Báo cáo khoa học: SHIP2 interaction with the cytoskeletal protein Vinexin

SHIP2 Interaction with the Cytoskeletal Protein Vinexin

Tác giả: Nathalie Paternotte, Jing Zhang, Isabelle Vandenbroere, Katrien Backers, Daniel Blero, Noriyuki Kioka, Jean-Marie Vanderwinden, Isabelle Pirson, Christophe Erneux

Lĩnh vực: Sinh học phân tử, Tế bào học

Nội dung tài liệu: Nghiên cứu này xác định protein khung xương Vinexin là một đối tác tương tác mới với SHIP2 (src homology 2 domain-containing inositol 5-phosphatase 2). SHIP2 có vai trò trong việc chuyển hóa phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) thành phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Sự tương tác giữa SHIP2 và Vinexin đã được xác nhận và cho thấy sự cùng định vị của chúng ở ngoại vi tế bào. Vinexin không ảnh hưởng đến hoạt động xúc tác 5-phosphatase của SHIP2. Tuy nhiên, cả việc biểu hiện quá mức SHIP2 hoặc Vinexin đều làm tăng khả năng bám dính của tế bào lên collagen I. Dữ liệu cho thấy sự tương tác giữa SHIP2 và Vinexin có thể thúc đẩy sự định vị của SHIP2 tại các vị trí bám dính của tế bào, góp phần vào việc hình thành các điểm tiếp xúc tiêu điểm và tăng cường khả năng bám dính của tế bào.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 25 August 2005, accepted 28 September 2005)
• doi:10.1111/j.1742-4658.2005.04996.x
• The src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We report the identification of the cytoskeletal protein Vinexin as a protein interacting with SHIP2. This was achieved by yeast two-hybrid screening using the C-terminal region of SHIP2 as bait. Vinexin has previously been identified as a vinculin-binding protein that plays a key role in cell spreading and cytoskeletal organization. The interaction between SHIP2 and Vinexin was confirmed in lysates of both COS-7 cells and mouse embryonic fibroblasts (MEF). The C-terminus was involved in the interaction, as shown by the transfection of a truncated C-terminus mutant of SHIP2. In addition, we showed the colocalization between Vinexin a and SHIP2 at the periphery of transfected COS-7 cells. When added in vitro to SHIP2, Vinexin did not affect the PtdIns(3,4,5)P3 5-phosphatase activity of SHIP2. Enhanced cell adhesion to collagen-I-coated dishes was shown upon transfection of either SHIP2 or Vinexin to COS-7 cells. This effect was no longer observed with either a catalytic mutant or the C-terminus mutant of SHIP2. It also appears SHIP2 specific; this was not seen with SHIP1. Adhesion to the same matrix was decreased in SHIP2-/- MEF cells compared with MEF+/+ cells. Our data suggest that SHIP2 interaction with Vinexin promotes the localization of SHIP2 at the periphery of the cells leaving its catalytic site intact. The complex formation between Vinexin and SHIP2 may increase cellular adhesion. The data reinforce the concept that SHIP2 is active both as a PtdIns(3,4,5)P3 5-phosphatase and as a modulator of focal contact formation.
• The ubiquitous src homology 2 (SH2) domain containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] in vitro [1,2]. The reaction product catalysed by SHIP2 is phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. SHIP2 is a member of the inositol 5-phosphatase family, and with the SH2 domain containing inositol 5-phosphatase 1 (SHIP1), is
• Abbreviations
• Fig. 1. Isolation of Vinexin as SHIP2 (SH2 domain containing inositol 5-phosphatase 2) partner using yeast two-hybrid screening.
• Fig. 2. Association of SHIP2 with Vinexin a in transfected COS-7 cells.
• Fig. 3. Endogenous Vinexin a and ẞ associate with SHIP2 in MEF+/+ cells.
• Fig. 4. Vinexin a and SHIP2 are colocalized at the periphery of transfected COS-7 cells.
• Vinexin a did not influence PKB activity in COS-7 cells
• Adhesion of MEF SHIP2 cells and COS-7 cells upon SHIP2 and Vinexin a overexpression
• Fig. 5. Adhesion assay on MEF SHIP2 cells and on COS-7 cells overexpressing SHIP2 and Vinexin a.
• Influence of SHIP2 proline-rich sequences and catalytic mutant on cell adhesion
• Fig. 6. Effect of overexpression of SHIP2, ASH2 SHIP2 and AProline SHIP2 on adhesion.
• Fig. 7. Effect of a catalytic mutant SHIP2 and SHIP1 overexpression on adhesion.
• Fig. 8. Interaction between Vinexin a and AProline SHIP2 or SHIP2 D607A.
• Discussion
• Experimental procedures
• Materials
• Yeast two-hybrid screening
• Expression vector constructs
• Cell lines
• Transfection of COS-7 cells
• Immunoprecipitation and immunoblotting
• Malachite phosphatase assay
• Cell adhesion assay
• Confocal immunofluorescence microscopy
• Acknowledgements
• References