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INTRODUCTION |
Inositol-containing phospholipids (phosphatidylinositols) are
found in all eukaryotes and constitute 2-8% of total cellular phospholipids. Phosphatidylinositol
(PtdIns)1 is synthesized in
the endoplasmic reticulum and accounts for more than 80% of the total
phosphatidylinositols (1). Phosphatidylinositol 4-phosphate
(PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) are synthesized from PtdIns by the
sequential actions of PtdIns 4-kinase and PtdIns(4)P 5-kinase. In
addition, PtdIns 3-kinase phosphorylates PtdIns(4,5)P2 and
PtdIns at the 3 position of the inositol ring and adds to the
complexity of PtdIns metabolism (1, 2).
PtdIns(4,5)P2 plays several fundamental roles in cell
physiology. Phospholipase C catalyzes the hydrolysis of
PtdIns(4,5)P2 to yield 1,2-diacylglycerol (DG) and inositol
1,4,5-trisphosphate, two prominent eukaryotic second messengers (3-8).
PtdIns(4,5)P2 regulates actin microfilaments on the basis
of its ability to bind to and regulate a host of actin severing,
capping, and bundling proteins in vitro (9).
PtdIns(4,5)P2 also binds to proteins with pleckstrin
homology domains, such as pleckstrin or all phospholipase C isozymes
(10, 11). In addition, PtdIns and its phosphorylated metabolites also
play important roles in the regulation of membrane traffic (12).
Since the PtdIns(4,5)P2 metabolism plays such crucial roles
in several aspects of cell physiology, it is logical that
PtdIns(4,5)P2 synthesis and degradation are stringently
regulated. Mammalian cells contain several isoforms of PtdIns(4)P
5-kinase with different biochemical properties. The type I PtdIns(4)P
5-kinase is stimulated by both heparin and spermine. On the other hand,
the type II PtdIns(4)P 5-kinase is inhibited by heparin, and inhibited
or not affected by spermine (13, 14). In addition, the type I and not
type II PtdIns(4)P 5-kinase is also stimulated by phosphatidic acid (15). This stimulation may be essential for resynthesis of
PtdIns(4,5)P2 in response to PtdIns(4,5)P2
hydrolysis by phospholipase C and subsequent conversion of DG to
phosphatidic acid. An additional mechanism for resynthesis of
PtdIns(4,5)P2 may involve protein kinase C-mediated
activation of the PtdIns 4-kinase and PtdIns(4)P 5-kinase. This
speculation is based on the ability of phorbol esters and DG to elevate
levels of PtdIns(4)P and PtdIns(4,5)P2 in intact cells
(16). Consequently, elevation of DG levels following activation of
phospholipase C may promote synthesis of PtdIns(4)P and
PtdIns(4,5)P2. However, direct evidence for involvement of protein kinase C in activation of PtdIns 4-kinase and PtdIns(4)P 5-kinase is missing. On the other hand, small GTP -binding protein Rho
was found to interact with and regulate PtdIns(4)P 5-kinase (17, 18).
In addition, cAMP was implicated in regulation of PtdIns(4)P and
PtdIns(4,5)P2 synthesis in Saccharomyces
cerevisiae (19). However, Buxeda et al. (20)
demonstrated that the 45- and 55-kDa forms of S. cerevisiae
PtdIns 4-kinase are neither phosphorylated nor regulated by
cAMP-dependent protein kinase.
Recently, type II (21, 22) and type I (23) PtdIns(4)P 5-kinases were
cloned, and two S. cerevisiae genes, MSS4 (24) and FAB1 (25), encoding PtdIns(4)P 5-kinase, were
characterized. Zhang et al. (26) showed that type I
PtdIns(4)P 5-kinases are able to utilize PtdIns(3)P to form
PtdIns(3,4)P2 and further phosphorylate PtdIns(3,4)P2 to generate PtdIns(3,4,5)P3. This
surprising finding identifies PtdIns(4)P 5-kinase as a key player
capable of generating several polyphosphoinositide signaling molecules.
In this study we show that at least one form of PtdIns(4)P 5-kinase in
Schizosaccharomyces pombe is associated with plasma membrane. We describe purification and characterization of this PtdIns(4)P 5-kinase and present evidence that this PtdIns(4)P 5-kinase
is phosphorylated and regulated by casein kinase I (CK1) in
vitro and in vivo.
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EXPERIMENTAL PROCEDURES |
Materials--
Protein purification media were from Pharmacia.
Calibration proteins for electrophoresis were from Bio-Rad.
Phospholipids were from Sigma and detergents from Pierce.
[
-32P]ATP was purchased from Amersham and
[3H]inositol from American Radiolabeled Chemicals.
Strains and Media--
S. pombe strain SP223 (h+
ade6-M216 leu1-32 ura4-D18) was grown at 32 °C in minimal medium
(27) supplemented with 75 mg/liter each of adenine, leucine, and uracil.
Purification of PtdIns(4)P 5-Kinase--
Preparation of the
membrane fraction was performed batchwise, starting from cells grown in
3 liters of YEA medium to A600 nm = 1.5. Cells
were harvested by centrifugation (2,000 × g × 10 min), washed once with water and once with spheroplast buffer (20 mM potassium phosphate, pH 7.5, 1.4 M sorbitol,
10 mM NaN3, 0.3% 2-mercaptoethanol). The
spheroplasts were prepared and lysed as described previously (28). The
crude extract (
150 ml) was centrifuged at 5,000 × g
for 10 min, supernatant was removed and centrifuged at 25,000 × g for 30 min. The pellet (P2) from this spin was resuspended
in 15 ml of sucrose solution (55% sucrose, 20 mM Tris-HCl,
pH 7.5, 1 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, and 20 µg/ml each of pepstatin, aprotinin, and leupeptin).
Aliquots of 2.5 ml were placed at the bottom of a 1.4 × 8.9-cm
centrifuge tubes (each batch was processed in 6 tubes) and subjected to
sucrose gradient fractionation as described previously (28, 29).
Briefly, the pellet fraction in centrifuge tubes was overlaid with the following volumes and concentrations of sucrose (containing 20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml each of
pepstatin, aprotinin, and leupeptin) at 0 °C: 1 ml × 50%, 1 ml × 47.5%, 1.5 ml × 45%, 1.5 ml × 42%, 1.5 ml × 40%, 1 ml × 37.5%, and 1 ml × 30%. After
centrifugation (16 h × 170,000 × g, SW41 rotor),
the gradients were fractionated from the top (600-µl fractions) and
labeled sequentially as fractions 1-19. The pellet was resuspended in
600 µl of the sucrose solution and taken as fraction 20. All
fractions in one tube were assayed in duplicate for marker enzymes,
which included vanadate-sensitive plasma membrane ATPase, NADPH
cytochrome c reductase, cytochrome c oxidase,
-D-mannosidase, and GDPase (29). The PtdIns(4)P 5-kinase
activity containing fractions (10-13) from all centrifuge tubes were
combined and stored at
80 °C until another 3 batches were
processed in the same way. The pooled fractions 10-13 from all batches
(total volume
60 ml) were diluted with 120 ml of buffer (20 mM Tris-HCl, pH 7.5, 1 M NaCl) and centrifuged
at 170,000 × g for 1 h. The supernatant was
supplemented with 0.01% Triton X-100 and concentrated by
ultrafiltration under N2 gas in a stirred pressure cell
(Amicon) to 20 ml. The concentrated sample was loaded onto
phenyl-Superose HR 5/5 (Pharmacia) column equilibrated in 10 mM Tris-HCl buffer, pH 7.5, containing 1 M
NaCl, 1 mM EGTA, and 0.1% 2-mercaptoethanol. The column
was washed with 10 ml of the above buffer and then developed with 30 ml
of linear gradient of decreasing NaCl (from 1 M to 0 M) in the same buffer and 0.5-ml fractions were collected.
PtdIns(4)P 5-kinase eluted at 0.5 M NaCl. Fractions
containing PtdIns(4)P 5-kinase activity were loaded on a Sephacryl
S-100 HR column (1.5 × 90 cm) equilibrated and developed with 10 mM Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl (buffer A). Fractions (5 ml each) with PtdIns(4)P 5-kinase activity were pooled and loaded on a Mono Q 5/5 FPLC column equlibrated in buffer A. The column was washed with 20 ml of buffer A and then
developed with 40 ml of linear gradient of 0.1-0.4 M NaCl in 10 mM Tris-HCl buffer, pH 7.5. PtdIns(4)P 5-kinase
activity elutes as a sharp peak centered at 230 mM NaCl.
The active fractions (1 ml each) were pooled and concentrated by
dialysis against storage buffer (50% glycerol, 20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Brij 35, 1 mM dithiothreitol) and stored at
20 °C.
PtdIns(4)P 5-Kinase Assay--
The PtdIns(4)P 5-kinase was
assayed essentially as described (13, 14). The reaction mixture (total
volume 50 µl) contained 20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 50 µM PtdIns(4)P, 2 mM Triton X-100, and 50 µM [
-32P]ATP (1 Ci/mmol). The reaction
was initiated by the addition of ATP, allowed to continue for 5 min at
30 °C, and terminated by an addition of 200 µl of 1 M
HCl. The reaction product was extracted with 600 µl of chloroform,
methanol, 12 M HCl (200:100:1). The phases were separated
by centrifugation, the aqueous phase was removed and discarded, and the
organic phase was washed once with 500 µl of methanol, 1 M HCl (1:1). The organic phase was then dried in a vacuum
concentrator (Savant), resuspended in 50 µl of chloroform, methanol,
12 M HCl (200:100:1), and spotted onto Whatman Silica Gel
150A plates. The plates were developed in chloroform, methanol, water,
15 M ammonium hydroxide (90:90:20:7). Standard
phospholipids were visualized with iodine vapor and the reaction
products by autoradiography. Radioactivity in the reaction product was
determined by scraping the silica gel corresponding to the labeled
product and quantitating by scintillation counting.
Renaturation of PtdIns(4)P 5-Kinase Activity on
Nitrocellulose--
The renaturation assay was performed according to
Ref. 13 with few modifications. Purified PtdIns(4)P 5-kinase (650 ng) was incubated in SDS sample buffer (1% SDS, 10 mM
dithiothreitol, 10% glycerol, 20 mM Tris, pH 6.8) for 15 min at room temperature. The sample was subjected to SDS-PAGE on a 8%
gel and electrophoresed at 4 °C. After transfer to nitrocellulose
using Mini-Trans-Blot Cell (Bio-Rad), the membrane was washed in 0.1%
Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM
dithiothreitol, 50 mM Tris, pH 7.4, for 30 min. The
membrane was then cut into 19 sections and each section was incubated
individually for 6 h in a buffer containing 200 µg/ml
PtdIns(4)P, 400 µg/ml phosphatidylserine, 1 mM Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM
dithiothreitol, 50 mM Tris, pH 7.5, and 100 µM [
-32P]ATP. The reaction was
terminated by adding 0.5 ml of methanol, 1 M HCl (1:1) and
extracted with 0.3 ml of chloroform. The organic layer was dried under
nitrogen, resuspended in chloroform:methanol:water (2:1:0.01), and
spotted onto Whatman Silica Gel 150A plates. The plates were developed
in chloroform, methanol, water, 37% ammonium hydroxide (45:35:8.5:1.5)
and PtdIns(4,5)P2 was visualized by autoradiography.
Radioactivity was determined by excising the appropriate piece of TLC
plate and counting it in a liquid scintillation mixture.
Phosphorylation of PtdIns(4)P 5-Kinase with
Cki1--
Recombinant Cki1 (one of the four homologs of casein kinase
I in S. pombe) was expressed in Escherichia coli,
purified, and assayed as described (28). Purified PtdIns(4)P 5-kinase
(0.2 µg) was incubated with the indicated amounts of Cki1 at 30 °C in 10 µl of the following buffer: 25 mM Mes, pH 6.5, 50 mM NaCl, 15 mM MgCl2, 2 mM EGTA, 100 µM ATP. Following the incubation
with Cki1, the reaction mixture was diluted to 100 µl with PtdIns(4)P 5-kinase assay buffer, and the activity of PtdIns(4)P 5-kinase was determined.
Phosphoamino Acid Analysis--
The 32P-labeled
PtdIns(4)P 5-kinase was subjected to phosphopeptide and phosphoamino
acid analysis essentially as described (30, 31). After electrophoresis,
the wet gel was exposed to film (Hyperfilm-MP, Amersham) at room
temperature for several hours. The phosphorylated bands corresponding
to PtdIns(4)P 5-kinase were excised, the protein extracted overnight in
50 mM NH4HCO3, pH 7.5, 0.1% SDS,
5% 2-mercaptoethanol, mixed with 40 mg of bovine serum albumin, and
precipitated in 20% trichloroacetic acid at
20 °C. Precipitated
proteins were washed in 95% ethanol (
20 °C) and subjected to
partial hydrolysis in 6 M HCl for 90 min at 105 °C. The
samples were lyophilized, resuspended in electrophoresis buffer (88%
formic acid/glacial acetic acid/water, 50:156:1780, pH 1.9), and
separated by electrophoresis on cellulose thin layer plates in a buffer
(pyridine/acetic acid/water, 10:100:1890, 0.5 mM EDTA, pH
3.5) for 40 min at 900 V. The marker phosphoamino acids were visualized
by staining with ninhydrin (0.25% in acetone).
Phosphopeptide Mapping--
Cleavage of proteins by
N-chlorosuccinimide was performed as described (32, 33). The
32P-labeled PtdIns(4)P 5-kinase was subjected to SDS-PAGE
and the wet gel was exposed to film at room temperature for 4 h.
The bands corresponding to the phosphorylated PtdIns(4)P 5-kinase were
excised and rinsed in 0.1% (w/v) solution of urea in acetic acid and
water (1:1). The gel slices were then incubated in 60 mM
N-chlorosuccinimide in the above solution. After 3 washes in
10 mM Tris-HCl, pH 8.0, the gel slices were equilibrated in
sample loading buffer before electrophoresis on 15% SDS-PAGE gel.
[3H]Inositol Labeling of S. pombe Cells--
The
cells were grown in minimal medium to A600 nm ~ 1.5, harvested, and washed with inositol-free minimal medium, and
cultured in 10 ml of the same medium containing 20 µM
[3H]inositol (5 µCi/ml) for 16 h. The cells were
then harvested, washed with water, resuspended in 2 ml of methanol, and
disrupted with glass beads. The homogenate was acidified with 2 ml of 1 M HCl and extracted 3 times with 2 ml of chloroform. The
combined extracts were washed with 2 ml methanol, 1 M HCl
(1:1) and concentrated under stream of nitrogen. TLC analysis was
performed as described above and radioactivity corresponding to PtdIns,
PtdIns(4)P, and PtdIns(4,5)P2 was determined by excising
the appropriate pieces of TLC plate and counting them in a liquid
scintillation mixture.
Analytical Methods--
Protein concentration was determined by
the method of Bradford (34). Marker enzymes for subcellular fractions
were assayed as described previously (28, 29).
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RESULTS |
PtdIns(4)P 5-Kinase Activity in Cell Lysates--
The PtdIns(4)P
5-kinase activity is easily detectable in cell lysates of S. pombe, however, the specific activity depends on the method of
cell breakage. Osmotic lysis of spheroplasts consistently yielded about
2-fold higher specific activity of PtdIns(4)P 5-kinase (~0.3
nmol/min/mg) than breakage with glass beads. Moreover, PtdIns(4)P
5-kinase activity was more stable in cell lysates prepared by the
former method, probably because osmotic lysis of spheroplasts minimizes
proteolysis. Therefore, we chose osmotic lysis of spheroplasts for
preparation of cell lysates with PtdIns(4)P 5-kinase activity. In pilot
experiments to determine the subcellular distribution of S. pombe PtdIns(4)P 5-kinase, we found that the PtdIns(4)P 5-kinase
activity was exclusively associated with particulate fractions, no
activity was detectable in soluble fraction. When we performed
differential centrifugation, almost 80% of the PtdIns(4)P 5-kinase
activity was recovered in pellet fraction sedimenting between
5,000 × g and 25,000 × g (fraction P2). Approximately 10% of the activity was recovered in pellet fractions sedimenting at 5,000 × g (fraction P1) and
about 10% of the activity was recovered in pellet fraction sedimenting
between 25,000 × g and 170,000 × g
(fraction P3).
