From the Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201
Received for publication, September 14, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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The mammalian phosphoinositide kinase PIKfyve
catalyzes the synthesis of phosphatidylinositol 5-P and
phosphatidylinositol 3,5-P2, thought essential in
cellular functions, including membrane trafficking. To discern the
intracellular loci of PIKfyve products' formation, we have examined
the localization of PIKfyve protein versus enzymatic
activity and a possible acutely regulated redistribution in 3T3-L1
adipocytes. Subcellular fractions of resting cells that were positive
for immunoreactive PIKfyve, such as cytosol (~76%), internal
structures (low density microsomal fraction (LDM), composed of
recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal
elements) (~20%), and plasma membrane (~4%),
expressed enzymatically active PIKfyve. While the presence of a FYVE
finger in PIKfyve predicts early endosome targeting, density gradient
sedimentation, immunoadsorption, and fluorescence microscopy analyses
segregated the LDM-associated PIKfyve from the membranes of the
recycling endosomes and GLUT4. PIKfyve fluorescence staining largely
coincided with trans-Golgi network/multivesicular body markers,
indicating PIKfyve's role in the late endocytic/biosynthetic pathways.
A subfraction of particulate PIKfyve resisted nonionic detergent
treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or
pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM,
which was coupled with a commensurate increase of PIKfyve lipid kinase
activity and an electrophoretic mobility shift. We suggest the
recruited PIKfyve specifies the site and timing of phosphoinositide
signals that are relevant to the acute insulin action.
The key function of phosphorylated derivatives of
phosphatidylinositol
(PtdIns),1 called
collectively PIs, in eukaryotic cell regulation has been recognized for
more than 2 decades with the discovery that PtdIns 4,5-P2
is converted by phospholipase C to two second messengers, inositol
trisphosphate and diacylglycerol. It has become increasingly clear,
however, that PIs have a signaling role in their own right, i.e. they do not require hydrolysis to set in motion
cellular processes. In most cases, PIs were found to serve as
site-specific signals on membranes that recruit/activate effector
protein complexes at the interface with the cytosol. Phosphoinositide
signals are used in this way by eukaryotic cells to modulate a large
number of responses, such as membrane ruffling, secretion, vesicular trafficking, insulin-regulated membrane translocation of the
fat/muscle-specific GLUT4 glucose transporter, cell adhesion,
chemotaxis, DNA synthesis, and cell cycle (1-4).
The intracellular PIs identified to date stem from the same precursor,
PtdIns, and differ by the degree and position of phosphorylation of
PtdIns' head group. Out of the five candidate phosphorylation positions, only the hydroxyls at positions D-3, D-4, and D-5 are found
phosphorylated intracellularly, separately or in all possible combinations, resulting in PtdIns 3-P, PtdIns 4-P, PtdIns 5-P, PtdIns
3,4-P2, PtdIns 4,5-P2, PtdIns
3,5-P2, and PtdIns 3,4,5-P3 (2). Among the PI
species, PtdIns 5-P and PtdIns 3,5-P2 have attracted
increasing attention. They have been identified only recently mainly
because PtdIns 5-P and PtdIns 3,5-P2 are poorly separated
from PtdIns 4-P and PtdIns 3,4-P2, respectively, under classical HPLC analysis (5-7). An enzyme activity, thought responsible for the main pathway of their biosynthesis in mammalian cells, called
PIKfyve (phosphoinositide kinase
for five position containing a fyve finger),
has been recently identified by molecular cloning (8). PIKfyve is a
large protein of 2052 amino acids, which displays an intrinsic,
wortmannin-resistant (ID50, 600 nM) lipid kinase activity to generate PtdIns 5-P and PtdIns
3,5-P2 in vitro (8, 9). While PIKfyve
intracellular distribution remains to be determined, of interest is
that PIKfyve contains two evolutionarily conserved, plausible
membrane-targeting, domains: a FYVE finger, found in other mammalian
proteins as a major determinant for localization to early endosomes
(10-12), and a DEP domain (named after Dishevelled, Egl-10, and pleckstrin; Ref. 13), reported
sufficient for targeting to specific membrane compartments (14, 15). In
Saccharomyces cerevisiae, only PtdIns 3,5-P2 and
not PtdIns 5-P is documented (16). The enzyme responsible for its
biosynthesis is the 2278-amino acid protein Fab1p (16, 17) that, with
the exception of the DEP region, shares similar domain architecture and
is related in sequence to PIKfyve (8). Fab1p is found associated with fractions enriched for Golgi/endosome, lysosomal, or cytosolic marker
proteins (16). Yeast strains defective in Fab1p, concomitantly with a
depletion of the PtdIns 3,5-P2 pool, display severe defects in cell growth, expanded vacuoles, which fail to acidify, and defects
in MVB formation. Thus, through PtdIns 3,5-P2 production, Fab1p is thought to regulate several steps in yeast membrane
trafficking such as internal vesicle formation or cargo selection
within the MVB and the recycling/turnover of membranes from the
vacuolar surface to earlier compartments (18). That PIKfyve may play a
role in membrane trafficking events in mammals is suggested by the
results of complementation experiments in fab1-deficient yeast mutants, demonstrating that PIKfyve exhibits the ability to
suppress the vacuolar defect and restore the basal PtdIns
3,5-P2 pool (19). Clearly, these studies underscore the
importance of PIKfyve in cellular regulation and suggest that local
generation of PtdIns 3,5-P2 and probably PtdIns 5-P may
have an important role in signaling and/or execution of membrane
trafficking in mammalian cells. Because PtdIns 5-P has never been
detected in yeast, it is conceivable this PI derivative, produced
presumably by PIKfyve enzymatic activity, has evolved over time to
serve specific mammalian cell functions.
A common feature among the cellular events regulated by PIs is that
they are spatially and temporally restricted. Therefore, the key to our
understanding of the molecular mechanism underlying the PI-regulated
signals rests upon knowledge of the precise site and timing of the PI
generation. Thus far, direct measurement of PI levels in
situ meets several restrictions mainly related to the fact that
PIs are in low abundance with a rapid turnover and therefore cannot be
readily approached by conventional techniques (20). An alternative
approach involves following the enzyme activity's intracellular
location with the premise that the latter will largely dictate where
localized synthesis of the phosphoinositide products can occur. In this
study, we undertook biochemical and morphological approaches to
characterize the intracellular localization of PIKfyve protein and to
relate it to PIKfyve enzymatic activity. We demonstrate here that
subcellular fractions of resting 3T3-L1 adipocytes that were found
positive for immunoreactive PIKfyve expressed an enzymatically active
protein. PIKfyve largely colocalized with markers for
Golgi-to-late-endosome traffic but was segregated away from recycling
endosomal pathway markers. A subpopulation of the PIKfyve particulate
pool was found to be detergent-resistant and cofractionated with
structures, possibly cytoskeletal, recently reported positive for key
elements of the insulin-signaling circuit relevant to GLUT4 exocytosis
in 3T3-L1 adipocytes. We further demonstrate that acute insulin
stimulation in this cell type induces spatially localized recruitment
of PIKfyve to inner membranes, thus implying PIKfyve lipid products as
regulated site-specific signals relevant to acute insulin action.
