From the Max Planck Institute of Molecular Plant
Physiology (MPI-MOPP), Karl-Liebknecht-Stra
e 25, Haus 20, D-14476 Golm/Potsdam, Germany, ¶ University of Lund, Plant
Biochemistry, P. O. Box 117, S.E. 22100 Lund, Sweden, and ** Department
of Plant Sciences, University of Cambridge,
Cambridge CB2 3EA, United Kingdom
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ABSTRACT |
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Phosphatidylinositol metabolism plays a central
role in signaling pathways in animals and is also believed to be of
importance in signal transduction in higher plants. We report here the
molecular cloning of a cDNA encoding a previously unidentified
126-kDa phosphatidylinositol (PI) 4-kinase (AtPI4K Synthesis and hydrolysis of phosphoinositides play an important
role in the transduction of physiological signals such as hormones,
growth factors, and neurotransmitters in animal cells (1, 2).
Sequential phosphorylation of the D4 and D5 positions of
L- PI 4-kinase catalyzes the phosphorylation of PtdIns to PtdIns-4-P,
the first committed step toward the synthesis of
PtdIns-4,5-P2, and therefore represents a potentially
crucial point of regulation of the
phosphatidylinositol-dependent pathways. Two types of PI 4-kinases, II and III, differing in size and sensitivity to detergents and adenosine, have been identified in a wide range of tissues and
cellular compartments (15, 16). Type I PI kinases phosphorylate the D3
position of the inositol ring and are therefore referred to as PI
3-kinases (17).
cDNAs encoding functional PI 4-kinases have been isolated from
animals and Saccharomyces cerevisiae. Putative PI 4-kinase clones have been identified in Schizosaccharomyces pombe,
Dictyostelium discoideum (18), and Caenorhabditis
elegans (19). The proteins encoded by these genes have now been
grouped into two distinct subfamilies, 1.1 and 1.2, based on sequence
and structure similarities (20). Subfamily 1.1 is represented by
proteins of 68-122 kDa and subfamily 1.2 by proteins of 200-230 kDa
(with the exception of human PI4K Studies conducted with plant tissues have demonstrated the presence of
PI 4-kinase activity in plasma membranes (23), the cytosol (24), the
cytoskeleton (13), and nuclei (25). The PI 4-kinase activity present in
the plasma membrane of carrot cells responded to osmotic stress, cell
wall-digesting enzymes, and light (26, 27). After solubilization from
plasma membranes, this activity could be activated by a soluble 49-kDa
protein, PIK-A49, which was also able to bind and bundle actin and was identified as an elongation factor 1 We are interested in the molecular and genetic analysis of cellular
processes involving phosphoinositides in higher plant cells. We
describe here the molecular cloning, heterologous expression, and
biochemical characterization of a PI 4-kinase, designated AtPI4K Material--
Enzymes used for DNA restriction and modification
were purchased from Boehringer Mannheim and New England Biolabs
(Danvers, MA). DNA primers for polymerase chain reaction (PCR) were
obtained from TibMolbiol (Berlin, Germany). [ Bacteria and Plants--
Escherichia coli strain XL-1
Blue (Stratagene, Heidelberg, Germany) was used for DNA cloning
procedures and for library screening. Strain DH10 Isolation of the AtPI4K
DNA sequencing was done by MWG Biotech (Munich, Germany). Computational
analysis was performed with the help of the programs of the Wisconsin
Genetics Computer Group (GCG Package, Version 8.1 (34)). The FASTA (35)
and BLAST (36) search programs were used for sequence comparisons on
DNA and amino acid sequences in GenBankTM, EMBL, dbEST, and
SwissProt data bases. Sequence alignments were performed using the
BESTFIT program. Short repetitive sequences within the AtPI4K Protein Expression in Insect Cells--
Insect (Spodoptera
frugiperda Sf21) cells (Invitrogen, Leek, The Netherlands)
were cultivated as monolayer cultures as described (38) at 27 °C in
TNM-FH medium (Sigma) supplemented with 10% fetal calf serum.
Expression of recombinant AtPI4K SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Proteins extracted from insect cells or obtained after
protein purification (see below) were separated on 6%
SDS-polyacrylamide gels (39). Western blot analysis was performed
essentially as described previously (40). The antiserum raised against
the polyhistidine tag (Arg-Gly-Ser-His6; Qiagen) was used
at a 1:1500 dilution in blocking buffer (0.3% lowfat milk powder
(Heirler, Radolfzell, Germany), 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20). Alkaline phosphatase-conjugated
secondary antibody (anti-mouse IgG; Promega, Madison, WI) was used at a
1:7500 dilution in TBST (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20, 1% bovine serum albumin). Blots
were developed using nitro blue tetrazolium in conjunction with
alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate.
Biochemical Analysis of Recombinant AtPI4K
PI 4-kinase activity was determined at 37 °C in 50 µl of reaction
buffer (50 mM Tris-HCl, pH 7.4, 80 mM KCl, 10 mM MgCl2, 2 mM EDTA, 5 µg PtdIns
(Sigma), 0.2% Triton X-100 (unless otherwise indicated)), and 0.1 µg
of affinity-purified protein. The reaction was started by the addition
of 50 nM [
To determine the effect of wortmannin on PI 4-kinase activity, 1 µl
of stock solution (10 µM to 1 mM, wortmannin
dissolved in ethanol) was added to the reaction mixture. Ethanol was
used in control experiments. All experiments were performed at least three times on independent preparations of purified protein. The data
presented are the mean ±S.D.
