(Received for publication, March 18, 1997)
From the Phosphatidylinositol-4-phosphate 5-kinases
(PIP5Ks) utilize phosphatidylinositols containing D-3-position
phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate.
In addition, type I PIP5Ks phosphorylate phosphatidylinositol
3,4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while
type II kinases have less activity toward this substrate. Remarkably,
these kinases can convert phosphatidylinositol 3-phosphate to
phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction.
Kinase activities toward the 3-position phosphoinositides are
comparable with those seen with phosphatidylinositol 4-phosphate as the
substrate. Therefore, the PIP5Ks can synthesize
phosphatidylinositol 4,5-bisphosphate and two
3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules.
Two distinct pathways have been characterized for
agonist-stimulated signal transduction involving phosphatidylinositol
(PtdIns).1 One pathway entails activation
of phosphatidylinositol-specific phospholipase C by extracellular
agonists resulting in the hydrolysis of phosphoinositides to generate
soluble inositol phosphates including inositol 1,4,5-trisphosphate and
diacylglycerol (reviewed in Refs. 1 and 2). The other pathway involves
receptor-mediated activation of phosphatidylinositol 3-kinase (PtdIns
3-kinase) to produce the second messengers, phosphatidylinositol
3,4-bisphosphate (PtdIns 3,4-P2) and phosphatidylinositol
3,4,5-trisphosphate (PtdIns 3,4,5-P3) (reviewed in Refs. 3
and 4).
A pathway for the formation of D-3-phosphatidylinositols, proposed
based on kinetic studies of intact human neutrophils, is through
phosphorylation of the D-3 position of the myo-inositol ring
of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) by
a PtdIns 4,5-P2 3-kinase and subsequent dephosphorylation
of PtdIns 3,4,5-P3 to produce PtdIns 3,4-P2
(5). This pathway has been supported by the existence of the
extensively characterized PtdIns 3-kinase enzyme family, which can
catalyze in vitro phosphorylation of phosphatidylinositol
4-phosphate (PtdIns 4-P) and PtdIns 4,5-P2. Evidence for a
different pathway for the formation of D-3 phosphatidylinositols has
been found in human platelets, NIH 3T3 cells, and plants in which
phosphorylation of the D-3-position of PtdIns to form PtdIns 3-P is
followed by phosphorylation of the D-4-position to give PtdIns
3,4-P2 and then of the D-5-position to form PtdIns
3,4,5-P3 (6-10). The importance of these various routes of
synthesis has been disputed. Indeed, until now, enzymes that catalyze
the direct phosphorylation of PtdIns 3-P and PtdIns 3,4-P2
have not been clearly identified.
Several phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) have
been discovered and characterized as enzymes synthesizing PtdIns
4,5-P2 (reviewed in Ref. 11). The best characterized isoforms are the type I and type II kinases that differ both
biochemically and immunologically (12-14). The sequence of a type II
PIP5K (PIP5KII None of the PIP5K isozymes have been examined for alternative
substrates except phosphatidylinositol for which there was no detectable activity (12-14). We report here that the type I and II
PIP5K isozymes also utilize the 3-phosphate-containing
phosphatidylinositides, forming PtdIns 3,4-P2 and PtdIns
3,4,5-P3. This supports the existence of an additional
pathway for the synthesis of 3-phosphate-containing phosphatidylinositol polyphosphates.
PtdIns and PtdIns 4-P were from
Boehringer Mannheim or Sigma. PtdIns 3-P and PtdIns 3,4-P2
dipalmitoyl esters were synthesized (22) according to Chen and
Prestwich,2 and Thum et al.
(24), respectively. PtdIns 3-P and PtdIns 3,4-P2 dipalmitoyl esters were also from Matreya. [3H]inositol
1-phosphate, [3H]Ins 1,4-P2,
[3H]Ins 1,3,4-P3 and
[ Recombinant human PIP5Ks were expressed in
Escherichia coli and purified by Ni2+-chelate
chromatography (15, 16, 19). These proteins were stored in 50 mM Tris, pH 7.5, 1 mM EGTA, 150 mM
NaCl, 0.01% sodium azide, and 20% glycerol for the type I isozymes or
50 mM Tris, pH 8.0, and 20% glycerol for the type II
isozymes. Polymerase chain reaction-based cloning of the recombinant
PIP5KI PIP5K isozymes were assayed in 50-µl reactions
containing 50 mM Tris, pH 7.6, 10 mM
MgCl2, 0.5 mM EGTA, 2-100 µM
substrate prepared in isotonic KCl solution, and 50 µM
ATP (4 µCi/nmol). The reactions, at 37 or 22 °C, ranged from 5 to
40 min. Immunoprecipitate kinase activities were tested for 10.5 min at
22 °C in 50 mM Tris, pH 7.5, 10 mM
MgCl2, 1 mM EGTA, 50 µM
substrates prepared in Tris buffer and 50 µM ATP (4 µCi/nmol). The reactions were stopped by the addition of 100 µl of
1 N HCl, and the lipid products were extracted using 200 µl of chloroform/methanol (1:1). The organic phase was washed at
least once in 80 µl of methanol, 1 N HCl (1:1). The
organic phase was then spotted on TLC plates and run as before (16).
After autoradiography for 1-12 h, the spots on the TLC plates
corresponding to products were scraped into vials and counted using
Cerenkov radioactivity in a Beckman scintillation counter.
Phosphatidylinositol
products scraped from TLC plates were treated with methylamine reagent
at 53 °C for 50 min (6). The glycerophosphorylinositol
phosphate products of deacylation reactions were mixed with
[3H]inositol 1-phosphate, [3H]Ins
1,4-P2, and [3H]Ins 1,3,4-P3 as
internal standards and separated by HPLC with a Partisil 10 Sax column
(Whatman) (25). The 1-ml fractions of eluate from the HPLC column were
counted in a Beckman liquid scintillation counter.
PtdIns
[32P]3-P, PtdIns [32P]3,4-P2,
and PtdIns [32P]3,4,5-P3 were prepared
using a recombinant mutant p110 subunit of PtdIns 3-kinase that is
constitutively active (26) (gift from L. T. Williams, Chiron). The Sf9
cell-expressed p110 mutant PtdIns 3-kinase was purified using the
nickel-nitrolotriacetic acid affinity purification procedure
(PharMingen). The kinase reactions were in 1 ml containing 20 mm
Hepes, pH 7.5; 0.2 mM EDTA; 5 mM
MgCl2; 1 mg of PtdIns, PtdIns 4-P, or PtdIns
4,5-P2; 1 mg of phosphatidylserine, 1 mCi of
[ Rabbit polyclonal antibodies were raised against the
hexahistine-PIP5KI PIP5KI After they
were rinsed with phosphate-buffered saline, COS-7 cells were lysed in
1.0% Nonidet P-40, 50 mM Tris, pH 7.5, 150 mM
NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 2 µg/ml leupeptin and 10 trypsin inhibitory units/ml
aprotinin. The clarified lysates (250 µg) were precleared with
Omnisorb cells (Calbiochem), the protein G matrix used to isolate the
immune complexes. Immunoprecipitations were performed with 2 µg of
antibody and the Omnisorb cells (28). For the transfection experiments,
immunoprecipitations were done from 200 µl of lysate with 6 µg of
The ability of four different PIP5K isozymes to phosphorylate
PtdIns 3-P, PtdIns 3,4-P2, and PtdIns 4-P is shown in Fig.
1A. When analyzed by TLC, the PtdIns 3-P
phosphorylation product migrated as a phosphatidylinositol bisphosphate
(PtdInsP2). The product migrated more slowly than
PtdIns 4,5-P2, probably because the PtdIns 3-P substrate is
a dipalmitoyl synthetic lipid (22). The two additional faint spots
above PtdIns 3-P-derived PtdInsP2 may be phosphorylatable
minor contaminants arising during the chemical synthesis of PtdIns 3-P.
The PIP5KII enzymes have similar or greater activity toward
PtdIns 3-P in comparison with PtdIns 4-P, whereas the PIP5KI isozymes
prefer PtdIns 4-P. In addition, the PIP5KI enzymes also have activity
toward PtdIns 3,4-P2, producing a product that migrates as
PtdInsP3 (Fig. 1A, lanes 5 and
6). Remarkably, both PIP5KI isozymes and PIP5KII
The initial rate of PtdIns 3-P phosphorylation using PIP5KII Table I.
Kinetic parameters of PIP5K isozymes
Division of Hematology-Oncology, Departments
of Internal Medicine and Biological Chemistry and Molecular Biophysics,
Washington University School of Medicine, St. Louis, Missouri 63100, the Departments of ¶ Pharmacology and ** Biomolecular Chemistry,
Program in
Cellular and Molecular Biology, University of Wisconsin, Madison,
Wisconsin 53706, and the
Department of
Medicinal Chemistry, University of Utah,
Salt Lake City, Utah 84112
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
) has established that the PIP5K enzymes belong to a
novel family (15). More recently, cDNAs encoding type I PIP5K
(PIP5KI) and an additional PIP5KII isoform have been isolated (16-19).
The translated sequences of PIP5KI and PIP5KII enzymes have only 35%
amino acid identity in their kinase homology domains, further
establishing the distinctiveness of these two subfamilies (16). The
PIP5KI
cDNA is identical to a product of the gene reported to be
mutated in Friedreich's ataxia, a common hereditary autosomal
recessive disease (16, 20, 21). All four recombinant type I and II PIP5Ks have PtdIns 4-P 5-kinase activity (15-17, 19, 21).
Materials
-32P]ATP were from NEN Life Science Products.
Lipofectamine, OPTI-MEM, and fetal bovine serum were obtained from Life
Technologies, Inc. All other chemicals were purchased from Sigma.
used in these experiments resulted in two changes from the
published sequence: glutamine 300 to arginine and serine 421 to
proline. Both changes lie outside of the conserved kinase homology
domain (16). The activity of PIP5KII
is less stable; thus, larger
amounts were used to achieve comparable activity in the kinase
assays.
-32P]ATP, and 10 µg of PtdIns 3-kinase for 1 h
at 37 °C. The lipid products were extracted and purified using
preparative TLC (27). Purified PtdIns [32P]3-P, PtdIns
[32P]3,4-P2, and PtdIns
[32P]3,4,5-P3 were dried under N2
and deacylated (6) to produce glycerophosphorylinositol phosphate
standards.
fusion protein (16) as before (13, 14). The antibodies were affinity-purified on the PIP5KI
fusion protein coupled to Sepharose, following established procedures (13, 14, 28). To
serve as negative controls, preimmune IgGs and a depleted IgG pool were
prepared using protein A-Sepharose (Sigma). The depleted IgG were those
IgG isolated from immune sera after it had been depleted of
anti-PIP5KI
antibodies by multiple cycles over the antigen affinity
column. PIP5KII antibodies (13) were affinity-purified on a recombinant
PIP5KII
column (15). Western blots were performed as described
previously (16).
and
PIP5KII
were N-terminally tagged with the FLAG epitope by subcloning
their coding regions into pcDNA3-FLAG vectors provided by Dr. Jon
Morrow (Yale University). The epitope is recognized by the anti-FLAG m2
monoclonal antibody (Eastman Kodak Co.). Plasmids were introduced into
COS-7 cells by lipofectamine-mediated transfection following the
manufacturer's procedures (Life Technologies, Inc.). Transfection of
pcDNA3-CAT (Invitrogen) was used as a control. Transfected
cells were harvested 17 h after adding the DNA and lysed in 250 µl of Nonidet P-40 lysis buffer.
