pulled down by GST and GST fusions with the SH3 domain and then visualized by CBB staining. (F) A schematic presentation of SHIP2 deletion and point mutants used in this study. The red text indicates introduced alanines instead of intact amino acids (proline and leucine). PRD, proline-rich domain; SAM, sterile α motif. (G) FLAG-SH3YL1 (full length) and the indicated Myc-SHIP2 mutants expressed in COS-1 cells were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies. (H) NIH3T3 cells transiently transfected with FLAG-SH3YL1 and Myc-SHIP2 were serum starved and then stimulated with 20 ng/ml PDGF for 0 and 5 min. Cells were harvested, and the lysate was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies.

Mentions: To determine the molecular mechanism by which SH3YL1 mediates dorsal ruffle formation, we attempted to identify its binding proteins. The SH3 domain of SH3YL1 was expressed as a GST fusion protein and was used in a pull-down assay with HeLa cell lysates. Several proteins were identified, including N-WASP and dynamin 2, which are typical binding partners for SH3 domains (Fig. 5 A).


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)

SH3YL1 forms a complex with SHIP2. (A) The GST or GST-SH3 domain of SH3YL1 was incubated with (+) or without (−) HeLa cell lysates. Bound fractions were visualized by CBB staining. Proteins identified by mass spectrometry are shown. (B) Lysates of COS-1 cells expressing Myc-SHIP2 were pulled down by GST, GST fusions with the SH3 domain, or full-length SH3YL1 and then immunoblotted with anti-Myc or anti-GST antibodies. (C) FLAG-SH3YL1 (full length, ΔSH3, or W322A) expressed in HeLa cells was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-SHIP2 antibodies. (D) Endogenous SH3YL1 was immunoprecipitated with anti-SH3YL1 antibodies using lysates of NIH3T3 cells and immunoblotted with anti-SH3YL1 or anti-SHIP2 antibodies. (E) Purified FLAG-SHIP2 was pulled down by GST and GST fusions with the SH3 domain and then visualized by CBB staining. (F) A schematic presentation of SHIP2 deletion and point mutants used in this study. The red text indicates introduced alanines instead of intact amino acids (proline and leucine). PRD, proline-rich domain; SAM, sterile α motif. (G) FLAG-SH3YL1 (full length) and the indicated Myc-SHIP2 mutants expressed in COS-1 cells were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies. (H) NIH3T3 cells transiently transfected with FLAG-SH3YL1 and Myc-SHIP2 were serum starved and then stimulated with 20 ng/ml PDGF for 0 and 5 min. Cells were harvested, and the lysate was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105542&req=5

fig5: SH3YL1 forms a complex with SHIP2. (A) The GST or GST-SH3 domain of SH3YL1 was incubated with (+) or without (−) HeLa cell lysates. Bound fractions were visualized by CBB staining. Proteins identified by mass spectrometry are shown. (B) Lysates of COS-1 cells expressing Myc-SHIP2 were pulled down by GST, GST fusions with the SH3 domain, or full-length SH3YL1 and then immunoblotted with anti-Myc or anti-GST antibodies. (C) FLAG-SH3YL1 (full length, ΔSH3, or W322A) expressed in HeLa cells was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-SHIP2 antibodies. (D) Endogenous SH3YL1 was immunoprecipitated with anti-SH3YL1 antibodies using lysates of NIH3T3 cells and immunoblotted with anti-SH3YL1 or anti-SHIP2 antibodies. (E) Purified FLAG-SHIP2 was pulled down by GST and GST fusions with the SH3 domain and then visualized by CBB staining. (F) A schematic presentation of SHIP2 deletion and point mutants used in this study. The red text indicates introduced alanines instead of intact amino acids (proline and leucine). PRD, proline-rich domain; SAM, sterile α motif. (G) FLAG-SH3YL1 (full length) and the indicated Myc-SHIP2 mutants expressed in COS-1 cells were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies. (H) NIH3T3 cells transiently transfected with FLAG-SH3YL1 and Myc-SHIP2 were serum starved and then stimulated with 20 ng/ml PDGF for 0 and 5 min. Cells were harvested, and the lysate was immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-Myc antibodies.
Mentions: To determine the molecular mechanism by which SH3YL1 mediates dorsal ruffle formation, we attempted to identify its binding proteins. The SH3 domain of SH3YL1 was expressed as a GST fusion protein and was used in a pull-down assay with HeLa cell lysates. Several proteins were identified, including N-WASP and dynamin 2, which are typical binding partners for SH3 domains (Fig. 5 A).

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