(Received for publication, July 23, 1996, and in revised form, October 17, 1996)
From the Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02215
Phosphatidylinositol (PtdIns) 4-kinases catalyze
the synthesis of PtdIns-4-P, the immediate precursor of
PtdIns-4,5-P2. Here we report the cloning of a
novel, ubiquitously expressed PtdIns 4-kinase (PI4K). The
2.4-kilobase pair cDNA encodes a putative translation product of
801 amino acids which shows greatest homology to the yeast
PIK1 gene. The recombinant protein exhibits lipid kinase
activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The
biochemical properties of PI4K
are characteristic of a type III
enzyme. Interestingly, both recombinant PI4K
and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that
we have cloned the previously described PtdIns 4-kinase that is
responsible for regulating the synthesis of agonist-sensitive pools of
polyphosphoinositides (Nakanishi, S., Catt, J. K., and Balla, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5317-5321).
The metabolism of phosphoinositides has long been acknowledged to
play a central role in the transduction of signals triggered by a
variety of growth factors and hormones. Both the enzymes and their
product phosphoinositides are present in virtually all eukaryotic
organisms and tissues that have been studied. Over the past several
years the complexity of phosphoinositide metabolism has become better
appreciated. In the classically defined phosphatidylinositol (PtdIns)1 turnover pathway, sequential
phosphorylation of the 4 and 5 positions yields PtdIns-4-P and
PtdIns-4,5-P2, the latter of which acts as a substrate for
phospholipase C producing inositol 1,4,5-trisphosphate, a stimulator of
intracellular Ca2+ release (2), and diacylglycerol, a
stimulator of certain protein kinase C isoforms (3). More recently
PtdIns-4-P and PtdIns-4,5-P2 have been shown to regulate
cytoskeletal rearrangement through the association with a variety of
actin binding proteins (4, 5). PtdIns-4,5-P2 has also been
shown to stimulate both phospholipase D (6, 7) and -adrenergic
receptor kinase (8). Finally, all of these lipids are substrates of
PtdIns 3-kinase, yielding an array of 3-phosphorylated products (9). It
is now clear that the synthesis of a variety of polyphosphoinositides
from the starting substrate PtdIns is catalyzed by at least three types of PtdIns kinases (10, 11).
PtdIns 3-kinase (a type I enzyme) catalyzes the phosphorylation of PtdIns at the D3 position of the inositol ring. This enzyme was initially identified through its association with viral oncoproteins and a number of growth factor receptors (12). More recently several additional classes of PtdIns 3-kinases have been identified including a G protein-activated enzyme (13) and VPS 34p, a protein involved in protein trafficking in yeast (14).
PtdIns 4-kinases catalyze the phosphorylation of PtdIns at the D4 position of the inositol ring and have been divided into two types (II and III) based on their size and sensitivity to various compounds (11). The type II enzymes were initially characterized as membrane-associated 55-kDa proteins whose lipid kinase activity is highly stimulated by detergent and inhibited by both adenosine and the monoclonal antibody 4C5G (11, 15). The type III enzymes are membrane-associated proteins predicted to be >200 kDa in size that are less stimulated by detergent and are not inhibited by adenosine or 4C5G antibodies. The PtdIns 4-kinases are highly abundant and have been identified in a large number of membrane structures (reviewed Ref. 16).
Recently several PtdIns 4-kinases have been cloned and found to be
homologous to PtdIns 3-kinases. They all contain both a lipid kinase
unique domain and a C-terminal catalytic domain with distant homology
to protein kinases. In yeast, the PIK1 gene encodes a
125-kDa protein that is indispensable for cell growth and plays a role
in cytokinesis (17). It contains the lipid kinase unique domain at its
far N terminus and the catalytic domain in the characteristic C-terminal position. Although it is intermediate in size, its biochemical properties suggest that it is more similar to the type III
enzyme (18). In Dictyostelium discoideum, a putative PtdIns
4-kinase has recently been cloned, whose domain structure is similar to
PIK1, extending the identification of these proteins across several
species (19). A second yeast gene, STT4, encodes a 200-kDa
protein that is dispensable for growth in the presence of osmotic
stabilizers and has been implicated in the protein kinase C pathway
through its isolation in a screen for mutants sensitive to the protein
kinase C inhibitor staurosporine (20). Finally, the first PtdIns
4-kinase from higher eukaryotes, PI4K, was cloned and shown to
encode a 100-kDa protein with significant homology to STT4 and
biochemical properties of a type II enzyme (21). This protein, as well
as STT4, contains adjacent lipid kinase unique and catalytic domains at
its C terminus. An alternative splice of the PI4K
gene
that generates a 230-kDa protein has also been recently reported
(22).
These three types of PtdIns kinases all show homology to an ever expanding family of protein kinases whose substrates have not yet been identified. This family includes the TOR/FRAP proteins that are the cellular targets of the FK506-binding protein-rapamycin complex and are involved in cellular signaling and cell cycle control (23-27). It is interesting to note that although yeast TOR2 and mammalian FRAP/RAFT1 have associated PtdIns 4-kinase activities, these activities are probably not endogenous to the protein kinase catalytic site (27). Other members of this extended family include the ATM/MEC1/DNA-PK proteins that are involved in both cell cycle progression and checkpoint control and chromosomal maintenance and repair (28-30). All these proteins share a conserved C-terminal catalytic domain found in both lipid and protein kinases.
Within this conserved domain are specific amino acid stretches that
distinguish the subfamily of PtdIns 4-kinases from PtdIns 3-kinases and
the other family members. We have taken advantage of this subfamily
specificity to design degenerate PCR primers for the use in cloning
novel PtdIns 4-kinases. We have identified and cloned one such gene and
analyzed the biochemical properties of the encoded protein, which we
call PI4K. Interestingly, PI4K
is wortmannin-sensitive and shows
great similarity to a recently described wortmannin-inhibitable PtdIns
4-kinase that was partially purified from bovine adrenal cortex (1).
Nakanishi et al. (1) demonstrate that this enzyme is
responsible for regulating the hormone-sensitive pools of inositol
phospholipids. Recent studies in which the effects of 100 nM to 1 µM wortmannin have been used to
implicate phosphatidylinositol 3-kinase in membrane trafficking, cytoskeletal rearrangement, and signal transduction must be
reconsidered in view of the nearly ubiquitous expression of
wortmannin-sensitive PI4K
.
Human placenta and heart cDNA libraries and
the TA cloning kit were purchased from Clontech. Taq
polymerase was purchased from Perkin-Elmer. Expand PCR kit was
purchased from Boehringer Mannheim. PtdIns was purchased from Avanti,
[-32P]ATP from DuPont NEN, silica plates from Merck,
and wortmannin was purchased from Sigma. Random prime
labeling kit was purchased from Pharmacia Biotech Inc.
A 32-fold degenerate primer
(GGIGA(T/C)GA(T/C)TG(T/C)(C/A)GICA(A/G)GA), corresponding to the sense
orientation of the conserved sequence GDD(C/L)RQ(D/E), as well as a
64-fold degenerated primer (AT(A/G)TTICC(A/G)TT(A/G)TGIC(G/T)(A/G)TCT/CTT) corresponding to the
antisense orientation of the conserved sequence KDRHNGNI were used in
PCR reactions containing ~1 × 107 plaque-forming
units of a human placenta cDNA library in GT10. Reaction
conditions were 30 cycles of 94 °C for 1 min, 55 °C for 1 min,
72 °C for 1 min and then a 10-min extension at 72 °C. The 312-bp
product was digested with SmaI to eliminate PI4K
clones from the population of PCR products, reamplified as indicated above,
and redigested with SmaI. Individual clones were sequenced following subcloning into a TA cloning vector (Clontech) using M13
forward and reverse primers.
The fragment, corresponding to a novel putative PtdIns 4-kinase, was
random prime-labeled with [-32P]CTP and used to screen
a human heart cDNA library in
GT10, under standard procedures
(32). ~5 × 105 plaques were screened, and 8 positive clones were obtained. Each was subcloned into Bluescript
pKS-(Strategene) at the EcoRI site, under standard
procedures (32). The longest clone (3.2) was 1.5 kb, contained 1.2 kb
of coding sequence, and no in-frame stop at the 5
end. To extend the
sequence, a second overlapping clone (13.1) was digested with
EcoRI/PstI, and a 350-bp fragment corresponding to the 5
-most end of the sequences obtained was labeled and used to
rescreen the library as described above, resulting in one additional clone that extended the coding sequence in the 5
direction by 700 bp.
To obtain the remainder of the full-length cDNA, 5-RACE PCR was
performed using human placenta cDNA supplied by Clontech in their
5
-RACE PCR kit, under the manufacturer's suggested conditions. PCR
was performed using adapter primer 1 (Clontech) and an antisense primer
recognizing nucleotides 820-840 in PI4K
in reactions containing the
Expand PCR Enzyme mix (Boehringer Mannheim). Individual clones were
sequenced following subcloning into TA cloning vector as described
above. The consensus of five independent clones confirms the sequence
of the 5
end of PI4K
.
A multiple human tissue blot (Clontech) was probed with the randomly primed 312-nucleotide PCR product from the original placenta cDNA library, as per manufacturer's instructions.
Bacterial Expression and Antibody ProductionA GST-fusion
protein was generated by PCR using oligonucleotide primers recognizing
amino acids (aa) 410-414 (STRSV) in the sense orientation and aa
534-538 (PYGHL) in the antisense orientation, both tailed with
appropriate restriction enzyme recognition sites for subcloning into
pGEX4T2 (Pharmacia). Recombinant clones were screened by
SDS-polyacrylamide gel electrophoresis of Escherichia coli
protein lysates after isopropyl-1-thio--galactopyranoside induction,
and the fusion protein (GST4K
5
) was purified using glutathione-agarose affinity chromatography using standard procedures (33). The purified 45-kDa GST fusion protein was injected into rabbits,
and antiserum was collected using standard procedures (Charles River
PharmServices). Affinity purified antibodies were prepared by first
incubating 5 ml of crude serum (diluted 1:10 in 10 mm Tris, pH
7.5), with 500 µl of a 2 mg/ml GST affinity column, for 2 h at
4 °C. Unbound antibody was then chromatographed over a 2 mg/ml
GST4K
5
affinity column, and specifically bound antibodies were
eluted under both acidic and basic conditions as described elsewhere
(34). To prepare GST-cleared blotting antibodies, a 1:10,000 dilution
of crude serum in TBST was blotted against a membrane containing 40 µg of GST, and the supernatant was collected. The removal of GST
antibodies was confirmed by Western blotting the supernatant against
unrelated GST fusion proteins as described below.
The N-terminally deleted 4KL and the full-length 4K
cDNAs
were prepared by fusing three DNA fragments together as follows. First
the backbone was prepared by digesting clone 3.2 in pKS
with
StuI/HindIII. Second, clone 8.1 was PCR-amplified
using a 5
sense primer containing a HindIII site and a 3
antisense primer containing a StuI site, digested, and
ligated into the 3.2 vector prepared above, yielding pKS4K
60.
pKS4K
60 was then fused to 5
-RACE products to yield 4K
L and 4K
as follows. 5
-RACE product p3 was amplified using Clontech sense
adapter primer (AP1) and an antisense primer recognizing aa 274-280,
yielding a 1.5-kb fragment. pKS4K
60 was PCR-amplified using a sense
primer recognizing aa 239-245 and a T7 antisense primer, yielding a
2-kb fragment. To generate 4K
L, these two fragments were
PCR-amplified using a sense primer recognizing aa 83-87 and an
antisense primer recognizing aa 797-801 both containing appropriate
restriction site for subcloning. To generate 4K
, these two fragments
were PCR-amplified using a sense primer recognizing aa 1-5 and the
same antisense primer. The 2.3-kb (PI4K
L) and 2.5-kb (PI4K
)
amplified products were digested and subcloned into pGEX4T-2.
Recombinant clones were screened by SDS-polyacrylamide gel
electrophoresis, and the fusion proteins were purified as described
above. To obtain reasonable amounts of active proteins, we used lower
concentrations of isopropyl-1-thio-
-galactopyranoside (0.1 mM) and overnight induction at 25 °C.
Frozen pelleted J77 Jurkat cells were resuspended in cold lysis buffer (0.3 M NaCl, 20 mM HEPES, pH 7.5, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.2% Triton X-100, 500 µM vanadate, 200 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µM NaF, and 1 µg/ml leupeptin, pepstatin, and aprotinin) at a concentration of ~ 5 × 107 cells/500 µl. Lysates were incubated for 20 min on ice and then spun at 14,000 × g at 4 °C for 15 min. Supernatant was removed and added to 3 volumes of equilibration buffer (20 mM HEPES, 7.5, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, and dithiothreitol and inhibitors as indicated above). Protein from ~5 × 106 cells was precipitated by incubating preimmune, affinity-purified, or crude immune serum for 1.5 h at 4 °C and then adding bovine serum albumin-precoated protein A beads for 1.5 h at 4 °C. Pelleted beads were washed 2 × with lysis buffer, 2 × with lysis/equilibration buffer (1/3), and 2 × with 20 mM HEPES, 1 mM EDTA, 0.1% Triton X-100. For immunoblot analysis, beads subjected to lipid kinase assay (see below) were washed 1 × with MeOH, 2 × with phosphate-buffered saline + protease inhibitors, and then resuspended in 2 × SDS loading buffer.
