The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.
SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.
Dyson
J M
JM
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800 Australia.
O'Malley
C J
CJ
Becanovic
J
J
Munday
A D
AD
Berndt
M C
MC
Coghill
I D
ID
Nandurkar
H H
HH
Ooms
L M
LM
Mitchell
C A
CA
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells / Q000187:drug effects / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D064448:Filamins; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D008545:Melanoma; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D015345:Oligonucleotide Probes; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D000199:Actins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D015345:Oligonucleotide Probes; D010716:Phosphatidylinositols; D004815:Epidermal Growth Factor; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; C098706:INPPL1 protein, human; C000606435:Inppl1 protein, mouse; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases