SHIP2 interaction with the cytoskeletal protein Vinexin

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3].
• Vinexin is a cytoskeletal protein involved in cell spreading and organization.
• SHIP2's role in cell adhesion and cytoskeletal dynamics is not fully understood.

Purpose of the Study:

• To identify proteins interacting with SHIP2.
• To investigate the functional consequences of SHIP2-Vinexin interaction on cell adhesion and SHIP2 activity.

Main Methods:

• Yeast two-hybrid screening to identify interacting proteins.
• Co-immunoprecipitation and Western blotting to confirm protein interactions in cell lysates.
• Transfection of wild-type and mutant SHIP2 constructs in COS-7 cells.
• In vitro enzyme activity assays.
• Cell adhesion assays on collagen-I-coated dishes.
• Analysis of SHIP2 knockout mouse embryonic fibroblasts (MEFs).

Main Results:

• Vinexin was identified as a SHIP2-interacting protein via yeast two-hybrid screening.
• The interaction was confirmed in COS-7 cells and MEFs, involving the C-terminus of SHIP2.
• SHIP2 and Vinexin colocalized at the cell periphery.
• Vinexin did not alter SHIP2's catalytic activity in vitro.
• SHIP2 and Vinexin individually enhanced cell adhesion; this effect required functional SHIP2.
• SHIP2 deficiency in MEFs reduced cell adhesion.

Conclusions:

• SHIP2 interacts with the cytoskeletal protein Vinexin, promoting SHIP2 localization at the cell periphery.
• This interaction enhances cellular adhesion to collagen-I, independent of SHIP2's catalytic activity.
• SHIP2 functions both as a phosphatase and a modulator of focal adhesion formation.