Western blots were performed under standard procedures (Promega) using TBST+ milk (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 5% nonfat dry milk) for blocking and TBST for antibody incubations and washes. Signal was detected using horseradish peroxidase-coupled secondary antibodies and chemiluminescence as described by the manufacturer (DuPont NEN).
PtdIns Kinase Assay and HPLC AnalysisPtdIns kinase assays
were performed essentially as described elsewhere (35). Briefly, GSH
beads or Protein A beads containing PI4K were incubated in 50-µl
reactions containing 0.3% Triton X-100, 50 µM ATP, 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.2 mg/ml sonicated PtdIns, and 30 µCi [
-32P]ATP (3000 Ci/mmol DuPont NEN) at 37 °C for 20 min. Reactions were stopped with
105 µl of 1 N HCl and extracted with 160 µl of 1:1
(v/v) mixture of CHCl3:MeOH. The organic layer was
collected and analyzed by both thin layer chromatography and HPLC, as
described elsewhere (36).
To isolate novel PtdIns 4-kinases, DNA from a human
placenta cDNA library was used as a template in PCRs primed with
degenerate oligonucleotides derived from two regions highly conserved
among PI4K, PIK1, and STT4. The 312-bp product generated by PCR was SmaI-digested to eliminate PI4K
products from the
population, and the reamplified PCR product was subcloned and sequenced
to reveal a novel DNA fragment with homology to the family of PtdIns 4-kinases. This fragment was random prime-labeled and used for Northern
blot analysis of human tissues (see below). Based on tissue
distribution, it was used to screen a human heart cDNA library. A
number of overlapping clones were isolated, and the longest clone (3.2)
was further analyzed. The 1.5-kb insert contained a 1.2-kb open reading
frame, a 3
stop, and 0.3-kb of 3
-untranslated region. Since no 5
stop was detected, the library was rescreened with a probe from the 5
end of clone 3.2 yielding clone 8.1 which contained 750 bp of
additional open reading frame and still no 5
stop codon. To obtain the
remainder of the open reading frame 5
-RACE was performed on human
placenta cDNA, yielding a number of overlapping clones, five of
which were analyzed and shown to be identical over >80% of their
length. The clones differed slightly at the 5
end but all contained an
identical open reading frame contiguous with that of clone 8.1 and all
had identical stop codons in all three reading frames 5
of a potential
initiating methionine.
The 2.4-kb full-length cDNA predicts a protein of 801 aa with a
predicted size of ~90-kDa, starting from an initiation codon with a
favorable Kozak consensus sequence for translation initiation (Fig.
1) (37). A second potential initiating methionine would result in a protein 104 amino acids shorter of predicted size ~80
kDa. Both initiating methionines are followed by glycines suggesting
myristoylation of the N terminus (Fig. 1). The predicted protein,
PI4K, contains an N-terminal domain (lipid kinase unique domain)
(Fig. 2A) that is shared by members of both
the PtdIns 3- and PtdIns 4-kinase families (22). Additionally, a
C-terminal catalytic domain (Fig. 2B) defines this protein
as a member of a much larger family of protein/lipid kinases that
includes the PtdIns 3- and PtdIns 4-kinases, the TOR proteins, ATM,
DNA-PK, MEC1/RAD3, and MEI41 whose members are involved in such diverse functions as mitogenic signaling, cell cycle regulation, and DNA repair
(reviewed in Ref. 38). Interestingly, the functionally related
PtdIns 4P5-kinase family appears to share no significant sequence homology in either the lipid kinase unique domain or the
catalytic domain (39, 40).
PI4K shares most significant sequence homology with yeast PIK1 (42%
identity in the catalytic domain and 17% in the lipid kinase unique
domain) and with the newly described D. discoideum gene
DdPIK4 (45% in the catalytic domain and 19% in the lipid kinase unique domain). This is consistent with the conserved domain structure among these three proteins.
Northern blot analysis was performed on
multiple human tissues and revealed a single ~4-kb message in a
variety of tissues (Fig. 3). Although PI4K is
ubiquitously expressed, the precise distribution is distinct from that
of the other human PtdIns 4-kinase, PI4K
(21), suggesting
nonredundant functions for these two enzymes. The highest level of
expression was detected in heart, pancreas, and skeletal muscle.
Lipid Kinase Activity of Recombinant and Endogenous PI4K
To confirm that PI4K encodes an active PtdIns
4-kinase, several GST fusion constructs were generated, and recombinant
protein was expressed in E. coli. One such construct,
lacking the N-terminal 82 aa of PI4K
(GST4K
L) was expressed,
purified, and assayed for lipid kinase activity in reactions containing
PtdIns as a substrate (Fig. 4A). In contrast
to the control (C) GST fusion protein that lacked the
catalytic domain, GST4K
L generated significant amounts of PtdIns-P
at both concentrations tested. The full-length PI4K
gave identical
results (data not shown) confirming that the N-terminal nonconserved
portion of PI4K
was not required for lipid kinase activity. Since
E. coli lacks endogenous PtdIns kinases, these results
confirm that PI4K
encodes a PtdIns kinase.
To identify the lipid products generated, we performed HPLC analysis on
the deacylated products of the PtdIns kinase assay. A single peak,
precisely comigrating with [3H]glycerophosphorylinositol
4-phosphate standard, was observed (Fig. 4B), supporting the
classification of PI4K as a PtdIns 4-kinase. We observed only modest
inhibition by 500 µM adenosine and the type II-specific
inhibitory monoclonal antibody 4C5G (15), suggesting that PI4K
is
not a type II enzyme (data not shown). To further explore the enzymatic
properties of PI4K
we next examined its inhibition by wortmannin, a
fungal metabolite that inhibits PtdIns 3-kinase at nanomolar
concentrations (41, 42). Although the PI4K
and PIK1 enzymes were
resistant to micromolar concentrations of this drug, a partially
purified PtdIns 4-kinase from bovine adrenal cortex was shown to be
inhibited by 100 nM wortmannin (1). We assayed GST4K
activity in the presence of various concentrations of wortmannin and
observed concentration-dependent inhibition with an
IC50 of ~120 nM (Fig. 4C). This
suggests that PI4K
may be the same enzyme as was previously shown to
be wortmannin-sensitive (1).
This wortmannin-sensitive enzyme was identified in several cell types
including the human Jurkat T-cell line. We therefore generated
PI4K-specific antibodies to investigate the properties of endogenous
PI4K
immunoprecipitated from Jurkat cells. Antibodies were raised
against a GST fusion of a 100-aa partial clone of PI4K
. To
demonstrate the specificity of these antibodies for PI4K
, the
GST-cleared polyclonal serum was used to blot protein lysates from wild
type DH5
cells or DH5
cells expressing GST4K
L (Fig.
5A). A signal corresponding to GST4K
L and
several breakdown products were detected only in lysates from
transformed bacteria (lane 2). Identical results were
obtained when protein lysates were blotted with the crude PI4K
antibodies (data not shown) suggesting that the observed signal was
generated by PI4K
-specific antibodies and not by the GST antibodies
also present in this crude serum.
Using either preimmune or affinity purified immune serum, we
immunoprecipitated PI4K from detergent-solubilized cell lysates of
Jurkat cells. The proteins were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blot using the PI4K
antibody. A single protein migrating at ~110 kDa was observed only in
precipitations using immune serum (Fig. 5B). This protein has a mobility slightly slower than that predicted by the 801-aa PI4K
. It is likely that post-translational modification, such as
myristoylation (see Fig. 1), accounts for the decreased mobility. Aliquots of these same immunoprecipitates were also assayed for lipid
kinase activity in the presence of PtdIns (Fig. 5C). Whereas very low levels of PtdIns-P could be detected in assays containing preimmune serum (Pre), a significant amount of PtdIns-P
(10-90-fold higher than preimmune) was routinely produced in assays
containing affinity purified antiserum (Im) or crude immune
serum (data not shown). When similar assays were performed using either
PtdIns-4-P or PtdIns-4,5-P2 as substrates, no
phosphorylated products were generated (data not shown). As expected,
HPLC analysis of the lipid products confirmed the immunoprecipitation
of a PtdIns 4-kinase (data not shown). Additionally, PI4K
was unable
to phosphorylate PtdIns-3-P (data not shown) suggesting that it is
distinct from the previously characterized PtdIns-3-P 4-kinase (43,
44). PI4K
lipid kinase activity was only modestly affected by
non-ionic detergents, adenosine and 4C5G (data not shown), but was
strongly inhibited by wortmannin (Fig. 5D), with an
IC50 of 140 nM. Taken together, these results
strongly suggest that the 110-kDa PtdIns kinase immunoprecipitated from
Jurkat cell lysates is PI4K
.