Cell Cultures, Antibodies, and Fusion
Proteins--
Differentiation of 3T3-L1 mouse fibroblasts into
insulin-sensitive adipocytes on plates or glass coverslips was
described previously (21). Cells were used between 7 and 14 days after the onset of the differentiation program. COS-7 cells were maintained in DMEM, containing 10% fetal bovine serum, 50 units/ml
penicillin, and 50 µg/ml streptomycin sulfate. Rabbit polyclonal
anti-PIKfyve antiserum (R7069; East Acres) was directed against a
recombinant GST fusion protein comprising the N terminus of PIKfyve
(amino acids 1-100). Polyclonal or monoclonal antibodies against GLUT4 (R1288 and 1F8) and IRAP were gifts of Drs. M. Czech, P. Pilch and K. Kandror. Monoclonal anti-TfR (HTRH68.4), anti- Cell Treatment and Subcellular Fractionation--
3T3-L1
adipocytes (100-mm dish) were serum-deprived for 14 h in DMEM
supplemented with 0.5% bovine serum albumin. Cells were stimulated
with or without insulin (100 nM) or pervanadate (100 µM) for 7 and 20 min, respectively, at 37 °C, washed
twice with PBS and once with HES buffer (20 mM Hepes-HCl,
pH 7.5, 1 mM EDTA, 255 mM sucrose, containing
1× protease inhibitor mixture (1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml
pepstatin, and 1 mM benzamidine) and 1× phosphatase
inhibitor mixture (25 mM Immunoadsorption of GLUT4 Vesicles--
Immunopurification of
GLUT4-containing vesicles was achieved on anti-GLUT4 IgG as described
previously (21). Briefly, LDM fractions derived from insulin-treated or
untreated 3T3-L1 adipocytes were resuspended in PBS, supplemented with
1× protease and phosphatase inhibitor mixtures to a final
concentration of 1 mg/ml, and then immunoadsorbed (18 h at 4 °C) on
anti-GLUT4 IgG (R1288 antiserum, purified on protein A-Sepharose 4 Fast
Flow; Amersham Pharmacia Biotech) or control rabbit IgG (25 µg each).
Protein A-Sepharose CL-4B (Sigma) was added for the final 2 h of
the incubation. Following washings, the pellets along with aliquots of
the supernatants were subjected either to SDS-PAGE and immunoblotting
or to lipid kinase assay, as described below. The potency of anti-GLUT4
to quantitatively immunoadsorb GLUT4 vesicles was analyzed in a
parallel experiment, in which the samples were mixed in Laemmli buffer (no boiling) for 30 min at 25 °C and processed further for
immunoblotting. Anti-GLUT4 antibodies eliminated ~80% of the
GLUT4-vesicle pool in LDM.
Equilibrium Centrifugation in Self-formed Iodixanol
Gradient--
LDM fractions prepared from 3T3-L1 adipocytes and
resuspended in HES buffer supplemented with 1× protease and
phosphatase inhibitor mixtures were mixed with iodixanol (OptiPrep;
Sigma) in a polyallomer Quick-Seal centrifuge tube (13 × 51 mm;
Beckman) to either 14 or 30% iodixanol and 196 or 128 mM
sucrose concentrations, respectively, resulting in isoosmotic
solutions. A self-generating gradient was formed by centrifuging to
equilibrium at 4 °C in a VTi 65.2 rotor (Beckman L8-M-55 centrifuge)
for 4 h at 53,000 rpm as described (24). Fractions of ~0.35 ml
were collected from the bottom of the tube and were analyzed for
refractive index (Bausch and Lomb refractometer), protein
concentration, and presence of PIKfyve or other proteins by immunoblotting.
Transient Transfection and Fluorescence Microscopy--
COS-7
cells seeded on 100-mm dishes (for biochemistry) or 22 × 22 cm
coverslips (for microscopy) were transfected with cDNA constructs
indicated in the figure legends by LipofectAMINE (Life Technologies,
Inc.) or the calcium phosphate precipitation method as described
previously (22) and then processed for immunoprecipitation (see below)
or fluorescence microscopy. Twenty h post-transfection with
pEGFP-HA-PIKfyveS by LipofectAMINE, the cells were
serum-starved for 3 h and then incubated (3 or 15 min at 37 °C)
with Texas red Tf (8 µg/ml; Molecular Probes, Inc., Eugene, OR)
filtered through a 0.22-µm filter in DMEM (dye-free). The dishes were
placed on ice, and the cells were washed with ice-cold PBS and fixed
for 1 h at 4 °C with formaldehyde (4%) in PBS prior to
rewashing and mounting. The lysosomal compartment was labeled with
Texas red-conjugated dextran (1 mg/ml; Molecular Probes, Inc.; size
10,000) in DMEM (dye-free) plus 10% fetal bovine serum for 20 h
in COS cells, 24 h post-transfection by calcium phosphate
precipitation. The cells were then chased (2 h) with a dextran-free
medium and, after washes in PBS, were fixed in paraformaldehyde
(3.7%), rewashed, and mounted. Double labeling with anti-CI-MPR or
Immunoblotting and Immunoprecipitation--
For immunoblotting,
protein samples were fractionated by SDS-PAGE and then transferred onto
nitrocellulose membranes as previously described (8). The blots were
saturated with blocking buffer and probed (16 h at 4 °C) with the
antibodies indicated in the figure legends. After washes, bound
antibodies were detected with horseradish peroxidase-bound anti-rabbit
IgG or anti-mouse IgG (Roche Molecular Biochemicals) and a
chemiluminescence kit (PerkinElmer Life Sciences). In some experiments,
the blots were stripped either for 16 h at 4 °C in 100 mM glycine buffer/HCl, pH 2.2, containing 20 mM
magnesium acetate and 50 mM KCl or for 30 min at 65 °C
with 2% SDS, 100 mM Lipid Kinase Assay, TLC, and HPLC Analyses--
Following
subcellular fractionation of 3T3-L1 adipocytes and subsequent
solubilization of the membrane fractions (the final concentration of
the detergents is as in the RIPA buffer), the fraction lysates were
clarified by centrifugation and immunoprecipitated with anti-PIKfyve or
preimmune sera under conditions described above. The immunoprecipitates
immobilized on protein A-Sepharose beads were washed as described
elsewhere (9) and subjected to lipid kinase assay in 25 mM
HEPES buffer, pH 7.4, containing 100 µM lipid substrate
(synthetic diC16 PtdIns 3-P as an ammonium salt; a gift
from Echelon), 2.5 mM MnCl2, 2.5 mM
MgCl2, 50 µM ATP, and
[ Other Methods--
Pervanadate was generated from vanadate and
H2O2, mixed prior to experiments in a molar
ratio of 1:2 as described (25). Fraction density was calculated from
the refractive index (26). Protein concentration in all samples was
determined by a bicinchoninic protein assay kit (Pierce). Protein
levels on the immunoblots were quantified on a laser densitometer
(Molecular Dynamics, Inc., Sunnyvale, CA) by area integration scanning.
Several exposures of each blot were quantified to ensure the linearity
of the chemiluminescence signal on the film.
Biochemical Fractionations Indicate Cytosolic,
Cytoskeleton-associated, and Inner Membrane-bound Components of
PIKfyve--
PIKfyve subcellular distribution was first analyzed by
immunoblotting utilizing different cell fractionation techniques in 3T3-L1 adipocytes and using anti-PIKfyve antibodies directed against a
glutathione S-transferase fusion of the PIKfyve N-terminal
100 amino acids. The ability of anti-PIKfyve antibodies to specifically recognize endogenous PIKfyve in 3T3-L1 adipocytes and other cells by
Western blotting or immunoprecipitation was characterized previously (8, 9). To determine the soluble versus particulate pools of
PIKfyve, 3T3-L1 adipocytes, rat1 fibroblasts, or COS-7 cells were
fractionated into cytosol, total membranes, and nuclear pellets. A
significant fraction of the immunoreactive PIKfyve was found to reside
in the cytosol under resting conditions (70-80%). The remaining
PIKfyve was found associated with the total membrane fractions; no
detectable levels were documented in the nuclear pellets of either cell
type (not shown). To gain insight into the nature of the
membranes/structures with which PIKfyve is associated, we took
advantage of previously well defined protocols for differential centrifugation of 3T3-L1 adipocytes. This fractionation enables isolation of five fractions enriched in marker proteins for PM-enriched fraction; M/N; endosomes, Golgi elements, and endoplasmic reticulum (HDM); recycling endosomes, Golgi and cytoskeletal elements, and intracellular GLUT4 storage compartment (LDM); and soluble fraction (cytosol) (21, 27). The immunoreactive PIKfyve was detected predominantly in cytosol and LDM and at low levels in the PM-enriched fraction (Fig. 1), with an average
relative distribution of 77, 20, and 3%, respectively, as expressed
per equal number of cells. HDM and M/N fractions showed no detectable
PIKfyve levels (Fig. 1). Lack of significant contamination of the
PM-enriched fraction with intracellular membranes was evidenced by the
absence of a signal for the endosomal marker protein Rab4 upon
immunoblotting (not shown).
The subcellular fractionation in 3T3-L1 adipocytes presented above
implies that particulate PIKfyve mainly populates the intracellular GLUT4 storage compartment, Golgi/endosomes, and/or the cytoskeleton. To
distinguish among these possibilities and to determine the relationship
of the PIKfyve compartment to the other compartments, we performed a
series of LDM fractionations using equilibrium sedimentation in
self-formed iodixanol gradients, shown previously to effectively
separate GLUT4 vesicles, endosomal membranes, the bulk of other
membranes, and cytoskeletal elements (24). Upon sedimentation in 14%
iodixanol, the GLUT4 storage compartment is reportedly resolved into
two distinct pools, containing different levels of the TfR recycling
endosomal marker, TGN markers, or core proteins of the transport
machinery (24). In agreement with these studies, we have obtained two
peaks of GLUT4 vesicles, evidenced by the Western blotting of the
fractions collected from LDM equilibrium sedimentation at a starting
concentration of 14% iodixanol (Fig.
2A). Under these conditions,
PIKfyve was found to slightly overlap with the fractions containing the
immunoreactive GLUT4 (Fig. 2A) or IRAP, which reside
exclusively on GLUT4 vesicles, and TfR (see below). Intriguingly,
PIKfyve almost fully cofractionated with the p85 subunit of class
IA PI 3-kinase detected in the denser bottom part of the
14% iodixanol gradient (Fig. 2A).
Higher starting concentrations of iodixanol generate shallower
gradients at a lower refractive index, resulting in a greater separation of the denser particles with small differences in density (26). Under a better resolution of the GLUT4/endosomal compartments versus the denser structures, we sought to examine first
whether the observed slight overlap between the first GLUT4 peak and
PIKfyve would persist and, second, the extent of overlap between p85 PI 3-kinase and PIKfyve. It is worth emphasizing that the combined phosphorylation of D-3 and D-5 in PtdIns is a result of a joint action
of two lipid kinases; hence, a colocalization of PI 3-kinase and
PIKfyve may facilitate the fidelity for the product's synthesis. Equilibrium sedimentation at 30% iodixanol distributed the LDM into
three main protein peaks (Fig. 2C). Immunoblotting of the collected fractions indicated that TfR, IRAP, and GLUT4 cofractionated with the third protein peak at the top of the gradient at a density of
1.13-1.08 g/ml (Fig. 2B; fractions 14-16), in agreement
with the reported density of GLUT4 vesicles (28). The immunoreactive PIKfyve peak appeared clearly segregated from the above fractions, spanning fractions 5-12 (density 1.21-1.16 g/ml), comprising parts of
the first and the second protein peaks (Fig. 2B). The
immunoreactive p85 cosedimented entirely with the first protein peak
(Fig. 2B; fractions 2-8; density 1.30-1.19 g/ml),
indicating that while the fractions enriched in PIKfyve and PI 3-kinase
largely overlap, they do not colocalize identically. Clearly, the
results of the equilibrium sedimentation in density gradients
demonstrate that PIKfyve is segregated from the membranes of the
recycling endosomal system and GLUT4 storage compartment, sedimenting
with denser structures enriched in class IA PI
3-kinases.
Electron microscopy studies in 3T3-L1 adipocytes document that a
subfraction of LDM structures that sediments at the bottom of density
gradients is composed of cytoskeleton and macromolecular protein
complexes and, to a much lesser extent, of membranes (27). Signaling
intermediates, including those related to the insulin signaling
cascade, such as class IA PI 3-kinase and IRSs (29, 30),
are enriched in this subfraction. Because they appear to be Triton
X-100-resistant, it has been suggested that these proteins associate
with cytoskeleton rather than with membranes (27, 31). The
sedimentation of PIKfyve within the denser structures and its large
cofractionation with p85, documented above, suggests that a pool of the
particulate PIKfyve can also be attached to the cytoskeletal elements.
We tested this hypothesis by two independent biochemical approaches. In
the first, we examined the resistance of LDM-associated PIKfyve to
Triton X-100 solubilization. About 60% of the LDM-associated PIKfyve
was recovered in the Triton-insoluble fraction (Fig.
3A). In the second approach,
we first liberated the soluble and membrane-associated proteins from
intact cells with Triton X-100-containing cytoskeletal buffer and then
collected the cytoskeletal network retained on the dish. When 3T3-L1
adipocytes were treated in this manner, a significant pool of PIKfyve
equal to 10% of the total immunoreactive PIKfyve was detected in the Triton-insoluble pellet (Fig. 3B). Collectively, these data
indicate that a subfraction of particulate PIKfyve resists Triton
solubilization, suggesting a potential PIKfyve attachment to
cytoskeletal elements.
Distribution of PIKfyve Lipid Kinase Activity among Adipocyte
Subcellular Fractions--
Often the presence of an enzyme in a
particular compartment does not necessarily imply a site of catalytic
action. Therefore, we examined in parallel the PIKfyve lipid kinase
activity in the subcellular fractions. 3T3-L1 adipocytes were
homogenized in HES buffer and fractionated to obtain M/N, LDM, PM, and
cytosol or treated with Triton X-100-containing cytoskeletal buffer to
collect the soluble proteins and the Triton-insoluble cytoskeletal
elements. Anti-PIKfyve immunoprecipitates of the solubilized fractions
were analyzed for lipid kinase activity in the presence of PtdIns 3-P and [ Characterization of PIKfyve Protein Localization by Confocal
Microscopy--
The biochemical studies presented above document that
particulate PIKfyve is composed of two pools: Triton-soluble,
membrane-associated; and Triton-insoluble, attached, presumably, to
cytoskeletal elements. To further discern and define the
PIKfyve-containing elements/membranes, fluorescence studies were
performed using confocal microscopy. We took advantage of a
heterologous cell system transiently expressing PIKfyve full-length
protein tagged on its N terminus with GFP. We first confirmed the
expression of the predicted protein by immunodetecting a 230-kDa band
corresponding to EGFP-PIKfyve only in the lysates derived from the
transfected cells (Fig. 5A).
Expressed HA-tagged PIKfyve served as a size marker control (Fig.
5A). We next verified that the localization of EGFP-PIKfyve
reflected that of the endogenous protein by several ways. First, we
compared the subcellular distribution of the endogenous PIKfyve with
that of ectopically expressed PIKfyveWT in COS-7 cells by
immunoblotting. Endogenous PIKfyve, EGFP-PIKfyveWT, or
HA-PIKfyveWT partitioned among cytosol, intracellular
membrane, and plasma membrane fractions at identical ratios of 70:28:2.
Second, because the low endogenous levels of PIKfyve in COS cells (32)
prevent a convincing detection by immunofluorescence microscopy, we
compared the immunofluorescence signals associated with the authentic
PIKfyve in adipocytes with that of expressed EGFP-PIKfyve in
transfected COS cells. In both cell types, PIKfyve displayed a
characteristic discrete punctate pattern in the cell periphery (Fig.
5B, b, e, h, k,
n, and p). Diffuse staining, indicative of
soluble PIKfyve populations, was also observed, consistent with the
biochemical detection of PIKfyve cytosolic populations. Finally, loss
of the peripheral puncta and appearance of an exclusive diffuse
staining was associated with the expression of a EGFP-PIKfyve mutant
with a deleted FYVE finger, determined as an intracellular localization signal in other studies.2
Together, these results imply that the localization of heterologously expressed EGFP-PIKfyve in COS cells reflects that of the endogenous protein.
We first examined the localization of EGFP-PIKfyve in the context of
the recycling endosomal pathway. To label the membranes of the
endosomal recycling system, transfected COS cells were allowed to
internalize Texas red-Tf for 15 min at 37 °C. The cells were then
chilled and processed for fluorescence microscopy. Irrespective of the
similar vesicular-like pattern of appearance of the fluorescent signals
associated with EGFP-PIKfyve and Texas red-Tf, yellow color was
practically undetectable upon overlay of the two images (Fig.
5B). Accordingly, and in agreement with our previous
observations (8), the punctate staining of the native PIKfyve
documented in 3T3-L1 adipocytes was largely negative for GLUT4 (Fig.
5B). Thus, consistent with the biochemical studies presented
above, PIKfyve is segregated from the recycling endosomal pathway,
defined by TfR and GLUT4.
We next determined whether PIKfyve localizes to compartments along the
endocytic pathway but distal to the early endosomal recycling
compartment. PIKfyve distribution to lysosomes was monitored by dextran
fluorescent conjugates, which reach the lysosomes by pinocytosis and
largely accumulate there, resisting enzymatic cleavage. COS cells
expressing EGFP-PIKfyve were allowed to take up Texas red-dextran and
then chased for 2 h and processed for fluorescence microscopy.
Upon merging of the EGFP-PIKfyve and Texas red-dextran images, it was
apparent that the two punctate patterns of distribution were largely
different (Fig. 5B). PIKfyve-positive/dextran negative
structures were principally observed, and vice versa.
In both mammalian and yeast cells, the endocytic pathway merges with
the biosynthetic pathway at MVBs (used here as a synonym of
prelysosomal or late endosomal compartments) and, subsequently, their
distinct cargo transit together to the lysosome (33). Possible
localization of PIKfyve to MVBs was examined using CI-MPR, which sorts
and delivers newly synthesized lysosomal enzymes from TGN to late
endosomes. Electron microscopy studies indicate that, at least in
kidney cells (as are the COS cells), the late endosomes are the
predominant CI-MPR-positive compartment, while the lysosomes themselves
are CI-MPR-negative (34). Intriguingly, on the merged image a
substantial population of vesicles positive for both CI-MPR and
EGFP-PIKfyve, as judged by the appearance of yellow, were visible (Fig.
5B). Distinct populations of single-positive green- and
red-staining vesicles were also observed (Fig. 5B),
indicating that while the two proteins largely overlap, a unique
localization pattern is associated with each one of them. Documented
overlap between PIKfyve and CI-MPR marker is consistent with the notion that a subfraction of membrane-bound PIKfyve localizes to TGN/MVB. In
line with this notion was the detection of few vesicles positive for
both PIKfyve and the Golgi marker
The biochemical characterization of a PIKfyve pool resistant to Triton
extraction (Fig. 3) was confirmed by fluorescence microscopy. We
observed a largely preserved punctate pattern of the fluorescence associated with EGFP-PIKfyve following brief Triton X-100 extraction prior to cell fixation (Fig. 5B). Collectively, the results
of these approaches support the conclusion that a subpopulation of an
active PIKfyve enzyme associates with detergent-resistant cytoskeletal structures through a binding mechanism, which is also
Triton-resistant.
PIKfyve LDM Recruitment in Response to 3T3-L1 Adipocyte
Stimulation--
Relocation upon cellular stimulation appears to
be a common theme in cellular regulation. Class IA PI
3-kinase(s) for example is recruited to a variety of intracellular
locations in response to cell stimulation with growth factors,
including insulin, where it increases the local concentration of key
lipid products (20, 30). In the case of PIKfyve, regulated specific
intracellular targeting may selectively facilitate its interactions
with PtdIns 3-P substrates, given the fact that its other favorite
substrate, PtdIns, is about 400 times more abundant (32). This
consideration, together with the apparent existence of cytosolic and
particulate PIKfyve pools, suggests the possibility that PIKfyve may
relocate upon cellular stimulation. To test this hypothesis, we
examined the levels of PIKfyve in the subcellular fractions (PM, HDM,
LDM, M/N, and cytosol) derived from serum-deprived 3T3-L1 adipocytes acutely stimulated with insulin. At the basal state, the immunoreactive PIKfyve was distributed mainly between LDM and cytosol as
nonphosphorylated or poorly phosphorylated forms (Fig.
6, and see below). Importantly, insulin
action in 3T3-L1 adipocytes resulted in a 2-fold increase (2.15 ± 0.29; mean ± S.E., n = 7) of the immunoreactive
PIKfyve in LDM with a commensurate decrease in the cytosol (Fig.
6A). Concordantly, similar recruitment (2.5-fold) was
triggered upon cell stimulation with pervanadate (Fig. 6B),
a well known insulin-mimetic agent shown to increase IR phosphotyrosine
content (and that of other proteins) (25). No insulin- or
pervanadate-dependent changes in the PIKfyve levels were
documented in the remaining fractions (not shown). Intriguingly, the
insulin- or pervanadate-dependent recruitment of PIKfyve
onto LDM was accompanied by a decrease in the PIKfyve electrophoretic
mobility, causing the immunoreactive PIKfyve to appear on the gels as a
clear cut doublet (Fig. 6, A and B). It is
noteworthy that this bandshift was observed only in LDM; no significant
changes in the electrophoretic mobility were detected in the
immunoreactive PIKfyve from cytosol (Fig. 6, A and
B) or PM fraction (not shown) upon 3T3-L1 adipocyte
stimulation.
Since this mobility shift is suggestive of an IR-induced covalent
modification such as phosphorylation, we next examined the phosphorylation status of PIKfyve in the fractions. The profile and
subcellular distribution of tyrosine-phosphorylated proteins observed
in response to insulin versus control was largely in agreement with previous observations in this cell type (27, 35). We
readily detected the 170-kDa phosphotyrosine band (IRSs) in LDM
and cytosol and 95-kDa phosphotyrosine band (IR
The observed insulin-dependent relocation/mobility shift of
a subfraction of the cytosolic PIKfyve onto LDM raises the question of
the impact of this effect on PIKfyve lipid kinase activity. Therefore,
we analyzed the PIKfyve enzymatic activity in the subcellular fractions
derived from basal versus insulin-stimulated 3T3-L1 adipocytes subsequent to immunoprecipitation with anti-PIKfyve antibodies. Results illustrated in Fig. 6E clearly indicate
that a short insulin treatment results in an ~2.2-fold increase of PtdIns 3,5-P2 generated by PIKfyve immunoprecipitates of
LDM and a commensurate decrease of this product in PIKfyve
immunoprecipitates of the cytosolic fraction. Insulin treatment did not
change significantly the levels of PtdIns 3,5-P2 production
in PIKfyve immunoprecipitates of PM (not shown). These results indicate
that the insulin-dependent changes of PtdIns
3,5-P2 production in PIKfyve immune complexes from cytosol
and LDM principally parallel the alterations in PIKfyve immunoreactive
levels. Quantitation of four separate experiments is shown in Fig.
6E with PtdIns 3,5-P2-generating activity
expressed as a fold of basal activity in the cytosolic fraction. We
have also examined the PtdIns 3,5-P2 formation from PtdIns
3-P substrate by directly subjecting aliquots of the subcellular
fractions to lipid kinase assay. The results of these experiments
reproducibly demonstrated that insulin causes a similar increase of the
in vitro generated PtdIns 3,5-P2 in LDM, a
decrease in the cytosol, and relatively little change in the PM (not
shown), as with the PIKfyve immunoprecipitation experiments, discussed
above. Together, these data indicate that concomitantly with the
insulin-induced alterations in the PIKfyve subcellular distribution,
PIKfyve lipid kinase activity is increased in the LDM fraction while
depleted in the cytosol. These results further imply that the observed insulin-dependent hyperphosphorylation of PIKfyve does not
affect significantly the PIKfyve specific enzymatic activity and most likely is a signal for PIKfyve targeting/anchoring to LDM.
The apparent insulin-dependent recruitment of PIKfyve on
adipocyte LDM, the subfraction containing most of the intracellular GLUT4 storage compartment, suggests the possibility that PIKfyve may be
recruited onto GLUT4 vesicles. Analysis of the fractions from the
iodixanol gradients of LDM, however, did not detect significant differences in PIKfyve distribution from basal versus
insulin-treated 3T3-L1 adipocytes (Fig. 2, and data not shown).
Considering the fact that PIKfyve is of relatively low abundance
(silver stain experiments, not shown) it is possible that its absence
in the GLUT4-positive gradient fractions is due to detection
limitations. Therefore, we addressed the possibility of PIKfyve
recruitment onto GLUT4 vesicles by an alternative approach exploring
GLUT4 vesicle immunoadsorption on anti-GLUT4 antibodies. PIKfyve
presence in the immunopurified GLUT4 vesicles was then examined by both immunoblotting and enzymatic activity. Irrespective of insulin treatment, the immunoadsorbed GLUT4 vesicles neither contained detectable levels of immunoreactive PIKfyve (Fig.
7) nor generated detectable amounts of
PIKfyve products, PtdIns 5-P or PtdIns 3,5-P2 (not shown).
Both the immunoreactive PIKfyve (Fig. 7) and the products of its
enzymatic activity were recovered only in the GLUT4 vesicle
supernatant. By contrast, known residents of GLUT4 vesicles, such as
IRAP and TfR, were readily detected, as was their
insulin-dependent decrease due to GLUT4 vesicle's
departure to PM (Fig. 7), in agreement with previous studies (reviewed
in Ref. 3). Together, the data indicate that while PIKfyve function may
be essential for insulin action on GLUT4 translocation, this is not
achieved by PIKfyve's direct presence in the GLUT4 compartment.
In this study, we examined the intracellular protein localization
and the enzymatic activity distribution of the recently cloned
phosphoinositide 5-kinase PIKfyve (8) as a first step toward assessment
of the plausible intracellular sites of generation and action of its
lipid products, PtdIns 5-P and PtdIns 3,5-P2. To this end,
we have applied biochemical fractionation and immunofluorescence microscopy and utilized specific anti-PIKfyve antibodies to detect the
protein and measure its lipid kinase activity in basal or stimulated
cells expressing native or recombinant PIKfyve. Our studies
unequivocally demonstrate presence of an enzymatically active PIKfyve
in resting cells, distributed in at least two pools: cytosolic and
associated with particulate structures/membranes. Because PIKfyve does
not contain a transmembrane domain (nor is there any evidence for
post-translational modification that would enable it to associate with
membranes), PIKfyve retention on the particulate fractions should
result from interactions with membrane proteins, lipids, or
cytoskeletal elements. Both authentic and heterologously expressed
PIKfyve displayed typical peripheral punctate staining reminiscent of
endosomes but were found largely excluded from the early endosomal and
recycling pathway. Rather, PIKfyve colocalized with a
Golgi-to-late-endosome marker, MPR, suggesting a role in the later
steps of the endocytic and/or biosynthetic pathway. The punctate
pattern of PIKfyve fluorescence appearance was remarkably stable to
agents known to disrupt cellular membranes, implying PIKfyve
attachment, at least in part, to the Triton-insoluble cytoskeletal
network. We show further that in fractionated resting 3T3-L1 adipocytes
PIKfyve protein and activity were present mostly in the cytosol and to
a lesser extend in LDM but upon insulin stimulation underwent an
acutely directed recruitment to LDM linked with hyperphosphorylation
that increased its levels and lipid kinase activity more than 2-fold.
These results provide support for the hypothesis that the regulated
local production of PtdIns 3,5-P2 and/or PtdIns 5-P at a
particular intracellular site is important for acute insulin action.
Particulate PIKfyve Is Segregated from the Early
Endosomal/Recycling Compartment--
Association of PIKfyve with the
early endosome recycling compartment was expected because most of the
mammalian FYVE finger-containing proteins, including EEA1, Hrs, and
Ankhzn, are localized to early endosomes (10-12). Moreover, the
morphological appearance of the native PIKfyve in adipocytes or
heterologously expressed PIKfyve in COS cells as discrete peripheral
puncta was highly reminiscent of endosomes (Ref. 8 and Fig. 5). Several
lines of experimental evidence presented in this study clearly exclude
the recycling endosomes as a principal site of PIKfyve residency. Thus,
upon LDM fractionation by equilibrium sedimentation in density
gradients, the immunoreactive PIKfyve was recovered in a distinct
population of dense structures, segregated from the recycling endosome
membrane markers TfR, IRAP, or GLUT4. Next, PIKfyve protein and
activity were undetectable in the immunopurified intracellular GLUT4
storage compartment, which contains recycling endosomal markers to some extent. Finally, membranes of the recycling endosomal pathway, defined
by fluorescently labeled Tf or anti-GLUT4, were largely negative for
the punctate pattern associated with EGFP-PIKfyve or immunoreactive
adipocytic PIKfyve. The possibility that PIKfyve may label a
functionally distinct compartment within the earlier stages of TfR
recycling was ruled out in an experiment in which Texas red-Tf was
allowed to internalize for only 3 min in EGFP-PIKfyve-transfected COS
cells (not shown). Together, these data are consistent with the notion
that, while PIKfyve and its lipid products may be operational in the
endocytic pathway, PIKfyve is not associated with the membranes of the
early endosomal/recycling pathway.
PIKfyve in MVB Function--
Genetic studies in yeast predict that
Fab1p function, while unessential for the anterograde protein traffic
to the vacuole (lysosome) along all known vacuolar transport pathways,
plays a certain role in MVB protein sorting (16, 18). Because a correct
MVB function is also dependent upon class E VPS gene
products, it is proposed that localized production of the Fab1p product PtdIns 3,5-P2 recruits/activates class E Vps proteins from
the cytoplasm to drive different stages in the MVB sorting pathway (18). A possible role for PIKfyve and, therefore, the localized production of PtdIns 3,5-P2 and/or PtdIns 5-P in the MVB
function in mammalian cells is predicted in the present study by the
demonstration that a large proportion of PIKfyve populates MVBs,
evidenced by several criteria. Thus, similarly to MVBs (36),
PIKfyve-containing structures are labeled poorly for recycling
receptors such as TfR. Next, MVBs are negative for lysosomal markers
(33) as are the PIKfyve-containing structures (Fig. 5B,
g-i). Finally, as with MVBs (33), PIKfyve containing
structures are positive for the TGN-to-late endosome marker CI-MPR.
These data indicate that a substantial population of PIKfyve is
localized to MVB and are consistent with the notion of PIKfyve's zole
in the late compartments of the endocytic and/or biosynthetic pathway
in mammalian cells, a hypothesis that should be rigorously tested in
future studies.