-FLAG antibody. After several washes, the pellets were resuspended
in 80 µl of 50 mM Tris, pH 7.5, and split into 20-µl
samples for kinase assays and Western blotting.
produce
products from PtdIns 3-P that migrate in the PtdInsP3
position (Fig. 1A, lanes 1-3).
Fig. 1.
Recombinant PIP5K isozymes phosphorylate
3-phosphate-containing phosphatidylinositols. A, the
activities of E. coli-expressed recombinant PIP5K isozymes
were assayed using 80 µM PtdIns 3-P, PtdIns
3,4-P2, or PtdIns 4-P for 10.5 min at 22 °C. The enzymes assayed were PIP5KI (0.2 µg), PIP5KI
(0.7 µg), PIP5KII
(2 µg), and PIP5KII
(180 µg). The positions of products of the
reaction are marked by arrows. All lanes were
from the same TLC plate with different autoradiograph exposures.
Exposures were for 5 min (lane 3) or 15 min (lanes
1, 2, and 9-11) at room temperature or
1.5 h at
80 °C (lanes 4-8 and 12).
B, time course of PIP5KII
activity. The kinase activity
of PIP5KII
(2.4 µg) toward 80 µM PtdIns 3-P (filled circles) or PtdIns 4-P (open squares) was
assayed for 5-40 min at 37 °C. C, time course of
PIP5KI
activity. The kinase activity of PIP5KI
(0.4 µg) toward
5 µM PtdIns 3-P (filled circles) or PtdIns 4-P
(open squares) was determined for 0-20 min at
37 °C.
[View Larger Version of this Image (21K GIF file)]
was
4-fold greater than that of its previously identified substrate PtdIns
4-P (Fig. 1B). In addition, the phosphorylation of PtdIns 3-P was linear for a longer time interval compared with PtdIns 4-P
(Fig. 1B). In a short reaction (5 min), comparison of
PIP5KII
kinase activity toward PtdIns 3-P and PtdIns 4-P at various
concentrations indicated that the activity toward both substrates was
dependent on their concentrations (data not shown). The kinetic
parameters for PIP5KII
phosphorylation of PtdIns 3-P and PtdIns 4-P
are shown in Table I. The apparent Km
value of PIP5KII
for PtdIns 3-P is 3-fold greater than PtdIns 4-P.
However, the Vm for PtdIns 3-P is also about 3-fold
greater than that for PtdIns 4-P. As a result, the catalytic efficiency
(Vm/Km) for these two substrates
is the same. Little activity toward PtdIns 3,4-P2 was
detected with the type II PIP5Ks (Fig. 1A, lanes 3 and
4).
Enzymes
Substrates
PtdIns
3-P
PtdIns 4-P
PtdIns 3,4-P2
Km
Vm
Vm/Km
Km
Vm
Vm/Km
Km
Vm
Vm/Km
PIP5KII
120
90
0.8
50
39
0.8
NDa
ND
ND
PIP5KI
65
1903
29.0
47
29653
631.0
80
438
5.5
PIP5KI
b
5
42
8.4
262
4753
18.0
6
39
6.5
a
ND, none detected.
b
Putative Friedrich's ataxia gene product.
The kinetic parameters of PIP5KI isozymes with their different
substrates are listed in Table I. The time dependence of PIP5KI activity using PtdIns 3-P and PtdIns 4-P is shown in Fig.
1C. The substrate preferences of the type I isozymes are
different from the type II isozyme in that the type I PIP5K (
and
) enzymes phosphorylate PtdIns 4-P with a much greater
Vm than PtdIns 3-P. However, the
Km values of the PIP5KI enzymes using PtdIns 3-P are
lower than that of PIP5KII
.
The products of the PIP5K reactions using PtdIns 3-P and PtdIns
3,4-P2 as substrates were identified by HPLC analysis (Fig. 2). It was anticipated that the PtdInsP2
products would be PtdIns 3,5-P2, given the specificity of
the PIP5K enzymes toward PtdIns 4-P. Surprisingly, HPLC analysis
demonstrated that the deacylated product of all of the PIP5K isozymes
using PtdIns 3-P as substrate co-chromatographed with GroPIns
3,4-P2, as illustrated for PIP5KII in Fig.