We have identified and characterized PI4K, a novel PtdIns
4-kinase that is widely expressed in a variety of tissues. The cDNA
encodes an 801-aa protein that exhibits lipid kinase activity when
expressed in E. coli. Both the bacterially expressed and the
endogenous proteins exhibit properties consistent with the characterization of PI4K
as a type III enzyme. Antibodies raised against PI4K
detect a ~110-kDa protein in a number of cell types across several species. Interestingly, PI4K
is the first cloned PtdIns 4-kinase that is inhibitable by wortmannin, potentially implicating PI4K
in a variety of wortmannin-sensitive cellular pathways.
Sequence analysis of PI4K places it within the PtdIns 4-kinase
family and more generally places it in the larger family of lipid/protein kinases. It contains a conserved C-terminal catalytic domain with distant homology to protein kinases as well as strong homology to the dual specificity kinases such as PtdIns 3-kinase. Within this conserved domain is lysine 549 which, based on homology to
PtdIns 3-kinase, is the likely site of wortmannin reactivity (45). All
members of this lipid/protein kinase family contain this conserved
lysine, yet many, including PIK1 and PI4K
, are not inhibited by
micromolar concentrations of the drug, suggesting that additional
residues within the active site confer wortmannin sensitivity.
Members of this extended family have diverse cellular functions. For example, the yeast protein MEC1 and its Drosophila homologue MEI41 are checkpoint control genes which appear to monitor the state of the genome at the G1/S and G2/M transitions (28, 46, 47). Another family member, DNA-PK, was originally identified as a DNA-dependent protein kinase (48) but was subsequently shown to function in immunoglobulin gene rearrangement and DNA repair (29).
PI4K has properties similar to the wortmannin-sensitive PtdIns
4-kinase described by Nakanishi et al. (1). The partially purified enzyme was inhibited by wortmannin with an IC50 of
~50 nM, not dissimilar to the 120-140 nM
IC50 observed for PI4K
. The wortmannin-sensitive enzyme
had an apparent molecular mass of 125 kDa, as judged by gel filtration,
in agreement with the 110-kDa molecular mass observed for PI4K
. The
reported enzymatic properties of this protein are also very similar to
those of PI4K
. It is likely that we have cloned the PtdIns 4-kinase
that regulates the formation of agonist-sensitive inositol
phospholipids that are required for intracellular signaling in some
cells.
It should be noted that a PtdIns 4-kinase from the particulate fraction
of Schizosaccharomyces pombe has been observed to be
sensitive to the wortmannin analogue demethoxyviridin (49). Curiously,
this enzyme was not inhibited by wortmannin. Additionally, attempts to
isolate a drug-sensitive PtdIns 4-kinase from rat brain particulate
fractions were unsuccessful (49). Although we have detected PI4K in
rat brain, both our experiments and those of Nakanishi et
al. (1) suggest that it is only loosely associated with the
membrane. Furthermore, PI4K
is not the major PtdIns 4-kinase present
in membrane fractions, and therefore lipid kinase assays on these crude
fractions would not be expected to show wortmannin sensitivity.
Taken together, these data suggest that we need to reevaluate the
interpretation of experiments employing wortmannin as an inhibitor in
biological assays. For example, recent experiments have demonstrated
that wortmannin inhibits the proper targeting of the lysosomal enzyme
procathepsin D in a variety of cell types (50, 51). The concentration
of wortmannin used was as high as 1 µM with an estimated
IC50 of ~100 nM. Clearly, these elevated levels of wortmannin could be inhibiting PI4K thereby implicating it
in protein trafficking. Furthermore, both wortmannin and
demethoxyviridin have been reported to inhibit phospholipase D,
PtdIns-phospholipase C, and phospholipase A2 in
vivo (41, 52). It is likely that this inhibition is a downstream
effect of the inhibition of PtdIns 3-kinase and possibly PI4K
in
these cells, as little direct inhibition of these enzymes was observed
in vitro at µM concentrations of wortmannin.
The assumption that PtdIns 3-kinase is a critical mediator of all the
myriad pathways inhibited by wortmannin is likely to be an
oversimplification.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81802[GenBank].
We thank Brian Duckworth and Alex Toker for assistance in the HPLC analysis. We thank Karen Wong for critical review of the manuscript, and we thank Lucia Rameh and the members of the Cantley lab for technical assistance, useful advice, and insightful discussions.