Identification of a novel domain in two mammalian inositol-polyphosphate 5-phosphatases that mediates membrane ruffle localization. The inositol 5-phosphatase skip localizes to the endoplasmic reticulum and translocates to membrane ruffles following epidermal growth factor stimulation.
SKIP (skeletal muscle and kidney enriched inositol phosphatase) is a recently identified phosphatidylinositol 3,4,5-trisphosphate- and phosphatidylinositol 4,5-bisphosphate-specific 5-phosphatase. In this study, we investigated the intracellular localization of SKIP. Indirect immunofluorescence and subcellular fractionation showed that, in serum-starved cells, both endogenous and recombinant SKIP colocalized with markers of the endoplasmic reticulum (ER). Following epidermal growth factor (EGF) stimulation, SKIP transiently translocated to plasma membrane ruffles and colocalized with submembranous actin. Data base searching demonstrated a novel 128-amino acid domain in the C terminus of SKIP, designated SKICH for SKIP carboxyl homology, which is also found in the 107-kDa 5-phosphatase PIPP and in members of the TRAF6-binding protein family. Recombinant SKIP lacking the SKICH domain localized to the ER, but did not translocate to membrane ruffles following EGF stimulation. The SKIP SKICH domain showed perinuclear localization and mediated EGF-stimulated plasma membrane ruffle localization. The SKICH domain of the 5-phosphatase PIPP also mediated plasma membrane ruffle localization. Mutational analysis identified the core sequence within the SKICH domain that mediated constitutive membrane association and C-terminal sequences unique to SKIP that contributed to ER localization. Collectively, these studies demonstrate a novel membrane-targeting domain that serves to recruit SKIP and PIPP to membrane ruffles.
Gurung
Rajendra
R
Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
Tan
April
A
Ooms
Lisa M
LM
McGrath
Meagan J
MJ
Huysmans
Richard D
RD
Munday
Adam D
AD
Prescott
Mark
M
Whisstock
James C
JC
Mitchell
Christina A
CA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D016895:Culture Media, Serum-Free; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004815:Epidermal Growth Factor / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; C471548:SKIP enzyme, human; D010744:Phosphoric Monoester Hydrolases