Subcellular Distribution of PtdIns(4)P 5-Kinase--
To determine
the subcellular localization of PtdIns(4)P 5-kinase, the P2 pellet
fraction was further purified on sucrose density gradient and assayed
for organelle-specific enzyme markers (28) as well as for the
PtdIns(4)P 5-kinase activity (Fig. 1).
PtdIns(4)P 5-kinase cofractionated with vanadate-sensitive ATPase,
which is a marker for the plasma membrane fraction. The peak fraction had 27- and 34-fold higher specific activities of the ATPase and PtdIns(4)P 5-kinase, respectively, than the crude lysate. PtdIns(4)P 5-kinase did not comigrate with marker enzymes for mitochondria (cytochrome c oxidase), endoplasmic reticulum
(NADPH-cytochrome c reductase), Golgi apparatus (GDPase), or
vacuole (
-mannosidase) (Fig. 1).

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Fig. 1.
The PtdIns(4)P 5-kinase co-migrates with the
plasma membrane fraction. The P2 pellet was subjected
to density gradient centrifugation as described under "Experimental
Procedures." After centrifugation, gradients were fractionated from
top (fraction 1) to bottom (fraction 20) and assayed for: A,
protein amount (mg/fraction) and refractive index; B,
PtdIns(4)P 5-kinase (nmol/min/fraction) and vanadate-sensitive plasma
membrane ATPase (µmol/min/fraction); C, GDPase
(nmol/min/fraction) and -mannosidase (µmol/min/fraction);
D, cytochrome c oxidase (nmol/min/fraction) and
NADPH-cytochrome c reductase (nmol/min/fraction). For the
purification of PtdIns(4)P 5-kinase, total of 4 batches, each
consisting of 6 tubes, were processed this way. Gradient profile of one
typical gradient tube is shown for illustration.
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To further characterize the PtdIns(4)P 5-kinase activity, we
investigated the interactions between the protein and the plasma membrane. The plasma membranes purified by sucrose density gradient were incubated with one of the following: 1 M NaCl, 1%
Triton X-100, 0.2 M Na2CO3, or 1 M NH2OH. After the incubation, membranes were
centrifuged (170,000 × g) and the supernatants and the
pellets were assayed for PtdIns(4)P 5-kinase activity. The best
extraction and the highest specific activity in the supernatant was
achieved by 1 M NaCl. More than 90% of the PtdIns(4)P
5-kinase was recovered in the supernatant. Because most of the other
proteins remained associated with the membrane, this simple extraction
procedure resulted in a further 10-fold purification of the kinase. The solubilization with other reagents resulted in a significantly reduced
recovery of PtdIns(4)P 5-kinase in the supernatant. However, in this
experiment it is difficult to distinguish between the effect of
dissociation of PtdIns(4)P 5-kinase from the membrane and the effect of
the solubilizing reagents on the activity. Nevertheless, this result
shows that PtdIns(4)P 5-kinase is a peripheral membrane protein and
that the association with plasma membrane is mediated by ionic interactions.
Purification of PtdIns(4)P 5-Kinase--
The salt extract of the
plasma membrane fraction was further fractionated by
hydrophobic-interaction chromatography on a column of phenyl-Sepharose
(Fig. 2A). This chromatography
yielded only modest 3-fold purification. However, when we omitted this
step, the quality of resolution in subsequent purification steps
suffered. The next purification step featured gel filtration on a
Sephacryl S-100 column (Fig. 2B). PtdIns(4)P 5-kinase eluted
as a sharp peak corresponding to molecular mass of 65 kDa. This step
efficiently removes high molecular weight contaminants that are
difficult to eliminate using other methods. It also removes sodium
chloride remaining from the previous step. The final chromatographic
step is an anion exchange chromatography over a Mono-Q column (Fig. 2C), and yields a homogenous enzyme preparation with a high
specific activity (Table I). Analysis of
the preparation by SDS-PAGE and Coomassie staining (Fig.
3) reveals that the purified enzyme
consists of a single polypeptide with an apparent molecular mass of 63 kDa.

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Fig. 2.
Column chromatography profiles.
Chromatography was performed as described under "Experimental
Procedures." A, purification of PtdIns(4)P 5-kinase on a
phenyl-Superose column. Fractions 10-13 from the sucrose gradient
centrifugation were extracted by 1 M NaCl and
chromatographed on phenyl-Superose column. Activity of PtdIns(4)P
5-kinase and A280 nm are shown against the
elution volume. The active fractions (total 3 ml) were pooled.
B, purification of PtdIns(4)P 5-kinase on a Sephacryl S-100
HR column. The pooled active fractions from phenyl-Superose column were
chromatographed on Sephacryl S-100 HR and the active fractions (total
15 ml) were pooled. C, purification of PtdIns(4)P 5-kinase
on a Mono Q column. The pooled active fractions from Sephacryl S-100 HR
column were chromatographed on Mono Q column and the active fractions
(total 3 ml) were pooled and analyzed by SDS-PAGE.
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Fig. 3.
SDS-PAGE and renaturation of PtdIns(4)P
5-kinase. A, the purified protein was analyzed by 8%
SDS-PAGE and transferred to a nitrocellulose membrane. The strip of
nitrocellulose was washed, cut into 19 sections and each section was
assayed separately for PtdIns(4)P 5-kinase activity (cpm).
B, SDS-PAGE analysis of purified PtdIns(4)P 5-kinase.
Lane 1, purified PtdIns(4)P 5-kinase (1.5 µg); lane
2, molecular weight standards.
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To prove that the enzymatic activity resides in this single band, we
performed a renaturation assay. The purified protein was analyzed by
SDS-PAGE and transferred to a nitrocellulose membrane. The
nitrocellulose strip was washed, cut into 19 sections, and each section
assayed separately for PtdIns(4)P 5-kinase activity. The only
detectable activity corresponded exactly to the position of the
Coomassie-stained band and therefore the purified band contains
PtdIns(4)P 5-kinase (Fig. 3).
Kinetic Characterization of PtdIns(4)P 5-Kinase--
The kinetic
characterization of the enzyme showed that PtdIns(4)P 5-kinase is
activated by nonpolar detergents and some phospholipids. Variety of
detergents (CHAPS, cholate, deoxycholate, digitonin,
-octylglucoside, Triton X-100, Zwittergent 3-14, and Lubrol PX) were
tested for their effect on PtdIns(4)P 5-kinase activity. The highest
stimulation of PtdIns(4)P 5-kinase activity was obtained in the
presence of Triton X-100, the optimal concentration was found to be 2 mM, followed by an apparent inhibition of activity at
concentrations above 2 mM. These results are indicative of surface dilution kinetics (35-37). Zwittergent 3-14 and Lubrol PX were
less effective in stimulating PtdIns(4)P 5-kinase activity. Other
tested detergents did not have any significant effect or inhibited
PtdIns(4)P 5-kinase activity. These results suggest that PtdIns(4)P
5-kinase activity is stimulated only in the presence of detergents
which have a long saturated hydrocarbon tail and are nonionic or zwitterionic.
We used the Triton X-100 detergent/phospholipid mixed micelle system to
perform the kinetic characterization of the enzyme. The function of
Triton X-100 in this system is to form a uniform mixed micelle with the
substrate PtdIns(4)P. The Triton X-100 micelle serves as a
catalytically inert matrix in which PtdIns(4)P is homogenously
dispersed. In addition, the Triton X-100/PtdIns(4)P mixed micelle
system allows an analysis of the enzyme in an environment that mimics
the physiological surface of the membrane (38-42), where PtdIns(4)P
5-kinase functions. We determined dependence of the PtdIns(4)P 5-kinase
activity on the surface PtdIns(4)P concentration using different set
molar concentrations of PtdIns(4)P (Fig.
4A). In these experiments the
molar concentration of PtdIns(4)P was kept constant, while the
concentration of Triton X-100 was varied in order to produce the
desired surface concentrations of PtdIns(4)P. PtdIns(4)P 5-kinase
followed the surface dilution kinetics (35-37) since its activity was
dependent on the surface concentration of PtdIns(4)P (Fig.
4A), and exhibited saturation kinetics with respect to the
surface concentration of PtdIns(4)P. In addition, the dependence of
PtdIns(4)P 5-kinase activity on PtdIns(4)P surface concentration was
independent on the molar concentrations of PtdIns(4)P used in this
experiment. The Vmax was determined to be 2.7 µmol/min/mg, and the Km value for PtdIns(4)P was
1.0 mol % (Fig. 4B). When PtdIns(4)P was kept at its
saturating surface concentration (4.8 mol %; bulk concentration of
PtdIns(4)P 0.1 mM, concentration of Triton X-100 2 mM), and the concentration of ATP was varied, the enzyme
behaved according to the Michaelis-Menten kinetics with respect to the
bulk concentration of ATP, and the Km for ATP was
determined to be 8 µM.

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Fig. 4.
Dependence of PtdIns(4)P 5-kinase activity on
the surface concentration of PtdIns(4)P. A, the
PtdIns(4)P 5-kinase activity was measured as a function of the surface
concentration of PtdIns(4)P (mol %). The molar concentration of
PtdIns(4)P was held constant at 0.1, 0.075, or 0.125 mM,
while the Triton X-100 concentration was varied. B,
reciprocal plot of the data using PtdIns(4)P at a molar concentration
of 0.1 mM. The line drawn is the result of a
least-squares analysis of the data.
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Using the standard PtdIns(4)P 5-kinase assay, we determined the optimal
pH for the PtdIns(4)P 5-kinase activity to be 7.5 (Fig.
5), and we tested several phospholipids
and other compounds as possible effectors of PtdIns(4)P 5-kinase
activity (Table II). Previously, spermine
was shown to be a potent stimulator of type I PtdIns(4)P 5-kinase (14).
Our results are consistent with this observation, 0.5 mM
spermine increased the activity of the kinase to 300% (Table II). On
the other hand, heparin at 30 µg/ml inhibited 50% of the activity of
S. pombe PtdIns(4)P 5-kinase (Table II). Mammalian
PtdIns(4)P 5-kinase, type I, was stimulated by heparin, whereas the
type II was inhibited (14). On the basis of these results it appears
that S. pombe PtdIns(4)P 5-kinase shares characteristics
with both type I and type II mammalian PtdIns(4)P 5-kinases.
Phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine,
and diacylglycerol did not significantly affect PtdIns(4)P 5-kinase
activity, and PtdIns(4, 5)P2 reduced the activity to 45%
of the control. Phosphatidylinositol, phosphatidylserine (PS), and
phosphatidic acid (PA) stimulated the kinase activity to 125, 160, and
185%, respectively. Since the regulation of PtdIns(4)P 5-kinase by PA
and PS may be physiologically most significant, we performed a detailed
kinetic analysis to explore the mechanism of phospholipid activation of
PtdIns(4)P 5-kinase. The activity of PtdIns(4)P 5-kinase was determined
at different surface concentrations of PtdIns(4)P at different set
surface concentrations of PA or PS (Fig.
6). The addition of PA or PS to the assay
system resulted in an increase of the apparent
Vmax but had no effect on the apparent Km for PtdIns(4)P.

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Fig. 5.
Effect of pH on PtdIns(4)P 5-kinase
activity. The reaction mixture was buffered with 20 mM
acetate ( ), MES ( ), MOPS (×), Tris ( ), or CAPSO ( ).
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Table II
Effect of various compounds on PtdIns(4)P 5-kinase activity
PtdIns(4)P 5-kinase was incubated with the following compounds for 20 min on ice and 5 min at 30 °C, and then assayed as described under
"Experimental Procedures." The data represent the average of three
assays which agreed within 5%.
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Fig. 6.
Effect of PA and PS on the kinetics of
PtdIns(4)P 5-kinase with respect to the surface concentration of
PtdIns(4)P. The PtdIns(4)P 5-kinase activity was measured as a
function of the surface concentration of PtdIns(4)P (mol %) in the
absence of additional phospholipid or in the presence of the indicated
concentrations of PA or PS. The molar concentration of PtdIns(4)P was
held constant at 0.1 mM, while the Triton X-100
concentration was varied. Reciprocal plots of the data are shown and
the lines drawn are the results of a least-squares analysis
of the data.
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Since it was shown that mammalian PtdIns(4)P 5-kinases are regulated by
small GTP-binding protein Rho (17, 18), we examined the effects of
GTP
S on PtdIns(4)P 5-kinase activity. GTP
S did not exhibit any
effect on the kinase activity either in crude cell-free extract or in
purified plasma membrane fraction. Therefore, it seems unlikely that
PtdIns(4)P 5-kinase from S. pombe is regulated by small
GTP-binding proteins.
Regulation of PtdIns(4)P 5-Kinase by Phosphorylation--
Since
both PtdIns(4)P 5-kinase and Cki1, one of the four homologs of casein
kinase I (CK1) in S. pombe, localize to the plasma membrane
(28), we directly addressed whether Cki1 can phosphorylate PtdIns(4)P
5-kinase in vitro. Surprisingly, phosphorylation of PtdIns(4)P 5-kinase by Cki1 was easily detectable by autoradiography (Fig. 7). We performed a detailed
analysis of the PtdIns(4)P 5-kinase phosphorylation by Cki1 (Fig.
8). Cki1 phosphorylated and inactivated PtdIns(4)P 5-kinase in a time- and dose-dependent manner.
When Cki1 or ATP were omitted from the phosphorylation reaction, the inactivation of PtdIns(4)P 5-kinase did not occur. Time to achieve maximum inhibition of PtdIns(4)P 5-kinase activity (to 36% of the
control) by incubation with Cki1 was dependent on the amount of Cki1
used for phosphorylation. The phosphorylation stoichiometry of
PtdIns(4)P 5-kinase was determined during the course of the phosphorylation by Cki1 (Fig. 8). While the full inactivation required
transfer of 0.8 mol of phosphate per mol of PtdIns(4)P 5-kinase, the
completely phosphorylated protein contained 3.5 mol of phosphate/mol of
PtdIns(4)P 5-kinase. The phosphoamino acid analysis of PtdIns(4)P
5-kinase containing 0.8 mol of phosphate/mol of protein revealed that
only serine was phosphorylated (Fig. 9).
However, PtdIns(4)P 5-kinase containing 3.5 mol of phosphate/mol of
protein contained, in addition to phosphoserine, also a detectable amount of phosphothreonine. Phosphotyrosine was not detected in either sample (Fig. 9).

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Fig. 7.
In vitro phosphorylation of PtdIns(4)P
5-kinase by Cki1. Purified recombinant S. pombe Cki1
(0.2 µg) was incubated with [ -32P]ATP either alone
(lane 1) or in a mixture with purified PtdIns(4)P 5-kinase
(0.2 µg, lane 2) and subjected to 8% SDS-PAGE and
autoradiography. Lane 3 shows the position of PtdIns(4)P
5-kinase (gel stained with Coomassie). Since Cki1 autophosphorylates
and migrates as a 58-kDa band (27) and PtdIns(4)P 5-kinase migrates as
a 63-kDa band, in this experiment we used stoichiometric amounts of
both proteins in order to identify them unambiguously.
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Fig. 8.
Inactivation of PtdIns(4)P 5-kinase by
Cki1-mediated phosphorylation. PtdIns(4)P 5-kinase (0.2 µg) was
incubated without Cki1 or with the indicated amounts of Cki1 for
different time periods. Following the incubations, samples were diluted
and PtdIns(4)P 5-kinase activity assayed in duplicate as described
under "Experimental Procedures." The samples incubated without ATP
contained 9 ng of Cki1. The phosphorylation stoichiometry was
determined as described under "Experimental Procedures" for samples
containing 9 ng of Cki1 per 0.2 µg of PtdIns(4)P 5-kinase. ,
without Cki1; , without ATP; , Ckil (9 ng); , Cki1 (4 ng); ×,
Cki1 (2 ng); , mol P/mol protein.
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Fig. 9.
Phosphoamino acid analysis of the
32P-labeled PtdIns(4)P 5-kinase. PtdIns(4)P 5-kinase
(0.2 µg) was phosphorylated with Cki1 (9 ng) for: A, 3 min
or B, 20 min and subjected to SDS-PAGE. The bands
corresponding to PtdIns(4)P 5-kinase were excised, the protein
extracted and subjected to phosphoamino acid analysis as described
under "Experimental Procedures." The positions of the carrier
standard phosphoamino acids are indicated.
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To address a question whether phosphorylation of only one or several
serine residues is responsible for inhibition of PtdIns(4)P 5-kinase,
we cleaved the enzyme containing either 0.8 or 3.5 mol of phosphate/mol
of protein with N-chlorosuccinimide.
N-Chlorosuccinimide cleaves specifically tryptophanyl
peptide bonds and generates discrete map of peptides (32, 33). Only a
single phosphopeptide band of 18 kDa was detected in the sample with
0.8 mol of phosphate/mol of protein (Fig.
10, lane A). Since the
maximum inactivation of PtdIns(4)P 5-kinase by Cki1 requires transfer
of 0.8 mol of phosphate/mol of protein (Fig. 8), this result supports
the interpretation that only a single serine residue is phosphorylated
by Cki1 in the sample with 0.8 mol of phosphate/mol of protein and that
phosphorylation of this serine residue regulates the activity of
PtdIns(4)P 5-kinase. However, we cannot exclude the possibility that
several serine residues within the 18-kDa
N-chlorosuccinimide-resistant phosphopeptide are
phosphorylated by Cki1, and that the phosphorylation events synergistically inactivate PtdIns(4)P 5-kinase. Resolution of this
issue will require a generation of specific PtdIns(4)P 5-kinase mutants
by site-directed mutagenesis, when the sequence of S. pombe
PtdIns(4)P 5-kinase becomes available. In the sample with 3.5 mol of
phosphate/mol of protein, in addition to the 18-kDa phosphopeptide,
several additional phosphopeptide bands were detected (Fig. 10,
lane B). Since phosphorylation beyond 0.8 mol of
phosphate/mol of protein does not affect the activity of the PtdIns(4)P
5-kinase (Fig. 8), phosphorylation of serine or threonine residues in
these additional phosphopeptides does not influence the activity of PtdIns(4)P 5-kinase.

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Fig. 10.
Phosphopeptide mapping of the
32P-labeled PtdIns(4)P 5-kinase. PtdIns(4)P 5-kinase
(0.4 µg) was phosphorylated with Cki1 (18 ng) for: A, 3 min or B, 20 min and subjected to SDS-PAGE. The bands
corresponding to PtdIns(4)P 5-kinase were excised, rinsed, and treated
with N-chlorosuccinimide as described under "Experimental
Procedures." The peptides were resolved on 15% SDS-PAGE and
visualized by autoradiography. The migration of the molecular mass
standards (trypsin inhibitor, 22 kDa; lysozyme, 15 kDa) is
indicated.
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We performed kinetic analysis to further characterize the effect of
phosphorylation on the activity of PtdIns(4)P 5-kinase. Again, we
measured the dependence of the PtdIns(4)P 5-kinase activity (phosphorylated to 0.8 or 3.5 mol of phosphate/mol of protein) on the
surface concentration of PtdIns(4)P (Fig.
11). While the Km of
the phosphorylated enzyme for PtdIns(4)P did not change, the
Vmax was decreased to 1.0 µmol/min/mg. Also,
the Km value of the phosphorylated enzyme for ATP
was not changed. We did not observe any difference in the above values
between PtdIns(4)P 5-kinase containing 0.8 or 3.5 mol of phosphate/mol
of protein.

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Fig. 11.
Effect of phosphorylation on the kinetics of
PtdIns(4)P 5-kinase. PtdIns(4)P 5-kinase (0.2 µg) was
phosphorylated with Cki1 (9 ng) for 3 min and the activity of
phosphorylated or not phosphorylated PtdIns(4)P 5-kinase was measured
in duplicate as a function of the surface concentration of PtdIns(4)P
(mol %) as described under "Experimental Procedures."
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To determine whether Cki1 regulates PtdIns(4)P 5-kinase activity also
in vivo, we measured PtdIns(4)P 5-kinase activity in the
plasma membrane fraction purified from the wild type strain and the
strain overexpressing Cki1 (Table III).
Overexpression of Cki1 lowered the PtdIns(4)P 5-kinase activity to
48%. Incubation of purified plasma membrane fractions from the
corresponding strains with recombinant purified Cki1 resulted in a
further decrease of PtdIns(4)P 5-kinase activity. This effect was most
pronounced with plasma membrane prepared from strain which did not
overexpress Cki1. When wild-type S. pombe cells and cells
overexpressing Cki1 were labeled in vivo with
[3H]inositol, and the total lipids were isolated and
fractionated by thin layer chromatography, we measured significant
differences in the amounts of PtdIns(4,5)P2 (Table III).
The differences in PtdIns(4,5)P2 synthesis were specific
because the cellular contents of other phosphatidylinositols (PtdIns
and PtdIns(4)P) were the same in both strains. The regulation of
PtdIns(4,5)P2 synthesis by Cki1 overexpression correlates
well with the PtdIns(4)P 5-kinase activity measurements in the wild
type and Cki1 overexpressing strains (Table III). These results suggest
that PtdIns(4)P 5-kinase is a downstream effector of Cki1 in S. pombe, and that phosphorylation by Cki1 inactivates PtdIns(4)P
5-kinase.
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Table III
Regulation of PtdIns(4,5)P2 synthesis and PtdIns(4)P 5-kinase
activity by overexpression of Ckil
The results represent average of duplicate experiments which agreed
within 10%.