2A. This indicated that these kinases synthesize PtdIns 3,4-P2. The HPLC analysis also revealed that the product of
PIP5KI
and PIP5KI
activity toward PtdIns 3,4-P2 was
PtdIns 3,4,5-P3. This is shown for PIP5KI
in Fig.
2B.
The structure of these products was further verified using specific inositol lipid phosphatases. The inositol polyphosphate 4-phosphatase (4-phosphatase) specifically hydrolyzes the 4-position phosphate of PtdIns 3,4-P2 (27). Treatment of the putative PtdIns 3,4-P2 products of PIP5K reactions with recombinant 4-phosphatase resulted in release of 32P-labeled inorganic phosphate, confirming that this was PtdIns 3,4-P2 labeled in the D-4-position (data not shown). In addition, treatment of the PtdInsP3 product with the Lowe oculocerebrorenal syndrome 5-phosphatase, an enzyme that specifically hydrolyzes the 5-position phosphate of PtdIns 3,4,5-P3 (29, 30), released 32P-labeled inorganic phosphate (data not shown). This result confirms that the product of this reaction was PtdIns 3,4,5-P3 labeled on the D-5-position.
Reactions using PtdIns 3-P as substrate also contained a product that
migrated as PtdIns 3,4,5-P3. This product was observed using PIP5KI isozymes and PIP5KII but not PIP5KII
(Fig.
1A, lanes 1-4). HPLC analysis confirmed that
this was PtdIns 3,4,5-P3, which comigrated with lesser
concentrations of lyso-PtdIns 3,4-P2 (20% for PIP5KIs,
45% for PIP5KII
). The amounts of PtdIns 3,4,5-P3 formed
are shown in Table II. Because the substrate
concentrations were 80 µM and the intermediate PtdIns
3,4-P2 product was nanomolar where PtdIns 3-P was the
substrate, the amount of PtdIns 3,4,5-P3 formed is
remarkable. Indeed, the amount of PtdIns 3,4,5-P3 formed from PtdIns 3-P using either PIP5KI
or PIP5KII
was similar to that using 80 µM PtdIns 3,4-P2 with
PIP5KI
. These results suggest that synthesis of PtdIns
3,4,5-P3 from PtdIns 3-P is a concerted reaction. In the
case of the type II enzymes, PtdIns 3,4-P2 was not
detectably phosphorylated by these enzymes. Yet, when type II kinases
use PtdIns 3-P as substrate, PtdIns 3,4,5-P3 is
produced.
|
These data were obtained using recombinant, E. coli-expressed PIP5K isoforms, but similar results were observed
using native PIP5Ks from mammalian cells. PIP5KII purified from
erythrocytes had similar activity to the PIP5KII presented above
(data not shown). The ability of PIP5KI
to phosphorylate the
3-phosphate-containing lipids was validated by immunoprecipitation of
the kinase from COS-7 cells. When COS-7 cell lysates were Western
blotted with anti-PIP5KI
antibody, a single 68-kDa protein was
detected, which was immunoprecipitated with the same antibody (Fig.
3A). The PIP5KI
was not immunoprecipitated
using an IgG depleted of PIP5KI
reactivity (Fig. 3A) or
preimmune IgG (data not shown). The native PIP5KI
was able to
phosphorylate both PtdIns 3-P and PtdIns 3,4-P2, and the
activity toward the former was only 4-fold lower compared with PtdIns
4-P (Fig. 3B). In addition, the production of PtdIns 3,4,5-P3 from PtdIns 3-P was also observed.
The same pattern of phosphorylation of D3-phosphatidylinositols was
observed using recombinant PIP5KI and PIP5KII
expressed in COS-7
cells (Fig. 3, C and D). PIP5KI
and PIP5KII
containing a FLAG epitope at their N termini were transiently
transfected into COS-7 cells, immunoprecipitated with an anti-FLAG
monoclonal antibody, and assayed for activity using PtdIns 3-P, PtdIns
3,4-P2 and PtdIns 4-P. No PIP5K immunoreactivity (Fig.
3C) or activity (data not shown) was immunoprecipitated from
the control transfected cells. The only difference between the PIP5Ks
expressed in E. coli and mammalian cells was that the native
and recombinant PIP5KI
expressed in COS cells had quantitatively
greater activity toward PtdIns 3-P.
Here we report that the PIP5K isozymes are kinases with dual substrate specificity. They can phosphorylate PtdIns 4-P on the adjacent D-5-position. They can also phosphorylate 3-phosphate-containing phosphatidylinositols including PtdIns 3-P and PtdIns 3,4-P2 on the adjacent D-4- or D-5-positions. None of the other characterized phosphatidylinositol kinases are known to have this ability. The uniqueness of these enzymes is further emphasized by the lack of sequence homology with known phosphatidylinositol, inositol phosphate, or protein kinases (15, 16).
Our data demonstrate that the PIP5Ks have the in vivo
potential to synthesize the signaling molecules PtdIns
3,4-P2 and PtdIns 3,4,5-P3, in addition to
PtdIns 4,5-P2, as summarized in Fig. 4. This
catalytic capacity was shown for both native and recombinant PIP5K
isozymes. The production of both PtdIns 3,4-P2 and PtdIns 3,4,5-P3 when PtdIns 3-P is the initial substrate suggests
that the product of the first reaction is retained on the enzyme and phosphorylated again in a concerted reaction. This result has important
biological implications because it suggests an additional mechanism for
PtdIns 3,4,5-P3 synthesis within cells.
There is evidence in the literature that a platelet PtdIns 3-P 4-kinase
activity is stimulated by the thrombin receptor and protein kinase C
activation (7, 8). PIP5KII is present in platelets (18, 31) and, as
the only currently identified PtdIns 3-P 4-kinase in platelets, is a
likely candidate for that PtdIns 3-P 4-kinase activity. Based on all of
the available data, PIP5KII
has properties indistinguishable from
the platelet PtdIns 3-P 4-kinase (7, 8). PIP5KII
is activated by its
association with the TNF receptor (19) and could generate PtdIns
3,4-P2 involved in proliferation. The PIP5Ks could also be
regulated by receptors that are known to stimulate PtdIns
3,4-P2 and PtdIns 3,4,5-P3 production upon
agonist activation. As discussed previously, a pathway leading to
synthesis of PtdIns 3,4-P2 and PtdIns 3,4,5-P3 has been proposed to occur by phosphorylation of PtdIns
4,5-P2 by an agonist-activated 3-kinase (5). However, these
arguments were based upon the observation that this PtdIns 3-kinase
will phosphorylate all phosphoinositides lacking a phosphate in the D-3-position and that PtdIns 3,4,5-P3 appears to be the
initial product. With the data presented here, an alternative pathway in which PtdIns is phosphorylated by PtdIns 3-kinase and then phosphorylated by a PIP5K isoform is plausible, and the concerted reaction could explain why PtdIns 3,4,5-P3 appears
first.
The PIP5Ks have the potential to produce three signaling molecules: PtdIns 4,5-P2, PtdIns 3,4-P2, and PtdIns 3,4,5-P3. It will be very interesting to determine if the substrate preferences of these PIP5K isozymes are altered by receptor activation or different regulators such as the small G-proteins Rac and Rho (23, 32, 33). It is conceivable that modulation of these activities will be both spatially and temporally regulated, and thus the PIP5K enzymes could participate in a plethora of cellular events by generating multiple messengers. Given the expanded substrate repertoire of these enzymes, we propose that they be designated as phosphatidylinositol phosphate 4/5-kinases.
The pcDNA3-FLAG vectors were a gift of Dr. Jon Morrow (Yale University).