(Received for publication, October 28, 1996, and in revised form, February 26, 1997)
From the Phosphatidylinositol (PtdIns) 4-kinase catalyzes
the synthesis of PtdIns-4-P, the precursor of an array of lipid second
messengers generated by additional phosphorylation by PtdIns-4-P
5-kinase and PtdIns 3-kinase. PtdIns 4-kinase activity is conserved
from yeast to higher eukaryotes. Multiple isoforms of mammalian PtdIns 4-kinase have been purified, and the activities have been detected in
almost all subcellular locations. We previously reported the cloning
and characterization of the first mammalian PtdIns 4-kinase named
PI4K The metabolism of phosphoinositides is a key event in
transmitting mitogenic and developmental signals in response to a
variety of hormones and growth factors. Two signal transduction
pathways, each utilizing distinct phosphoinositide derivatives, have
been characterized. In one pathway, phosphatidylinositol
(PtdIns)1-4,5-P2 is hydrolyzed
by receptor-activated phospholipase C enzymes to generate the signaling
molecules inositol 1,4,5-trisphosphate and diacylglycerol (4).
PtdIns-4-P and PtdIns-4,5-P2 have also been shown to
mediate actin rearrangement by directly regulating actin-binding
proteins (5, 6). In a distinct pathway, phosphoinositides are
phosphorylated at the C-3 position to generated a family of lipid
messengers (7). These lipids, PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3 are not substrates for phospholipase C
but are implicated in mitogenesis (8), intracellular trafficking (9) as
well as actin rearrangement (10, 11). Feeding into both PtdIns pathways
is the precursor PtdIns-4-P, synthesized by PtdIns 4-kinases.
The subcellular distribution of mammalian PtdIns 4-kinases has been
studied. The majority of PtdIns 4-kinase activity in human cells is
membrane-bound (12). PtdIns 4-kinase activity is detected in most
membrane structures, including plasma membrane (13), nuclear envelope
(14), lysosome, Golgi apparatus (15), and endoplasmic reticulum (16).
PtdIns 4-kinase activity is also detected in coated vesicles, glucose
transporter-containing vesicles (17), and several specialized
organelles, such as chromaffin granules (18) and secretory vesicles
from mast cells (19). In addition, cytosolic PtdIns 4-kinases have been
described (20). In view of such a wide subcellular distribution, it has
long been speculated that different isoforms, targeted to different
intracellular compartments, perform different physiological functions.
While there is some evidence suggesting that distinct isozymes of
PtdIns 4-kinase may be targeted to distinct organelles and be
independently regulated, previous studies have been limited by lack of
cDNA clones and isoform specific antibodies.
In Saccharomyces cerevisiae, only two PtdIns 4-kinase
isoforms are found in the entire genome. PIK1, the first PtdIns
4-kinase to be cloned, encodes a nuclear-associated PtdIns 4-kinase of 125 kDa that is indispensable for cell growth (21, 22). Mutants arrest
in G2 due to defects in cytokinesis. A second yeast gene, STT4, encodes a 200-kDa PtdIns 4-kinase that appears to be cytosolic and is dispensable for growth. Genetic studies of STT4 suggest its
involvement in the protein kinase C pathway (23, 24). In contrast, the
yeast PtdIns 3-kinase, Vps34, is required for vesicle trafficking from
the Golgi apparatus to the vacuole (9). Interestingly FAB1,
a yeast gene that is homologous to the mammalian PtdIns-4-P 5-kinase,
is required for normal vacuole function and morphology (25). Because
PtdIns 4-kinase generates PtdIns-4-P, the precursor for subsequent
phosphorylation by downstream PtdIns kinases, PtdIns 4-kinases must
play either a direct or an indirect role in vesicular trafficking
(26).
Previously, we reported the cloning and characterization of the first
mammalian PtdIns 4-kinase, named PI4K PtdIns, [ The
2.6-kilobase pair PI4K Adherent
parental CHO-IRS or CHO-IRS cells transfected with PI4K Fractionation
procedures were performed according to Storrie and Madden (29).
Briefly, CHO-IRS cells transfected with PI4K Alkaline
phosphodiesterase, cytochrome c oxidase, HeLa cells
grown on coverslips were fixed with 4% paraformaldehyde for 20 min.
Fixed cells were permeabilized and nonspecific reactive sites were
blocked for 30 min at room temperature in phosphate-buffered saline
containing 0.1% Triton X-100, 5% normal goat and donkey serum. Cells
were then incubated with the appropriate primary PtdIns 4-kinase
isoform-specific antibody for 1 h at room temperature. Primary
antibodies were detected by species-specific secondary antibodies,
namely CyTM3-conjugated goat anti-rabbit IgG and
fluorescein isothiocyanate-conjugated donkey anti-mouse IgG. ER was
identified by staining BiP, a resident ER protein. Golgi was visualized
using anti- PtdIns kinase assays
were performed as described previously (30). Briefly, the reaction
mixture contained 0.3% Triton X-100, 50 µM ATP, 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.2 mg/ml sonicated lipids, 20 µCi of [ Since the 97-kDa form of
PI4K
CHO cells were selected to investigate the locations of
PtdIns 4-kinases because these cells have been well characterized in
subcellular fractionation studies and because they are convenient for
transient gene expression. Since the PI4K
To further investigate the intracellular localization of PtdIns
4-kinase isoforms, cells overexpressing PI4K Table I.
Summary of organelle marker enzyme assays and the distribution of
PtdIns 4-kinase isozymes
The various subcellular fractions were also assayed for total PtdIns
4-kinase activity. Although half of the PtdIns 4-kinase activity was
detected in the upper two factions where cytosol, Golgi apparatus, and
endoplasmic reticulum partition, and where both PI4K
To
further discern the intracellular localization of PI4K
Immunofluorescent studies with anti-PI4K We compared the subcellular distribution of known mammalian
PtdIns 4-kinases. The results demonstrate the differential localization of different isoforms and provide support for the hypothesis that individual members of this family of PtdIns kinases are targeted to
distinct subcellular compartments and hence are performing different
cellular functions. PI4K While this work was in progress, Nakagawa et al. (27)
reported the cloning and characterization of an alternative splice of
rat PI4K Nakagawa et al. (27) reported that Flag-tagged versions of
both forms of PI4K By separating intracellular structures on a step gradient and assaying
each fraction for PtdIns kinase activity, we have shown that the
lysosomal and plasma membrane-associated PtdIns 4-kinase activities are
unlikely to be the result of PI4K The finding of the differential ER and Golgi localization of PI4K We thank Fernando Agarraberes for his
assistance in the cell fractionation procedures. We also thank Brian
Duckworth and Anthony Couvillon for help in HPLC analysis, Dr. Lucia
Rameh for advice in immunofluorescence studies, Dr. Richard Mitchell
for insightful discussions, and Dr. Kermit Carraway III for critical
review of the manuscript.
Department of Cell Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(Wong, K., and Cantley, L. C. (1994) J. Biol.
Chem. 269, 28878-28884). Alternatively spliced forms of PI4K
have also been identified from several sources including bovine brain
(Gehrmann, T., Vereb, G., Schmidt, M., Klix, D., Meyer, H. E.,
Varsanyi, M., and Heilmeyer, L. M., Jr. (1996) Biochim. Biophys.
Acta 1311, 53-63). Recently we isolated a distinct human PtdIns
4-kinase gene, named PI4K
, that encodes an enzyme that is wortmannin
sensitive (Meyers, R., and Cantley, L. C. (1997) J. Biol.
Chem. 272, 4384-4390). Here we report the locations of these
enzymes and provide evidence for other yet unidentified isoforms
present in specific organelles. PI4K
is mostly membrane-bound and
located at the endoplasmic reticulum; whereas PI4K
is in the cytosol
and also present in the Golgi region. Neither of these isoforms
accounts for the major type II PtdIns 4-kinase activity detected in the
lysosomes and plasma membrane fraction.
(1). This protein is highly
homologous to the yeast STT4 enzyme. Northern blot analysis of the
poly(A)+ mRNAs from human tissues and cell lines
revealed multiple alternatively spliced transcripts. A 230-kDa PtdIns
4-kinase made from an alternatively spliced form of the rat PI4K
has
recently been described (27). A bovine gene encoding a 170-200-kDa
PtdIns 4-kinase that shares more than 95% identity with the human
PI4K
in the overlapping region has also been reported (2). It
represents probably another splice variant of PI4K
. More recently,
we isolated another human cDNA that encodes a 110-kDa PtdIns
4-kinase (named PI4K
) that is more homologous to the yeast PIK1
gene. PI4K
is wortmannin-sensitive and may be the same enzyme
recently reported to be involved in the hormone sensitive pools of
inositol phospholipids (28). To determine the relative roles of these
enzymes in producing phosphoinositides for membrane trafficking,
hormone sensitive PtdIns turnover, growth factor-dependent
PtdIns-3,4,5-P3 production, and cytoskeletal rearrangement,
it is first important to determine the subcellular locations of these
enzymes.
Materials
-32-P] ATP, and silica
gel plates were purchased from Avanti (Alabaster, AL), DuPont NEN, and
E. Merck (Germany), respectively. Mouse monoclonal antibody 12CA5,
reactive to the influenza virus hemagglutinin, was purchased from
BabCo. Rabbit polyclonal anti-PI4K
antibody 3334 was raised to a
peptide corresponding to amino acids 501-512, KPYPKGDERKKA, coupled to
keyhole limpet hemacyanin (1). Peptide antibodies were
affinity-purified prior to immunoblotting and immunofluorescent
staining experiments. Anti-PI4K
antibody was raised against a
GST-fusion protein that was generated by polymerase chain reaction
using oligonucleotide primers that generate amino acids 410-538 of
PI4K
(3). Anti-BiP and anti-
-adaptin were purchased from
StressGen Biotechnologies Corp. and Sigma, respectively.
and PI4K
was tagged at the amino terminus with a
9-amino acid epitope (YPYDVPDYA) derived from influenza virus
hemagglutinin by insertion into Bluescript encoding the epitope
sequence. The tagged cDNA was subcloned into a mammalian expression
vector pRC/CMV (Invitrogen). The construct was transiently transfected
into CHO or HeLa cells using LipofectAMINE (Life Technologies, Inc.)
according to the manufacture's procedures. The tagged cDNA was
also subcloned into a baculoviral expression vector. Recombinant viruses were generated with the linear AcMNPV transfection module (Invitrogen) and were plaque-purified prior to use. The GST-PI4K
construct was generated by polymerase chain reaction as described in
detail elsewhere (3) and was expressed in Escherichia coli by standard procedures. The fusion protein is predicted to be 120 kDa
since it lacks the amino-terminal 82 amino acids of PI4K
.
were
harvested with phosphate-buffered saline containing 1 mM
EDTA and 1 mM EGTA. Cells were pelleted and then
resuspended in homogenizing buffer (10 mM Tris-HCl, pH 7.4, 20 mM NaCl, 1 mM phenylmethylsulfonyl fluoride,
1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin). After
swelling for 15 min on ice, the cells were Dounce-homogenized (25-35
strokes). Nuclei and unbroken cells were spun out at 1000 × g for 10 min at 4 °C. The postnuclear supernatant was
centrifuged at 100,000 × g for 1 h in a SW55Ti rotor (Beckman Instruments). The membrane pellet was solubilized with
1% Triton X-100 in homogenizing buffer. The cytosolic and particulate
fractions were adjusted to equal volume.
were disrupted by low
pressure nitrogen cavitation in 0.25 M sucrose, pH 7.4, containing various proteinase inhibitors. After centrifugation at
1300 × g for 10 min, the postnuclear supernatant was
collected and overlaid on a hybrid percoll/metrizamide discontinuous
density gradient (5 ml of 6% Percoll, 2 ml of 17% metrizamide, 2 ml
of 35% metrizamide). The step gradient was centrifuged at 20,000 rpm
for 30 min at 4 °C in a SW44Ti rotor (Beckman Instruments). Interfaces were sequentially removed from the top of the gradient as
follows: first the top of the gradient, followed by the postnuclear supernatant in 0.25 M sucrose, the sucrose/Percoll
interface, the 6% Percoll/17% metrizamide interface where lysosomes
sediment, and finally the 17%/35% metrizamide interface where
mitochondria sediment.
-hexosaminidase,
and
-mannosidase II were used as the marker enzymes for plasma
membrane, mitochondria, lysosomes, and Golgi apparatus, respectively.
Lactate dehydrogenase was used as a cytosol marker. Each of the enzyme
assays was performed as described previously (29).
-adaptin. The immunostained cells were observed by either
conventional light or confocal microscopy.
-32P]ATP (3000 Ci/mmol; DuPont NEN) per sample. Assays were performed at 37 °C for
20 min and then stopped with 25 µl of 5 N hydrochloric acid. The lipid was extracted with 160 µl of 1:1 (v/v)
chloroform:methanol. The organic layer was collected and analyzed by
both thin layer chromatography and HPLC as described in detail
elsewhere (31).
PtdIns 4-Kinase Antibody Specificity
and the PI4K
enzyme have similar mobilities on SDS-gels, it
is critical to verify that the antibodies raised against the highly
divergent regions of these two enzymes indeed do not cross-react. We
assessed the isozyme specificity of the PtdIns 4-kinase antibodies by
immunoblotting the recombinant PI4K
and PI4K
that were expressed
in Sf9 insect cells and in E. coli, respectively. Fig.
1 shows that PI4K
antibody detected a single band of
97-kDa protein only in Sf9 cells expressing PI4K
(left
panel, lane 3). No immunoreactivity was observed in either Sf9 cells expressing wild-type viral proteins (left
panel, lane 4) or in E. coli expressing
PI4K
(left panel, lane 2). Conversely, when
the blot was reprobed with anti-PI4K
antibody, it reacted with the
120-kDa GST-PI4K
only in cells subjected to the induction of
expression (right panel, lane 2) and not in the
nontransformed E. coli (right panel, lane
1). The immunoreactivity was specific to PI4K
and not to the
GST motif, since the GST-cleared antibodies still detected the 120-kDa
PI4K
fusion protein (3). These results demonstrate that the
antibodies are highly specific, and therefore suitable for
immunocytochemical localization of PtdIns 4-kinase isozymes.
Fig. 1.
Isozyme specificity of the PI4K and
antibodies. PI4K
was expressed in Sf9 insect cells via
baculovirus infection, and the carboxyl-terminal 723 amino acids of
PI4K
were expressed as a GST fusion protein in E. coli.
Lysates prepared from untransformed E. coli (lane
1) or E. coli expressing PI4K
(lane 2)
and lysates prepared from Sf9 cells expressing human PI4K
(lane 3) or wild-type viral proteins (lane 4)
were immunoblotted first with anti-PI4K
antibodies (left
panel), and then reprobed with anti-PI4K
antibodies (right panel).
[View Larger Version of this Image (46K GIF file)]
and
in CHO-IRS
Cells
and PI4K
genes are
almost ubiquitously expressed in human tissues (1, 3), and since the
antibodies raised against these enzymes react with the respective
proteins in other mammalian tissues (not shown), we first investigated
the ability of these antibodies to blot proteins in CHO cells. The
anti-PI4K
antibodies did not detect a 97-kDa PI4K
isoform in the
parental CHO cells (Fig. 2A, left panel), but strongly reacted with an approximately 180-kDa protein (p180) of the size expected for the high molecular weight alternative splice of PI4K
(2). This result is consistent with the ubiquitous expression of the 7.5-kilobase pair message for PI4K
(1, 27). The
97-kDa form of PI4K
was detected when this cDNA was introduced. The PI4K
antibody reacted with an approximately 110-kDa band (Fig.
2A, right panel), consistent with the size of
this protein previously detected in Jurkat cells (3). The postnuclear
supernatants from the PI4K
transfected cells were separated into
soluble and particulate fractions by centrifugation at 100,000 × g. The 97-kDa PI4K
was predominantly associated with the
particulate fraction (Fig. 2B, left panel), as
was p180. p110 PI4K
was found predominantly in the soluble fraction
(Fig. 2B, right panel).
Fig. 2.
A, immunoblot of postnuclear
supernatants prepared from parental CHO-IRS and PI4K-transfected
CHO-IRS using anti-PI4K
and anti-PI4K
antibodies. Equal
quantities of protein prepared from the transfected and the parental
CHO-IRS cells were analyzed by SDS-polyacrylamide gel electrophoresis,
and immunoblotted first with anti-PI4K
(left panel) and
then with anti-PI4K
(right panel). The immunoreactive
band of the exogenously expressed PI4K
(97-kDa) is indicated by a
solid arrow. The endogenous PI4K
(110 kDa) and a protein
of 180 kDa, which reacts with PI4K
antibody and probably represents
an alternative spliced form of PI4K
(see text), are indicated by
unfilled arrows. B, PI4K
and PI4K
have different distributions in high speed centrifugation. The supernatant (S) and particulate (P) fractions from a 1-h
100,000 × g spin of homogenized PI4K
-transfected
CHO-IRS cells were analyzed by immunoblotting with anti-PI4K
(left panel) and anti-PI4K
(right panel).
[View Larger Version of this Image (16K GIF file)]
were fractionated using
the hybrid Percoll/metrizamide discontinuous density gradient as
described previously (29). This gradient allows the isolation of
lysosomes, mitochondria, and partial separation of plasma membrane from
cytosol and organelles such as ER and Golgi. Interfaces sequentially collected from the top of the step gradient were adjusted to the same
volume. Equal portions of individual fractions were then used for
organelle marker enzyme assays, and for Western blot analysis with the
anti-PtdIns 4-kinase antibodies. Monitoring the
-hexosaminidase
activities, we found 85% of the lysosomes sedimented at the interface
of Percoll/17% metrizamide as expected (Table I). The
cytochrome c oxidase assays indicated that 69% of the
mitochondria sedimented to the 17%/35% metrizamide interface, and
22% remained in the Percoll/17% metrizamide interface. Western blot
revealed no significant amounts of anti-PI4K
or anti-PI4K
reactive protein present in these two fractions. Instead, p97 PI4K
,
PI4K
, and the anti-PI4K
reactive p180 concentrated at the top of
the gradient and in the 0.25 M sucrose fraction (Fig. 3), where over 95% of Golgi apparatus and 40% of
plasma membrane cofractionated (Table I). About 40% of the plasma
membrane marker activity was also present at the sucrose/Percoll
interface, but PI4K
and
were not detected in this fraction,
suggesting that they are not associated with this fraction of the
plasma membrane.
Top
fraction
Sucrose
Sucrose/Percoll
Percoll/17%
metrizamide
17%/35% metrizamide
%
Alkaline
phosphodiesterase I (plasma membrane)
18
22
39
18
3
-Mannosidase II (golgi apparatus)
44
54
1
1
0
-Hexosaminidase (lysosome)
0
3
12
85
0
Cytochrome c oxidase
(mitochondria)
1
2
6
22
69
Total PtdIns 4-kinase
activity
21
30
34
15
0
4C5G inhibitable
activity
15
21
26
11
0
97-kDa
PI4K
+
+
p180-kDa
form
+
+
PI4K
+
+
Fig. 3.
Distribution of PI4K and
in
subcellular compartments. Subcellular organelles were separated
using a hybrid Percoll/metrizamide discontinuous density gradient as
described under "Experimental Procedures." Each fraction
representing equal numbers of cells was blotted with anti-PtdIns
4-kinase antibodies. PI4K
(top panel) and PI4K
(bottom panel) are both concentrated in the top fraction of
the gradient (Top) and the sucrose fraction (S).
A minimal amount of PI4K
is detected in the sucrose/Percoll
(S/P) interface, the Percoll/17% metrizamide
(P/17%M), or 17%/35% metrizamide (17%/35%M) fractions, where plasma membrane, lysosomes, and mitochondria sediment,
respectively.
[View Larger Version of this Image (33K GIF file)]
and PI4K
fractionate (Fig. 3), a substantial amount of activity was detected in
the region where plasma membranes sediment (34%) and where lysosomes
sediment (15%) (Table I). The assay was carried out under conditions
in which PtdIns 3-kinase is inactive (0.3% Triton X-100). HPLC
analysis of the deacylated products generated under these conditions
verified the absence of PtdIns-3-P (Fig. 4). The PtdIns
4-kinase activity was not significantly inhibited by 1 µM
wortmannin, indicating that the wortmannin-inhibitable PI4K
is not a
major component of total activity in these fractions (data not shown).
Consistent with previous studies, an inhibitory antibody, 4C5G, raised
against the 55-kDa type II PtdIns 4-kinase caused about 75% inhibition
of the plasma membrane PtdIns 4-kinase (sucrose/Percoll fraction, Table
I). This antibody also substantially inhibited PtdIns 4-kinase activity
in other fractions. The 55-kDa type II PtdIns 4-kinase was previously
shown to be the major PtdIns 4-kinase in red cell plasma membrane (32)
and in other cells. Thus, the results in Table I indicate that the
membrane bound forms of PI4K
and PI4K
are in compartments of the
cell consistent with Golgi apparatus and/or endoplasmic reticulum, and
that the major PtdIns 4-kinase activity in plasma membrane and lysosome is due to a type II PtdIns 4-kinase distinct from the PI4K
isoforms and from PI4K
.
Fig. 4.
HPLC analysis of PtdIns-P. HPLC analysis
was performed on the PtdIns-P products generated from the lipid kinase
assay as described under "Experimental Procedures." The standards
[-3H]PtdIns-4-P and
[
-]3H]PtdIns-4,5-P2 are shown
(dotted line), and the migration position of
[
-3H]PtdIns-3-P is indicated. Presented here is the
profile of the PtdIns-P product generated from the top fraction of the
gradient (solid line). PtdIns-P products from other
fractions also comigrate exclusively with the PtdIns-4-P standard (not
shown).
[View Larger Version of this Image (27K GIF file)]
and
,
cytoimmunofluorescence studies were performed using both conventional
and confocal microscopy. We chose HeLa cells for these studies since
they grow better on coverslips and are hence more suitable for
immunofluorescent staining experiments. HeLa cells transiently
expressing HA-tagged PI4K
were double stained with anti-PI4K
(Fig. 5A) and anti-HA epitope antibodies
(Fig. 5B). The two antibodies yielded identical patterns,
suggesting that the staining obtained using anti-PI4K
antisera was
specific. The anti-HA antibody did not stain non-transfected cells (not shown). The anti-PI4K
antibodies revealed punctate staining near the
nucleus and in the cytoplasm, a pattern similar to that expected for
ER. To determine whether PI4K
is present in the ER, we double stained an ER resident protein Bip (Fig. 5C) and PI4K
(Fig. 5D) in parental HeLa cells. Overlap of PI4K
with
the ER marker was clearly observed after merging the two images by
confocal microscopy (Fig. 5E). However, the colocalization
was not complete; some punctate staining of PI4K
in the peripheral
regions was not observed to superimpose with BiP staining. As in CHO
cells, the major endogenous protein in HeLa cells that reacts with the
anti-PI4K
antibody is the p180 protein (not shown), so this protein
appears also to be located in the ER.
Fig. 5.
Immunocytofluorescent localization of and
isoforms of PtdIns 4-kinase. HeLa cells overexpressing
HA-tagged PI4K
were double stained with anti-PI4K
(A)
and anti-HA antibodies (B). Anti-PI4K
was detected by
CyTM3-conjugated goat anti-rabbit antibody. Anti-HA was
visualized by fluorescein isothiocyanate-conjugated donkey anti-mouse
antibody. C, D, and E, confocal
microscopy emphasizing the colocalization of PI4K
and the ER marker
BiP. A parental HeLa cell was double stained with anti-BiP antibody
(C) and anti-PI4K
(D). Partial overlap is
indicated in (E). Colocalization of PI4K
and the Golgi marker
-adaptin are shown by double-labeling parental HeLa cells with anti-
-adaptin (F) and anti-PI4K
(G).
Overlap in the Golgi region was revealed in (H). The scale
bars in (C) and (D) represent 20 µm, and those
in (F) and (G) represent 25 µm.
[View Larger Version of this Image (77K GIF file)]
antibody revealed a pattern
characteristic of the Golgi apparatus. Specifically, we noted a
crescent of staining on one side of the nucleus (presumably the TGN),
and some punctate staining within the cytoplasm which may represent the
buds from extended TGN tubules (Fig. 5G). The Golgi
localization was confirmed by double staining
-adaptin, which has
previously been shown to be localized in the TGN and the late endosomes
(Fig. 5F) (33, 34). In this case, complete colocalization
was observed (Fig. 5H). Staining could be eliminated by
preincubation of the primary antibody with the immunizing GST-PI4K
and not by GST alone (not shown), indicating that the observed signal
is generated by PI4K
-specific antibodies. Our observation that much
of the PI4K
protein is in the supernatant of a 100,000 × g spin (Fig. 2B) is consistent with several
possibilities. First, the lysis and homogenization procedures dislodge
some of this protein from the particulate fraction. Second, the
cytosolic fraction is partially lost during cell fixation, or last,
this fraction is not as easily visualized because of dilution.
is detected predominantly in the
particulate fraction, and PI4K
is present in the cytosol and to a
lesser extent in the particulate fraction. Fractionation of CHO-IRS
cells revealed that neither the
nor
isoform is located in the
lysosomes or mitochondria, and probably not in the plasma membrane.