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DISCUSSION |
The present paper describes the purification and characterization
of PtdIns(4)P 5-kinase from plasma membranes of S. pombe and
its regulation by CK1. The molecular mass of the S. pombe PtdIns(4)P 5-kinase (63 kDa) is similar to the 68-kDa type I PtdIns(4)P 5-kinase purified from bovine erythrocyte membranes (15) and 53-kDa
type II PtdIns(4)P 5-kinase purified from cytosolic fraction of human
erythrocytes. (13, 14). The Km values for PtdIns(4)P
and ATP, stimulation by spermine and phosphatidic acid, and inhibition
by heparin suggest that PtdIns(4)P 5-kinase purified from S. pombe shares characteristics with both type I and type II
mammalian PtdIns(4)P 5-kinases.
Biochemical comparison with S. cerevisiae PtdIns(4)P
5-kinase homologs encoded by MSS4 (24) and FAB1
(25) genes is not possible, since both were identified solely on the
basis of DNA sequence similarity with mammalian PtdIns(4)P 5-kinase
(20). MSS4 encodes protein of 89 kDa, which upon
overexpression suppresses mutations in PtdIns 4-kinase encoded by the
STT4 gene (24). FAB1 encodes protein of 257 kDa
which is associated with the vacuole and is involved in regulation of
vacuolar homeostasis. Neither MSS4 nor FAB1 were
shown to be PtdIns(4)P 5-kinases by biochemical criteria. Our results
do not suggest existence of additional PtdIns(4)P 5-kinase homologs in
S. pombe. Cell fractionation and chromatographic separations
always yielded only one peak of activity. However, we cannot exclude
the possibility that another S. pombe homolog(s) with low
activity escaped biochemical detection.
The presented data clearly show that Cki1 phosphorylates PtdIns(4)P
5-kinase in vitro and regulates its activity both in
vitro and in vivo. In addition, the incorporation of
[3H]inositol in PtdIns(4,5)P2 is regulated in
a Cki1-dependent manner, and the overexpression of Cki1
results in a decreased activity of PtdIns(4)P 5-kinase associated with
plasma membrane.
S. pombe contains four CK1 homologs encoded by
cki1+, cki2+, hhp1+, and
hhp2+. Cki1 (product of cki1+ gene)
associates with plasma membrane and its deletion or overexpression does
not result in any observable phenotype (28). Like other members of the
CK1 family (43-45), it is a constitutively active, monomeric protein
kinase, and phosphorylates Ser and Thr residues downstream of Asp, Glu,
or phosphorylated amino acid residues (28, 46, 47). Although many
proteins, such as RNA polymerase I and II (48), p53 protein (49),
regulatory subunit of protein phosphatase I (50), cytoskeletal proteins
myosin, troponin, and ankyrin (50), the cAMP response element modulator
CREM (51), and the 14-3-3 protein (52) were identified as substrates of CK1 in vitro, only in a very few cases has the
phosphorylation by CK1 been shown to correlate with functional changes
of the substrate (53-57). As we demonstrate here, PtdIns(4)P 5-kinase is one of the few identified targets regulated by CK1 phosphorylation.
What is the significance of PtdIns(4)P 5-kinase regulation by CK1?
Growing evidence obtained from a variety of experimental systems
supports a direct role for inositol phospholipids in vesicular traffic
events, distinct from their roles in classical signaling pathways (12).
In this context it is important to note that recent results implicated
CK1 of S. cerevisiae in vesicle transport (58, 59).
YCK1, YCK2, and YCK3 (isoforms of CK1 in S. cerevisiae; Refs. 60 and 61) suppress in an increased dosage a
growth defect of gcs1
mutant cells. GCS1 is a
GTPase-activating protein (GAP) for monomeric GTP-binding protein ARF,
which is involved in vesicle transport. The phosphorylation and
inactivation of PtdIns(4)P 5-kinase by CK1 may explain the mechanism of
this suppression. PtdIns(4,5)P2 regulates ARF in a complex
way: (i) it stimulates guanine nucleotide exchange factor and activates
ARF by increasing the level of GTP-ARF (62), and (ii)
PtdIns(4,5)P2, together with phosphatidic acid, stimulates
ARF GAP, which results in a lower level of GTP-ARF and lower activity
of ARF (63). The gcs1
mutation disrupts the GTP-GDP cycle
on ARF and results in higher activity of ARF. The growth defect of
gcs1
cells was suppressed by overexpression of CK1, which
inactivates PtdIns(4)P 5-kinase and lowers the level of
PtdIns(4,5)P2. This may result in reduced activities of ARF
guanine nucleotide exchange factor and GTP-ARF, since
PtdIns(4,5)P2 is an activator of ARF guanine nucleotide exchange factor (62). The overexpression of CK1 may thus balance the
activity of ARF and suppress the gcs1
mutation. As shown by Poon et al. (64), imbalance in the GTP-ARF level in
S. cerevisiae results in a severe growth inhibition.
However, the involvement of CK1 in vesicle trafficking may be more
complex. Panek et al. (59) isolated four mutations in
S. cerevisiae, which eliminate the requirement for activity
of YCK1 and YCK2. These mutations lie in four
proteins with similarity to the four subunits of clathrin adaptors.
Since clathrin adaptor proteins bind PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in mammalian cells, these results do not
exclude the possibility that CK1 regulates vesicle trafficking at the plasma membrane by modulating the synthesis of
PtdIns(4,5)P2. It will be of interest to determine whether
overexpression or depletion of YCK1 and YCK2 in
S. cerevisiae is reflected by changes in the activity of
PtdIns(4)P 5-kinase associated with the plasma membrane and by
synthesis of PtdIns(4,5)P2.