PIKfyve and Cytoskeleton in Insulin Action on GLUT4
Translocation--
In insulin-sensitive muscle and fat, GLUT4 is
responsible for transporting the vast majority of glucose into the cell
(3, 37). While it is now clear that insulin evokes GLUT4 translocation from the intracellular storage compartment to the fat/muscle cell surface, the exact molecular mechanism is still elusive. Resting upon
solid experimental support from numerous studies, it is now well
accepted that insulin-regulated GLUT4 exocytosis is directly dependent
on the enzymatic activity of class IA PI 3-kinase(s) and
the production of PtdIns 3,4,5-P3 (30, 37). A first clue that PIKfyve function may also be important in GLUT4 membrane dynamics
came from experiments demonstrating enriched expression of both PIKfyve
mRNA and protein in primary fat cells and adipocytes in culture (8,
32). In fact, PIKfyve was originally identified through a search of
fat/muscle molecular elements, which, similarly to the
fat/muscle-specific GLUT4, are expressed in a tissue-specific manner
(38). Furthermore, PIKfyve displays an intrinsic activity to generate
PtdIns 5-P and PtdIns 3,5-P2, and both 5-PIs are now identified in resting 3T3-L1 adipocytes (9, 39). Intriguingly, it has
recently become apparent that insulin-induced GLUT4 translocation is
extremely sensitive to dephosphorylation of position D-5 in PtdIns
3,4,5-P3 (40). Because the intracellular PtdIns
3,4,5-P3 production is apparently a result of joint actions
of class IA PI 3-kinase(s) and other lipid kinases with a
specificity for positions D-4 and D-5, a role for PIKfyve function in
insulin action on GLUT4 could be expected. Intriguingly, our recent
studies detected complexes of PIKfyve with the class IA PI
3-kinase(s) in lysates of 3T3-L1 adipocytes (41), suggesting plausible
collaboration of these two kinases in the generation of the
insulin-relevant lipid messengers. PIKfyve's role in acute insulin
actions is further corroborated by the present data, demonstrating an
acute insulin-regulated recruitment/bandshift of the soluble PIKfyve
onto the LDM pool to increase the local synthesis of PtdIns
3,5-P2 (Fig. 6). This phenomenon is observed not only with
insulin but also upon 3T3-L1 adipocyte stimulation with the
insulinomimetic agent pervanadate, which increases the
IR-phosphotyrosine content (25), implying that a signal issued by
tyrosine-phosphorylated IR is relayed to Ser/Thr-hyperphosphorylate and
recruit PIKfyve. It is tempting to speculate that hyperphosphorylated
PIKfyve tethers to key LDM structures specifying the site and timing of
high local PI production. Together, our findings are consistent with a
model whereby a fraction of the soluble PIKfyve becomes phosphorylated
in an insulin-dependent manner. Phosphorylated PIKfyve
molecules are then recruited to insulin-sensitive intracellular sites,
which harbor and/or receive other insulin-stimulated signaling
molecules, i.e. PI 3-Ks, possibly jointly delivered with
PIKfyve. Locally generated lipids then serve as signaling mediators of
insulin end point responses. Our recent observation that increased
levels of PIKfyve by adenovirus-mediated gene delivery in
insulin-sensitive 3T3-L1 adipocytes mimic typical insulin-regulated
physiological responses3 is
consistent with this model. The challenge remains to identify the
PIKfyve Ser/Thr kinase, which is predicted in this study, as well as
plausible partners of PIKfyve recruitment to LDM.
While a correlation of the insulin-dependent
recruitment/mobility shift of PIKfyve protein and activity on LDM
within the time course of insulin-regulated GLUT4 exocytosis could be
documented, the causal relationship is somewhat hampered by the fact
that PIKfyve is largely excluded from the intracellular GLUT4 storage compartment. This notion is supported here by a variety of approaches, discussed above. Moreover, a potential PIKfyve recruitment into the GLUT4 compartment upon acute cell stimulation with insulin was also below the detection limit of different approaches. However, recent independent experimental evidence lends support to the notion
that key elements of the insulin signaling cascade relevant to GLUT4
exocytosis, such as IRSs and PI 3-kinase activity, are in fact outside
the GLUT4 compartment. Thus, in 3T3-L1 adipocytes, both PI 3-kinase and
IRS proteins cofractionate with the cytoskeletal-like insoluble
proteinaceous particles of LDM but not with GLUT4 (27, 31), similarly
to PIKfyve. Next, microinjected probes that specifically interact with
the intracellular PI 3-kinase products profoundly label PM, but not the
cell interiors, in response to acute insulin (42). The association of
PIKfyve with members of the insulin signaling cascade and the role of a
cytoskeleton scaffold to hold them in place remains to be elucidated.
This is an important objective in light of the increasing experimental
evidence recognizing cellular mictrotubules and microfilaments as an
integral part of membrane trafficking events including
receptor-mediated endocytosis and insulin action on GLUT4
exocytosis (43, 44).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP (M3A5), and
rabbit polyclonal anti-human CI-MPR antibodies were gifts of Drs. S. White, I. Trowbridge, T. Kreis, and B. Hoflack, respectively. Anti-PI 3-kinase p85 and anti-phosphotyrosine (4G10) antibodies were
from Upstate Biotechnology, Inc. (Lake Placid, NY); anti-GFP antibody
was from CLONTECH; and human insulin was from
Lilly. The EGFP-HA-PIKfyveS construct was generated by
subcloning the XbaI/SalI digest of pBluescript II
SK(+)-PIKfyveS cDNA (8) together with the
double-stranded oligonucleotide encoding for HA epitope (designed with
EcoRI/XbaI restriction sites) into the EcoRI/SalI digest of pEGFP C2
(CLONTECH).
-glycerophosphate, 10 mM sodium pyrophosphate, 50 mM NaF, and 2 mM NaVO3)) at 25 °C, and then scraped at
4 °C in HES buffer, supplemented with the inhibitor mixtures. Cells were homogenized by passing the cell suspension six times through a
221/2-gauge needle at 4 °C. Subcellular fractionation was
performed following previous protocols (21) using an SS-34 rotor
(Sorvall Instrument Division) in the first spin and Beckman TLA 100.3 rotor (Beckman Instruments Inc.) in the next spins to obtain HDM, LDM, PM, M/N, and cytosolic fractions. Pellets were resuspended in HES
buffer containing the above inhibitors to a protein concentration of
~2 mg/ml. Aliquots of the fractions were analyzed by immunoblotting or, following solubilization in 1% Nonidet, 0.5% sodium deoxycholate, 150 mM NaCl, were subjected to lipid kinase assay (see
below). LDM digestion with alkaline phosphatase (Sigma) or
-phosphatase (Calbiochem) was performed for 40 min at 25 °C.
Evaluation of Triton X-100-soluble versus insoluble pools of
PIKfyve was performed in 3T3-L1 adipocytes as described previously (22)
except for CSK buffer (100 mM NaCl, 300 mM
sucrose, 10 mM Pipes, pH 6.8, 3 mM
MgCl2, 1 mM EGTA, and 0.5% Triton X-100; Ref.
23) in place of PBS-Triton.
-COP antibodies and detection by CY3-coupled goat anti-rabbit IgG
(Kirkegaard & Perry Laboratories) or Texas red-coupled goat
anti-mouse IgG (Molecular Probes), respectively, was performed in
formaldehyde-fixed transfected COS-7 cells (20 h post-transfection with
LipofectAMINE). Double staining with affinity-purified anti-GLUT4
monoclonal (1F8) and anti-PIKfyve polyclonal antibodies in 3T3-L1
adipocytes was as previously described (8). Coverslips were mounted on
slides using the Slow Fade Antifade Kit (Molecular Probes).
Fluorescence analyses were performed with a confocal microscope (Zeiss
LSM 310) using a 63/1.4 immersion lens.
-mercaptoethanol in 62.5 mM Tris-HCl, pH 6.8. After blocking, blots were reprobed
with antibodies. For immunoprecipitation, the RIPA (50 mM
Tris/HCl, pH 8.0, containing 150 mM NaCl, 1% Nonidet P-40,
0.5% sodium deoxycholate, and 1× protease inhibitor mixture) lysates,
collected from transfected cells (calcium phosphate precipitation method) or 3T3-L1 adipocytes were precleared by centrifugation at
4 °C, and the supernatants were then incubated (16 h at 4 °C) with the antibodies indicated in the figure legends or with preimmune serum. Protein A-Sepharose CL-4B was added in the final 1.5 h of
incubation. Immunoprecipitates were washed with RIPA buffer (unless
otherwise stated), solubilized in Laemmli buffer, and analyzed by
SDS-PAGE and immunoblotting.
-32P]ATP (12.5 µCi). Following incubation for 15 min at 30 °C, the lipids were extracted and analyzed by TLC under
previously specified conditions (9). Generated radioactive PtdIns
3,5-P2 product was detected by autoradiography and
quantified by radioactive counting. Its identity and purity was
confirmed by HPLC analysis performed in the presence of appropriate
internal standards, detailed elsewhere (9). In some experiments,
cytosol, LDM, or PM fractions were subjected directly to the lipid
kinase assay without immunoprecipitation. In this case, following
3T3-L1 adipocyte fractionation, described under "Cell Treatment and
Subcellular Fractionation," aliquots of the fractions (20 µl) were
mixed with 30 µl of the lipid kinase reaction mixture and processed
as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PIKfyve is distributed mainly between cytosol
and intracellular membranes/structures of LDM fraction in 3T3-L1
adipocytes. Serum-deprived 3T3-L1 adipocytes were subcellularly
fractionated to obtain M/N, HDM, PM, LDM, and soluble (Cyt)
fractions. Aliquots (50-150 µg of protein) representing 60% of PM
or LDM, 100% of HDM, and 15% from cytosol or M/N on the basis of
total cell number were resolved by SDS-PAGE (6% acrylamide),
transferred onto nitrocellulose membrane, and immunoblotted with
anti-PIKfyve antiserum (1:5000 dilution). Shown is a chemiluminescence
detection of a representative experiment out of six independent
subcellular fractionations. PIKfyve detection below the 200-kDa marker
is denoted.
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Fig. 2.
Iodixanol equilibrium gradient sedimentation
analysis detects PIKfyve at the denser part of the gradient, segregated
from recycling endosome markers. LDM fraction obtained from
serum-deprived 3T3-L1 adipocytes was subjected to equilibrium
sedimentation in 14% (A) or 30% iodixanol (B)
as described under "Experimental Procedures." Fractions were
collected from the bottom of the gradients. Aliquots were analyzed by
SDS-PAGE on 10.5% (GLUT4) or 6% gels and immunoblotting with the
indicated antibodies. The results shown are chemiluminescence
detections of blots from a single 14 or 30% iodixanol gradient and are
representative of four independent experiments with similar results.
C, refractive index and protein concentration
(A562) of the 30% iodixanol gradient
fractions. The protein amounts in peaks I, II, and III are 54, 27, and
19% of total protein, respectively.
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Fig. 3.
Particulate PIKfyve is enriched in the Triton
X-100-insoluble component of LDM and resists Triton extraction of
3T3-L1 adipocytes. A, LDMs, obtained from
serum-deprived 3T3-L1 adipocytes, were resuspended in HES buffer,
supplemented with protease and phosphatase inhibitors. Aliquots were
made 0.8% Triton X-100 and incubated for 30 min at 4 °C under
rotation. The Triton-insoluble pool of LDM was pelleted by
centrifugation for 24 min at 200,000 × g at 4 °C
and resuspended. Aliquots of the initial LDM (lane
1), Triton-soluble (lane 2), and
Triton-insoluble material (lane 3) were analyzed
by SDS-PAGE and immunoblotting. B, 3T3-L1 adipocytes on a
100-mm dish were treated with CSK extraction buffer as described under
"Experimental Procedures" to extract the Triton-soluble proteins
(Tr-sol), typically 70% of the total protein.
Triton-insoluble (Tr-insol) elements remaining on the dish
were washed and collected with RIPA buffer (~30% of the total
protein). Total proteins (total) were collected by direct
cell solubilization (100-mm dish) in RIPA buffer, and upon
centrifugation, the RIPA-insoluble material was pelleted
(pellet). Aliquots of the fractions (50 µg and the entire
pellet) were analyzed by SDS-PAGE and immunoblotting. Shown are
chemiluminescence exposures of representative blots from three
experiments each of A and B. WB,
Western blot.
-32P]ATP as substrates. Generated PtdIns
3,5-P2 was separated by TLC and confirmed by HPLC (Fig.
4). Control experiments revealed a linear
relationship between the protein amounts (25 µg to 2 mg) subjected to
immunoprecipitation with anti-PIKfyve and the PtdIns 3,5-P2
production for each one of the fractions (not shown). Except for the
PM-enriched fraction, the basal PIKfyve activity measured in the
individual fractions principally paralleled the immunoreactive PIKfyve
amounts. However, while the distribution ratio of the immunoreactive
PIKfyve among cytosol, LDM, and PM was 4:1:0.18 as normalized per cell
number (Fig. 1), the distribution ratio of the PIKfyve lipid kinase
activity among those fractions was 4:1:0.8 under these conditions (Fig.
4A). For yet-to-be identified reasons, the PM-enriched
fraction showed significantly higher PIKfyve specific activity
(~5-fold). The relative distribution of PIKfyve lipid kinase activity
between the Triton-soluble and Triton-insoluble pools of 3T3-L1
adipocytes principally mirrored the PIKfyve relative amounts (Fig.
4B). Thus, the majority of the activity (87%) was recovered
in the Triton-soluble pool. The Triton-insoluble cytoskeletal network
showed ~13% of the enzymatic activity (Fig. 4B), which
was equivalent to the estimated PIKfyve protein levels (10%; see
above). These results demonstrate that all fractions found positive for
immunoreactive PIKfyve express enzymatically active protein and
indicate that PIKfyve protein presence in a particular fraction most
probably reflects a site of its action.
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Fig. 4.
PIKfyve enzymatic activity principally
parallels PIKfyve immunoreactive levels. 3T3-L1 adipocytes (100-mm
dish) were fractionated to obtain M/N, LDM, HDM, PM, and cytosolic
fractions. Fractions were resuspended and solubilized, and the lysates
were clarified as described under "Experimental Procedures"
(A). 3T3-L1 adipocytes (60-mm dish) were extracted with GSK
buffer containing 0.5% Triton X-100 (Tr-sol) for 10 min at
4 °C as described under "Experimental Procedures." The dish was
then washed twice with PBS, and the remaining Triton-insoluble
(Tr-insol) elements were scraped with RIPA buffer (B).
One-fourth of each lysate in A or the entire lysates in
B were immunoprecipitated with anti-PIKfyve N-terminal
antibodies. Washed immunoprecipitates were analyzed for lipid kinase
activity in the presence of PtdIns 3-P, [ -32P]ATP, and
ions as described under "Experimental Procedures." Lipid products
generated during the reaction were extracted and analyzed by TLC with
65:35 (v/v) n-propyl alcohol plus 2 M
acetic acid solvent system (A and B). HPLC
analysis of deacylated PtdIns 3,5-P2 product
(arrow) scraped from TLC plates (detailed under
"Experimental Procedures") verified its identity and the lack of
by-products (C). Shown are autoradiograms or representative
TLC plates of the fractions positive for PIKfyve activity (A
and B, two different experiments out of three to six with
similar results) and a representative HPLC elution profile of scraped
PtdIns 3,5-P2 spots (C). PtdIns
3,5-P2 was quantified by radioactive counting of the TLC
scrapings (see "Experimental Procedures"). Retention times
of radiolabeled glycerophosphorylinositol (GroPIns)
standards phosphorylated at the indicated positions are denoted by
arrows (C).
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Fig. 5.
Confocal microscopy documents PIKfyve as
peripheral puncta, excluded from recycling endosomes and lysosomes,
partially overlapping with MPR marker and largely
Triton-resistant. A, COS-7 cells were transfected with
pEGFP-HA-PIKfyve, pCMV5-HA-PIKfyve, or the empty vectors, lysed and
immunoprecipitated as indicated. SDS-PAGE and immunoblotting reveals
expression of anti-GFP-reacting protein with the expected size (230 kDa) as related to HA-PIKfyve (200 kDa) (arrows).
B, details of cell processing for confocal microscopy are
given under "Experimental Procedures." a-c, double
immunofluorescence staining for GLUT4 (a), PIKfyve
(b), and composite (c) in 3T3-L1 adipocytes
reveals practically no overlap. d-f, COS-7 cells
transfected with EGFP-PIKfyve (e; shown are two cells
expressing PIKfyve at different levels) were allowed to take up Texas
red-Tf (15 min; 37 °C) (d). No colocalization is
documented upon overlap of both images (f); g-i,
COS-7 cells transfected with EGFP-PIKfyve (h) were allowed
to take up Texas red-dextran (g). Only a single yellow
particle could be observed on the composite image (i).
j-l, double staining of EGFP-PIKfyve-transfected COS cells
(k) with anti-MPR antibodies (j) reveals a
substantial overlap, indicated in l. m-o, double
staining of EGFP-PIKfyve-transfected COS cells (n; shown are
three cells expressing PIKfyve) with anti- -COP antibody
(m) reveals a limited overlap, indicated in o.
p and q, COS-7 cells transfected with
EGFP-PIKfyve were extracted for 60 sec with Triton X-100 (0.5%) prior
to fixation (p). A phase-contrast image illustrates the
peripheral distribution of the Triton-resistant EGFP-PIKfyve puncta
relative to the whole cell (q). Bar, 10 µm.
-COP (Fig. 5B).
However, the inability of brefeldin A, a drug that causes a rapid loss of Golgi as a distinct organelle (22), to change substantially the
EGFP-PIKfyve punctate pattern (not shown) places the majority of the
particulate PIKfyve at MVBs.
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Fig. 6.
Insulin and pervanadate induce a recruitment
of the cytosolic PIKfyve to LDM, coupled with PIKfyve
hyperphosphorylation and proportional changes in PIKfyve lipid kinase
activity. A and C, serum-deprived 3T3-L1
adipocytes treated or not with insulin (100 nM; 7 min) were
fractionated to obtain LDM and cytosol. Proteins (55 µg of LDM and
200 µg of cytosol) were resolved by SDS-PAGE and immunoblotted.
B, 3T3-L1 adipocytes treated or not with pervanadate
(PV; 100 µM; 20 min) were fractionated to
obtain LDM and cytosol. Proteins (200 µg) were resolved by SDS-PAGE
and immunoblotted. Both treatments render PIKfyve doublet form
(indicated). D, equal amounts of LDM fraction derived from
insulin-treated adipocytes were subjected or not to dephosphorylation
for 40 min at 25 °C with alkaline phosphatase (AP; 50 units) or -phosphatase (
P; 50 units) in 10 mM Tris-HCl buffer, pH 7.4, containing 50 mM
NaCl, 10 mM MgCl2 and 1 mM
dithiothreitol (AP) or 50 mM Tris-HCl,
containing 2 mM MnCl2 and 5 mM
dithiothreitol (for
P). Proteins were analyzed by SDS-PAGE and
immunoblotting. A-D, representative immunoblots of 3-7
independent experiments for each panel. E, serum-deprived
3T3-L1 adipocytes treated or not with insulin (100 nM; 7 min) were fractionated to obtain LDM and cytosol. Detergents were added
to each fraction as described under "Experimental Procedures," and
after centrifugation, aliquots of the clear lysates (cytosol (400 µg)
and LDM (50 µg), equivalent to one-fourth of a 100-mm dish), were
immunoprecipitated with anti-PIKfyve. The immunoprecipitates were
subjected to lipid kinase assay with PtdIns 3-P substrate as described
in the legend to Fig. 4. Shown are an autoradiogram of a TLC plate of a
representative experiment (upper panel) and
quantitation of the PIKfyve activity in four independent cellular
fractionations (lower panel). PIKfyve activity in
the indicated fractions is expressed as -fold alteration of the basal
activity in cytosol. For each experiment, the activity was expressed
per equal number of cells.
-subunit) in PM and,
to a lesser extent, in LDM by probing with two anti-phosphotyrosine antibodies, 4G10 and PY20 (Fig. 6C, and data not shown).
However, neither one of the antibodies documented convincingly an
insulin-dependent tyrosine phosphorylation of PIKfyve in
several experiments, in which the insulin-regulated increase in
phosphotyrosine content of IRSs and IR was dramatic (Fig.
6C). These results indicate that PIKfyve LDM
recruitment/bandshift triggered by the tyrosine-phosphorylated IR most
probably involves phosphorylation of PIKfyve on Ser/Thr. To test this
hypothesis, LDMs derived from insulin-stimulated 3T3-L1 adipocytes were
treated with alkaline phosphatase or
-phosphatase prior to their
analysis by SDS-PAGE and immunoblotting. Both phosphatases rendered
PIKfyve a single migrating band (Fig. 6D) consistent with
the notion that the higher molecular weight band represents mobility-shifted, Ser/Thr-hyperphosphorylated PIKfyve and the lower
band is nonphosphorylated or poorly phosphorylated PIKfyve. In
addition, LDM dephosphorylation resulted in a significant decrease in
the PIKfyve immunoreactive levels (Fig. 6D), implying
possible dissociation of dephosphorylated PIKfyve from LDM. This was
confirmed by immunoblotting the supernatants of the reactions indicated in Fig. 6D and recovering the immunoreactive PIKfyve only in
the phosphatase-treated LDM samples (not shown). Collectively, these results indicate that PIKfyve recruitment on and dissociation from LDM
correlates with its hyperphosphorylation and dephosphorylation, respectively.
View larger version (90K):
[in a new window]
Fig. 7.
PIKfyve is undetectable in immunopurified
GLUT4 compartment. LDM fractions (330 µg) obtained from 3T3-L1
adipocytes, treated or not treated with insulin (100 nM; 7 min at 37 °C), were resuspended in PBS supplemented with 1×
protease and phosphatase inhibitors, immunoadsorbed on anti-GLUT4 IgG
(lanes 5 and 6) or rabbit IgG
(lanes 3 and 4), and immobilized on
protein A-Sepharose CL-4B beads. Washed immune complexes, together with
aliquots of the supernatants from the nonimmune IgG (lanes
1 and 2) were subjected to SDS-PAGE and
consecutive immunoblotting with antibodies as indicated. Shown are
chemiluminescence detections from a single immunoadsorption experiment
out of two with similar results. PIKfyve increase in the LDM fraction
upon insulin is visible in the supernatants (lane
2 versus lane 1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Mike Czech, Paul Pilch, Kostya Kandror, Bernard Hoflack, Ian Traubridge, Sue White, and Tomas Kreis (posthumously) for the kind gifts of antibodies. The assistance of L. Mayernik in confocal microscopy is appreciated.
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FOOTNOTES |
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* This work was supported by Juvenile Diabetes Research Grant 1-1999-40 and a Morris Hood Jr. Research Award (to A. S.).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.
To whom correspondence should be addressed: Dept. of
Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-5674; Fax: 313-577-5494;
E-mail: ashishev@moose.med.wayne.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M008437200
3 O. C. Ikonomov and A. Shisheva, unpublished data.
2 A. Shisheva, D. Sbrissa, and O. C. Ikonomov, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PtdIns, phosphatidylinositol; CI-MPR, cation-independent mannose 6-phosphate receptor; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; HDM, high density microsomal fraction; IRS, insulin receptor substrate; IRAP, insulin-regulated aminopeptidase; LDM, low density microsomal fraction; M/N, mitochondria/nuclei; MVB, multivesicular body; PBS, phosphate-buffered saline; PM, plasma membrane; PI, phosphoinositide; P2, bisphosphate; P3, trisphosphate; Tf, transferrin; TfR, transferrin receptor; TGN, trans-Golgi network; HPLC, high pressure liquid chromatography; HA, hemagglutinin; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; IR, insulin receptor.
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