Immunocytofluorescence studies demonstrate that PI4K
is present in
the endoplasmic reticulum, and PI4K
is localized to the Golgi
region.
gene that generates a protein predicted to be 230 kDa.
Gehrmann et al. (2) also isolated a partial bovine cDNA encoding a 170-200-kDa PtdIns 4-kinase, that is almost certainly another splice variant of PI4K
. The carboxyl-terminal half of both
high molecular weight proteins contains the domains necessary for lipid
catalysis, and share greater than 95% identity with the human p97
PI4K
. We also noted an alternative splice of PI4K
from human
tissues that is predicted to encode a larger protein (1). Nakagawa
et al. (27) found that the 97-kDa PI4K
and the higher
molecular weight form colocalize, and the later is predominantly
associated with the particulate fraction. These results are consistent
with our finding that the p180 and p97 cofractionate and colocalize. It
is unlikely that they are integral membrane proteins, since neither
splice variant contains a predicted transmembrane domain. However, it
is clear from our studies that the membrane association does not
require the extended amino-terminal sequence of the higher molecular
weight form of PI4K
.
localize to the Golgi membrane in COS cells. In
our study, both the exogenously expressed p97 PI4K
and the endogenous p180 localize to the ER rather than the Golgi of HeLa cells.
The discrepancy cannot be explained by the cell type, since we also
obtained ER staining when PI4K
was expressed in COS cells (not
shown). The possibility that the HA-tag affected localization is
unlikely, since the endogenous p180 exhibited the same location as the
HA-tagged p97.
or any splice variants of PI4K
.
A plausible candidate is the previously described 55-kDa type II PtdIns
4-kinase. Historically, PtdIns 4-kinases are classified into two
categories, types II and type III, on the basis of their molecular
sizes, and the differences in their sensitivity to adenosine and
nonionic detergent (13). The type II PtdIns 4-kinase is dramatically
activated by nonionic detergent but is potently inhibited by both
adenosine and the 4C5G monoclonal antibody (32). Type II PtdIns
4-kinase has been purified from erythrocyte membrane (32) and also
copurified with the epidermal growth factor receptor (35), as well as
with the lymphocyte CD4 antigen (36). Additional findings have also
demonstrated that it is recruited to and activated by epidermal growth
factor receptor upon ligand stimulation (37). We have extensively
investigated the possibility that the type II 55-kDa enzyme is a
proteolytic product of the 97-kDa PI4K
since they share similar
enzymatic properties. However, we are unable to detect proteins of 55 kDa resulting from the processing of the 97-kDa PI4K
. Thus, the
55-kDa type II PtdIns 4-kinase appears to be encoded by a distinct but related gene.
and PI4K
suggests a direct role of PI4K
and
in ER and Golgi
function, as opposed to the ligand-stimulated phosphoinositide turnover
at the plasma membrane. The finding that PI4K
is
wortmannin-sensitive (IC50, 140 nM) and is
located in the Golgi apparatus should caution future studies employing
wortmannin inhibition as the standard of implicating the role of PtdIns
3-kinase in targeting proteins from Golgi apparatus to lysosome.
Finally, our results do not exclude other sites of PtdIns 4-kinase
function. For example, the colocalization of ER marker was not complete
for PI4K
. We also noticed a significant amount of PI4K
in the
particulate fraction that could not be extracted with detergent,
implicating cytoskeletal association. Future studies with dominant
negative forms or gene knockouts will elucidate the roles of these
enzymes in mammalian cell function.
*
This research was supported by National Institutes of Health
Grant GM 36624.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
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1734 solely to indicate this fact.
To whom correspondence should be addressed: Beth Israel
Hospital, Division of Signal Transduction, Harvard Institutes of
Medicine, 10th Floor, 330 Brookline Ave., Boston, MA 02215. Tel.:
617-667-0947; Fax: 617-667-0957.
1
The abbreviations used are: PtdIns,
phosphatidylinositol; GST, glutathione S-transferase; CHO,
Chinese hamster ovary; ER, endoplasmic reticulum; HPLC, high
performance liquid chromatography; HA, hemagglutinin; BiP, binding
protein; TGN, trans-Golgi network; IRS, insulin receptor
substrate.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.