title: SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V: localization of SHIP-2 to the activated platelet actin cytoskeleton.
abstract: The platelet receptor for the von Willebrand factor (VWF) glycoprotein Ib-IX-V (GPIb-IX-V) complex mediates platelet adhesion at sites of vascular injury. The cytoplasmic tail of the GPIbalpha subunit interacts with the actin-binding protein, filamin, anchoring the receptor in the cytoskeleton. In motile cells, the second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3) induces submembraneous actin remodeling. The inositol polyphosphate 5-phosphatase, Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2), hydrolyzes PtdIns(3,4,5)P3 forming phosphatidylinositol 3,4 bisphosphate (PtdIns(3,4)P2) and regulates membrane ruffling via complex formation with filamin. In this study we investigate the intracellular location and association of SHIP-2 with filamin, actin, and the GPIb-IX-V complex in platelets. Immunoprecipitation of SHIP-2 from the Triton-soluble fraction of unstimulated platelets demonstrated association between SHIP-2, filamin, actin, and GPIb-IX-V. SHIP-2 associated with filamin or GPIb-IX-V was active and demonstrated PtdIns(3,4,5)P3 5-phosphatase activity. Following thrombin or VWF-induced platelet activation, detection of the SHIP-2, filamin, and receptor complex decreased in the Triton-soluble fraction, although in control studies the level of SHIP-2, filamin, or GPIb-IX-V immunoprecipitated by their respective antibodies did not change following platelet activation. In activated platelets spreading on a VWF matrix, SHIP-2 localized intensely with actin at the central actin ring and colocalized with actin and filamin at filopodia and lamellipodia. In spread platelets, GPIb-IX-V localized to the center of the platelet and showed little colocalization with filamin at the plasma membrane. These studies demonstrate a functionally active complex between SHIP-2, filamin, actin, and GPIb-IX-V that may orchestrate the localized hydrolysis of PtdIns(3,4,5)P3 and thereby regulate cortical and submembraneous actin.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Dyson
authors.forename: Adam D
authors.initials: AD
authors.lastname: Munday
authors.forename: Anne M
authors.initials: AM
authors.lastname: Kong
authors.forename: Richard D
authors.initials: RD
authors.lastname: Huysmans
authors.forename: Maria
authors.initials: M
authors.lastname: Matzaris
authors.forename: Meredith J
authors.initials: MJ
authors.lastname: Layton
authors.forename: Harshal H
authors.initials: HH
authors.lastname: Nandurkar
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.forename: Christina A
authors.initials: CA
authors.lastname: Mitchell
mesh_terms: D000199:Actins / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D048429:Cell Size; D003285:Contractile Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000199:Actins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D064448:Filamins; D008840:Microfilament Proteins; D018129:Phosphatidylinositol Phosphates; D019038:Platelet Glycoprotein GPIb-IX Complex; C060974:phosphatidylinositol 3,4,5-triphosphate; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases