From the Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia
Received for publication, September 30, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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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.
Phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2)1
serves as a precursor to two major signaling pathways. This central phosphoinositide is phosphorylated by phosphoinositide 3-kinase, forming PtdIns(3,4,5)P3, which transiently accumulates
at the plasma membrane of growth factor-activated cells and is
subsequently rapidly metabolized by PtdIns(3,4,5)P3 5- or
3-phosphate-specific lipid phosphatases. PtdIns(3,4,5)P3
recruits various effector proteins that contain PH domains, such as the
serine/threonine kinases Akt and PDK1 (1), and Rho, Rac, and
ADP-ribosylation factor guanine nucleotide exchange factors,
including P-Rex1 (2) and SWAP-70 (3), which in turn regulate many
signaling pathways, including inhibition of cell death, cell cycle
progression, actin polymerization, membrane ruffling, cell migration,
and secretion (4-7). In addition, PtdIns(4,5)P2 is
hydrolyzed by phospholipase C to produce inositol 1,4,5-trisphosphate
(Ins(1,4,5)P3) and diacylglycerol, which mobilize
intracellular calcium and activate protein kinase C,
respectively. PtdIns(4,5)P2 itself regulates the activity
of actin-binding proteins by suppressing the function of vinculin, cofilin, gelsolin, and profilin and by activating the
actin-gelatinizing activity of The inositol-polyphosphate 5-phosphatases (referred to as
5-phosphatases) are a large family of signal-modifying enzymes that hydrolyze the 5-phosphate from the inositol ring of both inositol phosphates, such as Ins(1,4,5)P3 and
Ins(1,3,4,5)P4, and/or PtdIns-derived messenger
molecules, including PtdIns(4,5)P2,
PtdIns(3,4,5)P3, and PtdIns(3,5)P2 (9). Ten
mammalian and four yeast homologs have been identified and
characterized. Two recent studies using a bioinformatics approach (10)
and x-ray crystallography (11) have demonstrated that the
5-phosphatases belong to the family of apurinic/apyrimidinic
endonucleases and share the same catalytic mechanism of action.
However, clarification of the substrate specificity of the various
5-phosphatases, in particular their in vivo substrates, awaits further delineation.
Many recent studies have demonstrated that the mammalian 5-phosphatases
play critical roles in regulating phosphoinositide 3-kinase and
PtdIns(4,5)P2 signals. Gene-targeted deletion of SHIP-1
results in a phenotype of myeloid cell expansion, splenomegaly, and
lung infiltration (12, 13), whereas SHIP-2 knockout mice demonstrate
insulin hypersensitivity and die from hypoglycemia (14).
Synaptojanin-1-deficient mice show accumulation of clathrin-coated vesicles in post-synaptic neurons and early death (15). Humans deficient in OCRL 5-phosphatase (16) demonstrate growth and mental
retardation, renal tubular acidosis, and cataracts, although mice that
lack this enzyme surprisingly show no phenotype (17). Homozygous male
5-phosphatase II-deficient mice are infertile due to disrupted sperm
function (18).
Two novel mammalian 5-phosphatases designated SKIP for
skeletal muscle and kidney enriched
inositol phosphatase (19) and PIPP for
proline-rich inositol-polyphosphate
5-phosphatase (20) have recently been cloned, but not
extensively characterized. The 5-phosphatase SKIP is a 51-kDa enzyme
that contains a central catalytic 5-phosphatase domain with no other
reported motifs. Kinetic analysis using recombinant 5-phosphatase
indicates that the enzyme's major substrates in vitro are
PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Ectopic
overexpression of SKIP in COS-7 cells results in loss of actin stress
fibers in the regions of overexpression, presumably via regulation
of PtdIns(4,5)P2 and thereby the actin cytoskeleton
(19). PIPP is a 107-kDa 5-phosphatase that hydrolyzes PtdIns(4,5)P2, Ins(1,4,5)P3, and
Ins(1,3,4,5)P4 and localizes to membrane ruffles (20).
Here we investigate the intracellular localization of SKIP and show
that, in the unstimulated cell, this 5-phosphatase localizes to the ER.
Following growth factor stimulation, SKIP translocated to plasma
membrane ruffles, mediated by the novel C-terminal domain SKICH, for
SKIP carboxyl homology.
Bioinformatics analysis demonstrates that this C-terminal domain is
also present in PIPP and several other signaling proteins. We propose
that the SKICH domain directs plasma membrane ruffle localization in
these 5-phosphatases.
Materials--
Restriction and DNA-modifying enzymes were
obtained from New England Biolabs Inc. or MBI Fermentas. The human
testis Marathon-Ready cDNA kit was from
Clontech. Synthetic oligonucleotides were from Geneworks (Adelaide, Australia) or the Department of Microbiology, Monash University (Melbourne, Australia), and the BigDye terminator cycle sequencing kit was from PE Applied Biosystems. Synthetic peptides
were ordered from Mimotopes (Melbourne). [ Isolation of the Human SKIP cDNA--
The 5'-end of human
SKIP was isolated using the Marathon-Ready cDNA kit by rapid
amplification of cDNA ends-PCR using the primer
5'-gggaacaaaggtggagtcaacatctgcctg, designed based on the expressed
sequence tag U45973 sequence, and the primer supplied with the kit.
This fragment was ligated with U45973 using a unique HindIII
site in the overlapping region to obtain the full-length SKIP cDNA.
Identification of the SKICH Domain--
The C-terminal domain of
human SKIP was used as a probe in a PSI-BLAST search (21) of the NCBI
Non-redundant Protein
Database.2 The search was
performed using h = 0.005 and converged after three
rounds. The alignment in Fig. 5A was generated using
ClustalW (22), and the figure was generated using Alscript (23).
Generation of Anti-peptide Antibodies--
Anti-peptide
antibodies to SKIP were generated against the synthetic peptide
comprising the N-terminal seven amino acids fused to the C-terminal
seven amino acids with a central cysteine
(NH2-MSSRKLSCGEAQPQI). The diphtheria toxoid-conjugated
peptide was injected subcutaneously into two New Zealand White rabbits.
Antibodies were affinity-purified on specific peptide coupled to
thiopropyl-Sepharose resin following the peptide manufacturer's protocols.
Immunoblotting of Various Cell Lines--
Expression of SKIP was
investigated in COS-7 and U87 cells, which were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum (FCS). Sol8 myoblasts were grown in DMEM supplemented
with 20% FCS and differentiated into myotubes in 5% horse serum. The
cells were scraped in 40 mM Tris (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM
benzamidine; the lysates were extracted in 1% Triton X-100 for 4 h at 4 °C; and soluble and insoluble fractions were isolated by
centrifugation at 16,000 × g. 100 µg of
Triton-soluble and -insoluble fractions were subjected to immunoblot
analysis using the affinity-purified anti-SKIP antibodies. Horseradish
peroxidase-conjugated anti-rabbit IgG was used as the secondary
antibody, and the blots were developed using enhanced chemiluminescence
(ECL, Amersham Biosciences).
Subcellular Fractionation--
Human glioblastoma U87 cells were
homogenized with 15 strokes of a motor-driven Teflon pestle, and the
subcellular fractions were obtained by sequential differential
centrifugation as described previously (24). Serum-starved or epidermal
growth factor (EGF; 100 ng/ml)-stimulated cells were fractionated into
high density microsomal, cytosolic, plasma membrane/ER, and high speed
pellet fractions, which were immunoblotted with affinity-purified
anti-SKIP antibodies. The fractions were also analyzed by
immunoblotting with organelle-specific markers: calnexin for the
endoplasmic reticulum (ER), Intracellular Localization of Endogenous SKIP--
COS-7 or U87
cells were grown on glass coverslips in DMEM supplemented with 10%
FCS. Cells were washed with phosphate-buffered saline and then fixed
and permeabilized for 10 min in phosphate-buffered saline with 3.7%
formaldehyde and 0.2% Triton X-100. Affinity-purified preimmune and
anti-peptide sera specific for SKIP were incubated with the cells for
1 h at room temperature. The cells were washed and incubated with
Alexa 488-conjugated anti-rabbit IgG. For colocalization studies, the
cells were incubated with anti- Transient Expression of GFP- or HA-tagged
SKIP/SKICH and SKICH Domain Mutants--
The open
reading frame of SKIP was PCR-amplified and cloned in-frame into the
pEGFP-C2 (Clontech) or pCGN vector, adding an N-terminal GFP or HA tag, respectively, to SKIP. The SKIP truncation mutants SKIP Intracellular Localization of Recombinant 107-kDa PIPP--
The
full-length cDNA encoding mouse PIPP or the SKICH domain (aa
767-836) of PIPP was PCR-amplified and cloned in-frame into the pCGN
vector, adding an N-terminal HA tag. The constructs were transiently
transfected into COS-7 cells as described for the SKIP constructs. The
HA-tagged recombinant proteins were stained with anti-HA antibody, and
fluorescence analyzed by confocal microscopy as described above.
Redistribution of Endogenous or Recombinant SKIP upon EGF or
Insulin Stimulation--
COS-7 and U87 cells were grown on coverslips
in DMEM supplemented with 10% FCS, and C2C12 cells were grown in DMEM
supplemented with 20% FCS. The cells were serum-starved by placement
in DMEM containing 0.1% FCS for ~24 h. COS-7 or C2C12 cells
transfected with various SKIP constructs were grown in normal medium
for 24 h before serum starvation. Following overnight serum
starvation, the cells were stimulated with EGF (100 ng/ml) or insulin
(100 nM), fixed, permeabilized, and stained with
affinity-purified anti-SKIP antibodies or anti-HA antibody as described
above; and where indicated, the cells were also stained with
phalloidin. The localization of the wild-type and truncation fusion
proteins at the plasma membrane was quantified by scoring >200
transfected cells over three independent transfections. For all
constructs, cells demonstrating plasma membrane staining were expressed
as a percentage of total transfected cells.
Phosphoinositide 5-Phosphatase Assays--
The open reading
frame of SKIP was PCR-amplified and cloned in-frame into the pTrcHisB
vector (Invitrogen), adding an N-terminal hexahistidine tag. The
recombinant protein was expressed in and purified from
Escherichia coli as described previously (10). 32P-Labeled PtdIns(3,4,5)P3 and
PtdIns(3,5)P2 were synthesized, and the 5-phosphatase
assays were performed using purified His-tagged recombinant SKIP or the
His tag alone as described (25, 26).
Characterization of the Intracellular Localization of
SKIP--
To investigate the subcellular distribution of SKIP
isoforms, we developed anti-peptide antibodies raised against a fusion peptide representing the N- and C-terminal seven amino acids of human
51-kDa SKIP (19). Immunoblot analysis using this affinity-purified antibody detected a 51-kDa polypeptide in both undifferentiated Sol8
myoblasts and differentiated myotubes. A weakly staining polypeptide of
36 kDa was also detected (Fig.
1A), which may represent the
smaller spliced isoform of SKIP predicted by cloning studies (19). In
U87 cells, a 51-kDa peptide and a faint 36-kDa peptide were detected in
the Triton-soluble fraction.
The intracellular localization of endogenous SKIP was investigated by
indirect immunofluorescence microscopy of several cell lines, including
COS-7 and U87 cells. In all cell lines, the predominant staining
pattern was punctate, localizing to the perinuclear region (Fig.
1B). Preimmune antiserum was non-reactive. Similar results were obtained when the recombinant 51-kDa isoform of SKIP fused to
either HA or GFP was expressed in COS-7 cells. Immunoblot analysis of
SKIP-transfected cells using antibodies specific to each tag indicated
that HA- or GFP-tagged recombinant 51-kDa SKIP was expressed intact
with minimal proteolysis (Fig. 1C).
The localization of endogenous 51-kDa 5-phosphatase was further
investigated by comparing the SKIP staining pattern in U87 cells with
that of antigens commonly used as markers of specific intracellular
compartments (Fig.
2A). Specific
antibodies were used in separate samples to stain the lysosomes
(LAMP2), clathrin-coated vesicles (
We also determined the SKIP localization in subcellular fractions of
serum-starved and EGF-stimulated U87 cells, isolated using a simplified
differential centrifugation that enables enrichment of fractions with
markers for the plasma membrane/ER, endosomes and clathrin-coated
vesicles (high density microsomes), and intracellular membranes
comprising clathrin-coated vesicles and other membranes in the high
speed pellet (24). The supernatant designated the cytosol contained all
elements that did not sediment at 177,000 × g (Fig.
2C, CYT). In whole cell lysates of U87 cells, two
isoforms of SKIP migrating at ~51 and 36 kDa, respectively, were
detected, consistent with the two predicted isoforms identified by
cloning studies. Following subcellular fractionation, the 51-kDa SKIP isoform was found in the plasma membrane/ER fraction, colocalizing with
markers specific for the plasma membrane (EGF receptor) and the ER
(calnexin). Markers for the early endosomes (EEA1) also co-sedimented
with this fraction. Neither SKIP isoform was detected in the cytosolic
fraction. However, the smaller SKIP isoform was consistently found in
the high speed pellet, which co-sedimented with the marker for
trans-Golgi-derived clathrin-coated vesicles, SKIP Hydrolyzes PtdIns(3,4,5)P3, but Not
PtdIns(3,5)P2--
Previous analysis of the substrate
specificity of SKIP has demonstrated that the enzyme hydrolyzes
PtdIns(3,4,5)P3, forming PtdIns(3,4)P2, and
PtdIns(4,5)P2, forming PtdIns(4)P (19). In this study, we
were unable to demonstrate any enzyme activity against
Ins(1,4,5)P3 or Ins(1,3,4,5)P4 (data not
shown). Because PtdIns(3,4,5)P3 has not been localized to
the ER, we also investigated whether SKIP hydrolyzes the more recently
identified phosphoinositide PtdIns(3,5)P2, which is
implicated in regulating endomembrane integrity in mammalian cells and
in yeast vacuolar function (27). To date, hydrolysis of
PtdIns(3,5)P2 by SKIP has not been reported. His-tagged
recombinant SKIP was expressed in E. coli and
affinity-purified, and enzyme assays were performed using
32P-labeled PtdIns(3,5)P2 as the substrate. The
identity of PtdIns(3,5)P2 was verified as described
previously (25). No hydrolysis of PtdIns(3,5)P2 was
detected using purified recombinant enzyme. However, SKIP actively
hydrolyzed PtdIns(3,4,5)P3, forming
PtdIns(3,4)P2 (Fig. 3),
indicating that the enzyme is active, but unlikely to regulate
PtdIns(3,5)P2.
SKIP Translocates to the Plasma Membrane of EGF- or
Insulin-stimulated Cells--
A significant in vitro
substrate for SKIP is PtdIns(3,4,5)P3 (19). Because it is
not clear if PtdIns(3,4,5)P3 is present on the ER, we
investigated the subcellular distribution of SKIP in COS-7 cells
following EGF stimulation, conditions under which PtdIns(3,4,5)P3 is transiently synthesized on the inner
surface of the plasma membrane (28, 29). COS-7 cells were serum-starved for 24 h and then stimulated with 100 ng/ml recombinant EGF for up
to 20 min. The localization of endogenous SKIP was detected by indirect
immunofluorescence (Fig. 4A).
In >90% of unstimulated cells, SKIP showed a predominantly
perinuclear distribution. However, within 1 min of EGF stimulation,
SKIP staining was detected at the plasma membrane initially at membrane
ruffles, areas at the leading edge of the cell (lamellipodia) where the
plasma membrane detaches from the support and rolls up (Fig.
4A, arrows). By 5 min of stimulation, SKIP
localized diffusely at the plasma membrane. Despite the increase in
plasma membrane staining, there was no obvious decrease in perinuclear
staining. The localization of SKIP at the plasma membrane was only
transient; and within 20 min, the 5-phosphatase relocalized to a
perinuclear distribution, with little plasma membrane staining
detected. Co-staining of COS-7 cells with phalloidin, which stains
polymerized actin, demonstrated no colocalization of SKIP with
actin in unstimulated cells. However, following EGF stimulation,
conditions under which membrane ruffles and actin are actively formed
and remodeled (30), SKIP colocalized with submembranous actin (as shown
by co-staining with phalloidin) at the leading edge of the cell,
specifically at membrane ruffles (Fig. 4B,
arrow). As previous studies have demonstrated that SKIP is
most highly expressed in skeletal muscle, we repeated these studies by
staining endogenous SKIP (Fig. 4C) or by expressing HA-tagged recombinant SKIP in the skeletal muscle cell line C2C12 (Fig.
4D). In unstimulated cells, SKIP localized in a
perinuclear distribution, with minimal colocalization with
submembranous actin detected by phalloidin staining. Following insulin
stimulation, conditions that promote membrane ruffling (31), both
endogenous (Fig. 4C) and recombinant (Fig. 4D)
SKIP staining was detected at areas of increased submembranous actin
staining, consistent with localization at membrane ruffles
(arrows in merged images). Membrane staining was not
detected in cells expressing empty vector (data not shown).
Identification of a Novel C-terminal Domain Designated
SKICH--
To identify the possible domains mediating membrane ruffle
localization, we undertook a bioinformatics analysis of the SKIP amino
acid sequence. We have identified a novel domain in human SKIP, a
128-amino acid region C-terminal to the 5-phosphatase domain. Data base
searching revealed that this region is highly conserved in mouse SKIP
(68% identity, EXPECT score of 2 × 10 Role of the SKICH Domain in Plasma Membrane Localization--
To
investigate the role of the SKICH domain in regulating intracellular
localization, the wild-type 5-phosphatase (HA-SKIP), mutant recombinant
SKIP lacking the SKICH domain (aa 1-324) (HA-SKIP
We further characterized the SKIP SKICH domain, in particular, the role
the regions surrounding the conserved motifs
DWXGX3VGX6YX4W and GX3PF play in mediating plasma membrane
and/or ER localization. The SKICH domain truncation mutants included
SKIP-(321-431), which lacks the SKIP C-terminal region beyond the
highly conserved motifs; SKIP-(360-448), which lacks the N-terminal
region of the SKICH domain prior to the conserved motifs; and
SKIP-(360-431), which comprises only the conserved regions within the
SKICH domain common to both SKIP and PIPP (Figs. 5 and
7A). For clarity, we have also shown the localization of the intact SKIP SKICH domain. We expressed these various recombinant proteins and determined the percentage of
cells demonstrating plasma membrane expression in resting and growth
factor-stimulated cells. These recombinant proteins were expressed at
the predicted size with minimal proteolysis. In serum-starved cells,
expression of SKIP-(321-431), which lacks the C-terminal region of the
SKICH domain, and expression of SKIP-(360-448), which lacks the
N-terminal region of the SKICH domain, demonstrated that 33% of cells
showed plasma membrane staining, an increased percentage compared with
the intact SKICH domain (Fig. 7B versus Fig.
6C). Following growth factor stimulation, these mutant
recombinant proteins behaved comparable to the intact SKICH domain,
with 75-80% of cells showing plasma membrane staining (Fig.
7B). SKIP-(360-431), which contains conserved SKICH
sequences, but lacks the N- and C-terminal regions of the SKICH domain,
demonstrated plasma membrane staining in the majority of serum-starved
(>60%) and EGF-stimulated (78%) cells (Fig. 7, A and
B).
The SKICH domain mutational analysis demonstrated that SKIP sequences
C-terminal to residue 431 might play a role in preventing constitutive
SKICH plasma membrane localization. We therefore investigated the
localization of the C-terminal 18 amino acids of SKIP. This sequence is
not found in the 5-phosphatase PIPP (Fig. 5), which did not localize to
the ER, but rather localized constitutively to the plasma membrane (see
Fig. 8). The SKIP C-terminal 18 amino acids (SKIP-(431-448)) were
fused to GFP and expressed in serum-starved and EGF-stimulated COS-7
cells (Fig. 7C). This fusion protein demonstrated a
perinuclear expression under both resting and stimulated conditions,
with little plasma membrane localization under either serum-starved or
stimulated conditions. We also noted intense nuclear expression of this
fusion protein, consistent with the passive diffusion of the
recombinant 28-kDa fusion protein into the nucleus. Collectively, this
analysis identified a "core sequence" within the SKICH domain
that mediates plasma membrane localization in both serum-starved and
growth factor-stimulated cells.
We noted, within the core sequence of the SKICH domain, a number
of highly conserved aromatic amino acids, which, in other proteins such
as the annexins, play a significant role in regulating membrane
association (36). To investigate the role of conserved residues within
the SKICH domain, we generated, by site-directed mutagenesis, mutations
of three of the highly conserved aromatic residues within the intact
SKICH domain (SKIP aa 321-448), Y349A or Y349F, W362A, and Y376A or
Y376F, and one other highly conserved residue, D361A. Mutation of the
conserved tyrosines (Y349A or Y349F and Y376A or Y376F) had no effect
on SKICH EGF-stimulated membrane ruffle localization (Fig.
7D). However, the D361A mutant and mutation of the adjacent
conserved aromatic residue (W362A) resulted in a significant decrease
in SKICH membrane association following EGF stimulation. These data
support the contention that the SKICH domain interacts in a specific
manner with a specific partner (either protein or lipid) within the
plasma membrane. However, we have not yet identified the precise nature
of the partner and are currently investigating both possibilities.
The SKICH Domain Mediates Membrane Ruffle Localization of the
107-kDa 5-Phosphatase PIPP--
Our bioinformatics analysis predicts
that the recently identified 5-phosphatase PIPP also contains a SKICH
domain. Studies by Mochizuki and Takenawa (20) have shown that PIPP
localizes constitutively to membrane ruffles. PIPP contains extensive
N- and C-terminal proline-rich domains. Overexpression of Myc-tagged recombinant PIPP and various deletion mutants in COS-7 cells by these
investigators demonstrated that deletion of the N-terminal proline-rich
domain does not result in loss of 5-phosphatase plasma membrane
expression. However, deletion of the C-terminal 277 amino acids (aa
725-1001 of rat PIPP), which includes the SKICH domain followed by the
C-terminal proline-rich domain, results in loss of localization of the
5-phosphatase to membrane ruffles (20). The sequences mediating
membrane localization were not further delineated. Our studies
predict that the PIPP C-terminal region contains a SKICH domain located
within aa 725-837, which encodes the analogous sequence in the
SKIP-(360-431) mutant and may constitutively associate with the plasma
membrane (Fig. 7A). We investigated whether this region in
PIPP mediated localization to membrane ruffles. HA-tagged wild-type
PIPP and the HA-tagged PIPP SKICH domain alone (aa 767-836)
(PIPP-SKICH) were expressed in resting and EGF-stimulated COS-7 cells.
In >90% of cells, wild-type PIPP localized in both the resting and
EGF-stimulated cells to membrane ruffles and also demonstrated
perinuclear staining (Fig. 8,
A and B). The isolated PIPP SKICH domain
localized to the plasma membrane ruffles in both resting (70%) and
EGF-stimulated cells (86%), comparable to the analogous region in the
SKIP domain (SKIP- (360-431)).
This study reports novel findings for two recently identified
signal-terminating 5-phosphatases, SKIP and PIPP. First, SKIP, a
PtdIns(4,5)P2 and PtdIns(3,4,5)P3
5-phosphatase, localizes to the ER in the unstimulated cell. Second,
SKIP translocates to membrane ruffles mediated by a novel domain we
have designated SKICH, which is found in another 5-phosphatase (PIPP)
and members of the TRAF6-binding protein family. Third, we have
delineated the core sequences within the SKICH domain that mediate
constitutive membrane association and the surrounding sequences
specific to SKIP that contribute to ER localization. Fourth, the
107-kDa 5-phosphatase PIPP also contains a SKICH domain that
constitutively localizes to membrane ruffles in both resting and
EGF-stimulated cells. The SKICH domain may thereby provide a mechanism
for localizing these 5-phosphatases to their specific substrates on the
inner wall of the plasma membrane.
Intracellular Localization of SKIP at the ER--
In the
unstimulated cell, SKIP is predominantly localized to the ER.
PtdIns(3,4,5)P3 has not been localized to the ER; however, it is of interest that another PtdIns(3,4,5)P3 phosphatase,
a PTEN homolog designated TPIP The SKICH Domain--
We have identified the SKICH domain as a
novel plasma membrane-targeting domain. In addition to SKIP and PIPP,
the SKICH domain occurs in several other proteins, including NDP52 and
TXBP151 (T6BP). TXBP151 was originally identified as a Tax1-binding
protein that binds the zinc finger protein A20, which acts as an
inhibitor of cell death. Overexpression of TXBP151 inhibits tumor
necrosis factor-induced apoptosis by an unknown mechanism (32). More recently, TXBP151, also called T6BP, has been shown to associate with
TRAF6 in response to interleukin-1 stimulation. However, the
interaction between TRAF6 and T6BP does not appear to regulate TRAF6-induced NF-
The lipid microenvironment in which a signaling molecule is located can
direct the activation of downstream signaling cascades and interaction
with other signaling components. The SKICH domain may mediate either
protein/protein or protein/lipid interactions. It may function as a
lipid-binding domain, as has been shown for PH domains, PX domains, and
C2 domains; and this is the subject of ongoing laboratory
investigations. However, we have noted that, although the PIPP SKICH
domain constitutively associates with the plasma membrane, the SKIP
SKICH domain mediates plasma membrane localization predominantly in
response to growth factor stimulation. Deletion mutant analysis
identified the highly conserved core sequence within the SKICH domain
that mediates constitutive association with the plasma membrane in both
SKIP and PIPP. These data suggest that the proteins or lipids to which
the SKICH domain binds are constitutively present in the plasma
membrane. Because PtdIns(3,4,5)P3 is not detected in
serum-starved cells, this implies that this region of the SKICH domain
does not act like the PH domain and bind to
PtdIns(3,4,5)P3 or to other signaling proteins that are localized to the plasma membrane only in stimulated cells.
Localization of SKIP and PIPP at Membrane Ruffles--
The
translocation of SKIP to membrane ruffles in response to EGF
stimulation was not reported by Ijuin et al. (19). However, this may relate to the transient nature of the 5-phosphatase
association with membrane ruffles, as the enzyme rapidly relocalizes to
the ER within 20 min of stimulation. Recent studies have demonstrated that the spatial and temporal accumulation of
PtdIns(3,4,5)P3 at membrane ruffles correlates with actin
repolymerization at this site (46). In contrast to SHIP-2, which also
localizes to membrane ruffles and inhibits membrane ruffling (47),
overexpression of SKIP did not alter the membrane ruffling response
(data not shown). However, we were unable to express SKIP at high
levels due to the accumulation of the recombinant protein in
intracellular vesicles. It is noteworthy that Ijuin et al.
(19) reported that overexpression of SKIP results in decreased actin
stress fiber formation in regions of the cell where SKIP accumulates,
but these authors also did not report any effect on the membrane
ruffling response. Furthermore, PIPP, which also contains a SKICH
domain and localizes to membrane ruffles, does not regulate membrane ruffling (20). PtdIns(4,5)P2 acts to restrict new actin
polymerization at the plasma membrane, whereas
PtdIns(3,4,5)P3 has been proposed to induce local actin
polymerization at membrane ruffles (46). Therefore, the downstream
effects of SKIP on actin polymerization at membrane ruffles will depend
on the expression and localization of these two signaling molecules and
on which of these lipids is the preferred SKIP substrate in
vivo. Collectively, these studies have shown that SKIP localizes
to two discrete membranes, the ER in resting cells and the plasma
membrane in stimulated cells, and may hydrolyze distinct substrates at
each site (PtdIns(4,5)P2 and PtdIns(3,4,5)P3,
respectively) to regulate discrete cellular functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was
from PerkinElmer Life Sciences. Cell lines were from the American Type
Culture Collection. The pCGN vector and anti-EEA1 (early
endosomal autoantigen-1) antibody
were gifts from Drs. T. Tiganis and Ban-Hock Toh (Monash University),
respectively. Monoclonal antibodies were from the following sources:
anti-hemagglutinin (HA; Babco), anti-green fluorescent protein (GFP;
Roche Molecular Biochemicals), anti-calnexin (Affinity Bioreagents
Inc.), and anti-LAMP2 (Hybridoma Bank, University of Iowa). Alexa
fluor/Texas Red-conjugated secondary antibodies were from Molecular
Probes, Inc., and horseradish peroxidase-conjugated secondary
antibodies were from Silenus Laboratories Pty., Ltd. (Victoria). All
other chemicals were from Sigma unless stated otherwise.
-adaptin for
trans-Golgi-derived clathrin-coated vesicles, the EGF
receptor for the plasma membrane, and EEA1 for early endosomes.
-adaptin, anti-calnexin, anti-LAMP2,
anti-
-COP (coatomer protein), or anti-EEA1 antibody for 1 h at
room temperature, followed by incubation with Alexa 594-conjugated
anti-mouse IgG, or with Texas Red-conjugated concanavalin A for 5 min
at 4 °C. Coverslips were mounted using SlowFade (Molecular Probes,
Inc.) and visualized by confocal microscopy (Leica).
SKICH (amino acids (aa) 1-324) and SKIP-SKICH (aa 321-448) and the SKICH domain mutants SKIP-(321-431),
SKIP-(360-448), SKIP-(360-431), and SKIP-(431-448) were also
generated by PCR amplification and cloned in-frame into the pCGN or
pEGFP-C2 vector. Site-directed mutagenesis of the SKICH domain (SKIP aa
321-448) to obtain Y349A or Y349F, D361A, W362A, and Y376A or Y376F
was performed using sequential PCRs. The sequences of all constructs and mutants were confirmed by dideoxy sequencing of both strands. COS-7
cells were transiently transfected by electroporation with 1-2 µg of
full-length SKIP or its truncation constructs. Approximately 3 × 106 cells were electroporated in DMEM in a total volume of
200 µl in a 0.4-cm sterile cuvette at 200 V and 975 microfarads using the Bio-Rad Gene Pulser II. After 48 h, the cells were fixed and permeabilized as described above. The HA-tagged proteins were localized
with anti-HA antibody and detected with Alexa 488-conjugated anti-mouse
IgG. The expression of the GFP- or HA-tagged recombinant protein was
confirmed by immunoblot analysis of the transfected cell lysates with
anti-GFP or anti-HA antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Intracellular localization of SKIP.
A, Sol8 myoblasts (100 µg) differentiated for 0, 1, 2, or
3 days or whole cell lysate (L) or the Triton-soluble
(TS) or Triton-insoluble (TI) fraction of U87
cells were immunoblotted with affinity-purified anti-peptide antibodies
to SKIP. B, COS-7 and U87 cells were fixed, permeabilized,
and stained with anti-peptide antibodies to SKIP. The protein was
visualized with Alexa 488-conjugated anti-rabbit IgG, and
fluorescence was analyzed by confocal microscopy. Preimmune sera were
non-reactive. C, COS-7 cells were transiently transfected
with constructs for HA- or GFP-tagged SKIP. 48 h
post-transfection, the cells were fixed, permeabilized, processed for
direct (GFP) or indirect (anti-HA, Alexa 488-conjugated anti-mouse IgG)
immunofluorescence, and analyzed by confocal microscopy. 100-µg
lysates of transfected cells were subjected to 10% SDS-PAGE, followed
by immunoblotting with anti-GFP or anti-HA antibody.
-adaptin), ER (antibodies to the
ER-specific protein calnexin or concanavalin A), or polymerized actin
(phalloidin). Little colocalization was observed with the lysosomes
(Fig. 2A). Some areas of colocalization were detected
between SKIP and
-adaptin, as shown in the merged images; however,
5-phosphatase expression also appeared in areas distinct from
-adaptin staining. The majority of SKIP staining coincided with the
ER, as shown by colocalization with calnexin and concanavalin A. No
colocalization of SKIP with phalloidin staining or with the endosomal
marker EEA1 was demonstrated (data not shown). Little expression of
SKIP was detected at the plasma membrane. Analysis of the expression of
HA-tagged recombinant 51-kDa SKIP demonstrated colocalization with the
ER-specific marker concanavalin A when the recombinant protein was
expressed in COS-7 cells, but no colocalization with early endosomes
(EEA1) and only partial colocalization with the Golgi marker
-COP
(Fig. 2B).
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Fig. 2.
Colocalization of SKIP with ER-specific
markers. A, U87 cells were labeled with
affinity-purified anti-peptide antibodies to SKIP and antibodies to
organelle-specific markers as indicted: LAMP2 (lysosomes), -adaptin
(Golgi), calnexin (ER), or concanavalin A (ER). Anti-SKIP antibodies
were visualized with Alexa 488-conjugated anti-rabbit IgG, and
organelle-specific markers were detected with Alexa 594-conjugated
anti-mouse IgG or Texas Red-labeled concanavalin A. Fluorescence was
analyzed by confocal microscopy. Regions of colocalization appear yellow in overlay images. B,
COS-7 cells were transiently transfected with construct encoding
HA-tagged SKIP and processed for indirect (anti-HA, Alexa
488-conjugated anti-mouse IgG) immunofluorescence. The transfected
cells were also counterstained with antibody to the Golgi marker
-COP, anti-EEA1 antibody, or Texas Red-labeled concanavalin A as
indicated. Areas of colocalization appear yellow in overlay
images. C, serum-starved (0 min) and EGF (100 ng/ml)-stimulated (5 min) U87 cells were fractionated using sequential
differential centrifugation. Fractions including the whole cell lysate
(WCL), high density microsomes (HDM), cytosol
(CYT), plasma membrane/ER (PM/ER), and high speed
pellet (HSP) were analyzed by immunoblotting with
anti-peptide antibodies to SKIP. The fractions were also immunoblotted
with organelle-specific markers: calnexin (ER),
-adaptin (Golgi),
EGF receptor (EGFR; plasma membrane), and EEA1 (early
endosomes). The migration positions of molecular mass markers are shown
on the left.
-adaptin,
consistent with the colocalization data shown in Fig. 2A.
The smaller isoform of SKIP may have a discrete membrane localization; however, this isoform is unlikely to express 5-phosphatase activity, as
it lacks the 57 amino acids of the N-terminal 5-phosphatase domain (19)
and therefore lacks critical conserved residues that have recently been
shown to be essential for 5-phosphatase catalytic activity (10).
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Fig. 3.
SKIP phosphoinositide 5-phosphatase
activity. Purified His-tagged recombinant SKIP in duplicate
samples (lanes 2 and 3) or the His tag alone
(lane 1) was used in 5-phosphatase enzyme assays and
analyzed by thin-layer chromatography using the 32P-labeled
phosphoinositide substrates PtdIns(32P3,5)P2
(a) and PtdIns(32P3,4,5)P3
(b). The migration positions of phosphoinositides are shown
on the right.
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Fig. 4.
Translocation of SKIP to the plasma membrane
upon EGF stimulation. A, COS-7 cells were serum-starved
for 24 h and stimulated with EGF (100 ng/ml) for the indicated
periods. The cells were fixed and stained with affinity-purified
anti-peptide antibodies to SKIP. Arrows indicate membrane
ruffles. B, COS-7 cells were serum-starved for 24 h and
stimulated with EGF (100 ng/ml) for 10 min. The cells were labeled with
anti-peptide antibodies to SKIP and Texas Red-conjugated phalloidin.
C, C2C12 cells were serum-starved for 24 h and
stimulated with insulin (100 nM) for 10 min and then
double-stained with anti-SKIP antibodies and Texas Red-conjugated
phalloidin. D, C2C12 cells were transiently transfected with
the HA-tagged SKIP construct. Following 24 h of serum starvation,
the cells were stimulated with insulin (100 nM) for 10 min.
The cells were stained with anti-HA antibody and Texas Red-conjugated
phalloidin. Arrows in the merged images demonstrate membrane
ruffle localization.
49) and is
also present C-terminal to the 5-phosphatase domain in 107-kDa PIPP
(38% sequence identity, EXPECT score of 2 × 10
36).
Our searches revealed that the C-terminal region of human SKIP shares
significant sequence similarity (20-23% identity, EXPECT scores
ranging from 5 × 10
36 to 2 × 10
48) with an ~130-amino acid region in the N terminus
of the recently described protein T6BP
(TRAF6-binding
protein), which has also been characterized as Tax1 (human
T-cell leukemia virus type I)-binding protein-1, TXBP151 (32, 33). We
have also noted this domain in a chicken (Gallus gallus)
protein homologous to T6BP (GenBankTM accession
number BAA94854). We also identified this domain in a cytoplasmic
protein of unknown function, NDP52 (34). All EXPECT scores are well
below the significance threshold of 1 × 10
6 as
described by Park et al. (35). A sequence alignment of all domains revealed that this novel domain is characterized by the highly
conserved motif
DWXGX3VGX6YX4W
and a second motif, GX3PF (Fig.
5A). We suggest that this
region in the C terminus of SKIP thus represents a novel domain, which
we have designated SKICH for SKIP carboxyl
homology. The original cloning studies reported that the
SKIP amino acid sequence contains a central 5-phosphatase domain and no
other signaling or targeting motifs; however, our analysis demonstrates
that this domain is present in the C terminus of 2 of the 10 mammalian
5-phosphatases and in the N terminus of a distinct family of proteins,
the TRAF6/Tax1-binding protein family (Fig. 5B).
Hydrophobicity analysis demonstrated no obvious transmembrane region or
C-terminal anchoring tail within the SKICH domain (data not shown).
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Fig. 5.
Amino acid sequence alignment of the SKICH
domain. A, multiple sequence alignment of SKICH
domain-containing proteins: human SKIP (beginning at aa 321;
GenBankTM/EBI accession number XP_008491), mouse SKIP
(beginning at aa 340; accession number NP_032942), rat PIPP (beginning
at aa 725; accession number BAA90553), human PIPP (beginning at aa 779;
accession number AAD15618), human T6BP (beginning at aa 1; accession
number AAG03025), and human NDP52 (beginning at aa 11; accession number
A56733). Conserved residues are boxed and in
boldface. Hydrophobic residues are yellow; polar
non-charged residues are green; acidic residues are
red; and basic residues are blue. Human SKIP
residues are numbered in red above the alignment.
hsa, Homo sapiens; mmu, Mus
musculus; rno, Rattus norvegicus.
B, schematic representation of the predicted domain
structures of SKICH domain-containing proteins.
SKICH), and the
SKICH domain alone (aa 321-448) (HA-SKIP-SKICH) were expressed as
fusion proteins with an HA tag in COS-7 cells (Fig.
6A). Immunoblot analysis using
antibodies to the HA tag of transfected cell lysates demonstrated that
these mutant recombinant proteins were expressed intact and migrated at
their predicted molecular mass (Fig. 6B). To quantitate the
localization of the wild-type and mutant fusion proteins at the plasma
membrane, >200 transfected cells were scored (over three independent
transfections for each construct), and the percentage of cells
demonstrating SKIP plasma membrane expression was determined in both
serum-starved and EGF-stimulated cells (Fig. 6C). In resting
cells, wild-type recombinant SKIP localized in a perinuclear
distribution, as shown for the endogenous protein, consistent with an
ER localization; and 25% of cells demonstrated plasma membrane
staining (Fig. 6, A and C). We noted that
expression of recombinant SKIP resulted in a greater percentage of
cells with plasma membrane staining in serum-starved cells than
observed upon staining the endogenous protein using anti-peptide
antibodies, when <5% of cells demonstrated plasma membrane staining
(data not shown). In resting cells, the recombinant 5-phosphatase that lacked the SKICH domain (HA-SKIP
SKICH) localized in a perinuclear distribution (Fig. 6A); however, in some cells, a more
diffuse perinuclear localization was demonstrated compared with
wild-type SKIP localization. The SKICH domain alone (HA-SKIP-SKICH)
localized in serum-starved cells in a perinuclear distribution, with
<25% of cells showing plasma membrane staining (Fig. 6, A
and C). Following growth factor stimulation, wild-type SKIP
expression at the plasma membrane was significantly increased, with
76% of cells demonstrating plasma membrane staining. In contrast,
cells expressing mutant SKIP lacking the SKICH domain (HA-SKIP
SKICH)
localized with a perinuclear distribution, and there was no increase in
the number of cells demonstrating plasma membrane expression (<25% of
cells) following growth factor stimulation (Fig. 6, A and
C). The SKICH domain alone (HA-SKIP-SKICH) behaved like the
wild-type 5-phosphatase, with 80% of stimulated cells demonstrating
significant plasma membrane staining.
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Fig. 6.
Role of the SKICH domain in plasma membrane
translocation. COS-7 cells were transiently transfected with
constructs encoding HA-SKIP, HA-SKIP SKICH, or HA-SKIP-SKICH as
shown. A, transfected cells were serum-starved for 24 h, stimulated with EGF (100 ng/ml) for 10 min, and prepared for
indirect immunofluorescence (anti-HA, Alexa 488-conjugated anti-mouse
IgG). B, 100-µg lysates of cells transfected with
HA-SKIP
SKICH (lane 1) or HA-SKIP-SKICH (lane
2) were analyzed by immunoblotting with anti-HA antibody.
C, transfected cells were scored for staining of recombinant
fusion proteins at the plasma membrane in serum-starved (white
bars) versus EGF-stimulated (gray bars)
cells, and the results are expressed as the mean percentage ± S.D. of all cells examined. A minimum of 200 cells were counted for
three independent transfections.
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Fig. 7.
Characterization of SKICH domain
mutants. A, the indicated SKICH domain mutants of SKIP
were expressed in COS-7 cells. Serum-starved ( EGF) and
EGF-stimulated (+EGF) cells were fixed, permeabilized, and
processed for confocal microscopy (anti-HA, Alexa 488-conjugated
anti-mouse IgG), or lysates were immunoblotted for the recombinant
fusion proteins using anti-HA antibody. B, transfected cells
were scored for staining of recombinant fusion proteins at the plasma
membrane in serum-starved (white bars) versus
EGF-stimulated (gray bars) cells, and the results are
expressed as mean percentage ± S.D. of all cells examined. A
minimum of 200 cells were counted for three independent transfections.
C, the C-terminal 18 amino acids of SKIP (SKIP-(431-448))
were expressed as a fusion protein with GFP in COS-7 cells.
Representative images are shown. D, the Y349A, D361A, W362A,
and Y376F SKICH domain (SKIP aa 321-448) mutants were expressed with
an HA tag in COS-7 cells, and the localization of mutant SKICH was
determined in serum-starved or EGF-stimulated cells (5 min).
Representative images are shown.
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Fig. 8.
Cellular localization of
PIPP-SKICH. A, COS-7 cells were transiently
transfected with constructs encoding HA-PIPP or HA-PIPP-SKICH.
Serum-starved ( EGF) or EGF-stimulated (+EGF)
cells were processed for confocal microscopy (anti-HA, Alexa
488-conjugated anti-mouse IgG). B, transfected cells were
scored for staining of recombinant fusion proteins at the plasma
membrane in serum-starved (white bars) versus
EGF-stimulated (gray bars) cells, and the results are
expressed as the mean percentage ± S.D. of all cells examined. A
minimum of 200 cells were counted for three independent
transfections.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TPEP (transmembrane
phosphatase with tensin homology)) and PTEN homologous inositol
lipid phosphatase
) that hydrolyzes the 3-phosphate from
PtdIns(3,4,5)P3, PtdIns(3,5)P2, PtdIns(3,4)P2, and PtdIns(3)P, also localizes to the ER
(37). Although PtdIns(3,4,5)P3 has not been identified in
the ER, recent studies using the PH domain of phospholipase
C
1 have identified, by electron microscopy, that
PtdIns(4,5)P2, in addition to its prominent localization
on the plasma membrane, is also detected on the ER, Golgi stack,
endosomes, and nucleus (38). SKIP also hydrolyzes
PtdIns(4,5)P2, forming PtdIns(4)P. Several recent studies have implied a potential role for PtdIns(4)P and
PtdIns(4,5)P2 in ER vesicular function. In yeast, there
exists a substantial pool of the precursor of
PtdIns(4,5)P2, PtdIns(4)P, which is present in the ER (39).
The mammalian PtdIns 4-kinase, the yeast homolog Stt4p (40, 41), and a
novel PtdIns(5)P 4-kinase localize to the ER (42). PtdIns(4)P promotes
Golgi exocytic trafficking and inhibits ER-to-Golgi transport. Yeast
sac1 null mutants demonstrate accumulation of PtdIns(4)P,
and trafficking from the ER to the Golgi is slowed (39). In a manner
analogous to SKIP, the yeast PtdIns(4,5)P2 5-phosphatase
Inp54p localizes to the cytosolic surface of the ER, anchored to this
site via its C terminus (43). Furthermore, inp54 null
mutants demonstrate enhanced secretion of reporter proteins from the
ER, consistent with the contention that PtdIns(4,5)P2 may
play a positive role in the regulation of secretory transport from this
compartment (43). The localization of SKIP to the ER may play a role
analogous to Inp54p in regulating PtdIns(4,5)P2 levels and
thereby vesicular trafficking from this compartment.
B or JNK (c-Jun N-terminal
kinase) activation (33). In addition to the N-terminal
SKICH domain, the T6BP family contains three coiled-coil regions that
are proposed to be involved in TRAF6 binding (Fig. 5B).
Other T6BP family members that contain an N-terminal SKICH domain
include a 62-kDa muscle-derived protein from chicken, which promotes
neurite outgrowth (44). Our bioinformatics analysis predicts that
NDP52, whose function is currently unknown, and T6BP are structurally
related (Fig. 5B). NDP52 is a protein that was originally
identified as a component of nuclear domain 10 (34), but was
subsequently shown to have a cytosolic localization (45). The
intracellular localization of T6BP has not been reported; however, the
results of our studies suggest that this protein and NDP52 may localize
to the plasma membrane in resting or growth factor-activated cells,
mediated by the SKICH domain.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Harshal Nandurkar for the PIPP 5-phosphatase cDNA and Dr. Susan Brown for advice and helpful discussions.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Monash University, Wellington Rd., Clayton, Victoria 3800, Australia. Tel.: 61-39-905-1245; Fax: 61-39-905-3726; E-mail: christina.mitchell@med.monash.edu.au.
Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M209991200
2 Available at www.ncbi.nlm.nih.gov.
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ABBREVIATIONS |
---|
The abbreviations used are: PtdIns, phosphatidylinositol; Ins, inositol; HA, hemagglutinin; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; EGF, epidermal growth factor; ER, endoplasmic reticulum; aa, amino acid(s); PH, pleckstrin homology; PX, Phox homology.
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REFERENCES |
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