HPLC Analysis of the Reaction Product--
The products of
enzyme assay were deacylated, mixed with 3H standards, and
resolved by Partisphere SAX HPLC according to Brearley et al. (42).
RNA Blot Analysis--
Material for RNA blot analysis of steady
state mRNA levels was harvested from well watered A. thaliana plants (Fig. 6, top left) or from plants
cultivated in liquid medium (Fig. 6, top right and
bottom). RNA was prepared according to the protocol of
Logemann et al. (43). Total RNA was quantitated
spectrophotometrically at 260 nm. RNA (30 µg/lane) was
electrophoretically separated in denaturing 15% (v/v) formaldehyde,
1.5% (w/v) agarose gels and blotted onto Hybond-N+
membranes (Amersham Pharmacia Biotech). RNA was fixed to the membrane
via UV-cross-linking (Stratalinker, Stratagene). An 0.8-kb-long 5'
fragment of the AtPI4K Cloning of AtPI4K Structural Organization of the A. thaliana PI 4-Kinase
Protein--
The deduced AtPI4K
Further sequence analyses showed that AtPI4K
Hydropathy analysis identified a relatively hydrophilic segment within
the N-terminal half of AtPI4K Biochemical Characterization of Recombinant
AtPI4K
When analyzed in vitro, recombinant AtPI4K
Triton X-100 activates animal PI 4-kinases (15) and is also able to
activate the partially purified enzymes from carrot (45) and C. roseus suspension culture cells (44). The activity of recombinant
AtPI4K
Adenosine inhibits mammalian PI 4-kinases previously classified as type
II more than it does type III enzymes (20). When tested on AtPI4K
Wortmannin, a hydrophobic steroid-related compound from the fungus
Talaromyces wortmannii, is a potent inhibitor of mammalian PI 3-kinases (46). Although PI 4-kinases were not first believed to be
inhibited by wortmannin, it has recently been demonstrated that the
yeast PI 4-kinase encoded by the STT4 gene is inhibited by
approximately 95% at 10 nM wortmannin (47).
Wortmannin-sensitive PI 4-kinases have also been cloned from human (48)
and bovine (49), although sensitivity of the PI 4-kinase is at least 10 times lower than that of P13-kinase. As is shown in Fig. 5C,
wortmannin had little effect at concentrations up to 0.1 µM but inhibited the plant recombinant enzyme by
approximately 90% at 10 µM.
Northern Blot Analyses Indicate Almost Constitutive Expression of
the AtPI4K Although PI 4-kinase activity has long been detected,
characterized, and partially purified from higher plants, cloning and functional expression of a plant PI 4-kinase has not yet been reported.
Here we describe the cloning of a cDNA from A. thaliana that encodes a functional 126-kDa PI 4-kinase. The size of this protein
is similar to the size of the smaller of the PI 4-kinases isolated from
yeast, D. discoideum, and animals, which is in the range of
90-122 kDa. Recently, Okpodu et al. (45) partially purified
a soluble PI 4-kinase from carrot suspension culture cells. The
molecular mass of this protein was estimated to be 83 kDa. Two distinct
PI 4-kinase activities have recently been partially purified from
spinach plasma membranes, with estimated sizes of 65 and 120 kDa.4 No peptide sequences
for any of the partially purified plant PI 4-kinases are available.
Therefore it is not possible to know whether any of the partially
purified plant PI 4-kinases corresponds to homologs of AtPI4K Sequence analyses showed that AtPI4K The partial PI 4-kinase isolated from A. thaliana, AtPI4K The catalytic properties of the recombinant plant protein,
i.e. its stimulation by low concentrations of Triton X-100
as well as its moderate inhibition by adenosine, although they do not match exactly, still resemble those of the PI 4-kinases previously classified as type III more than those classified as type II. As
recently observed with other PI 4-kinases, the AtPI4K The sequences available so far indicate that in animals each of the two
types of PI 4-kinases is represented by several splice variants of one
single gene. The whole genome of S. cerevisiae contains only
two PI 4-kinase genes. It is now clear that plants also contain
subfamily 1.1 and subfamily 1.2 PI 4-kinases. Because PI 4-kinase
activities have been detected in most cellular compartments, it is
probable that the domains identified in PI 4-kinases are involved in
the targeting of PI 4-kinase isoforms to various cellular compartments
through interactions with other proteins or lipids. The similarity in
structure of the PI 4-kinases found in the different phyla contrasts
markedly with the current data available for PI-PLC. Three to four
genes have been isolated from A. thaliana (56-58), potato
(59), and soybean (60). Based on protein structure and biochemical
properties, all plant PI-PLC isoforms appear to belong to a single
family, which is most closely related to the ) from the higher
plant Arabidopsis thaliana. The novel protein possesses the
conserved domains present in animal and yeast PI 4-kinases, namely a
lipid kinase unique domain and a catalytic domain. An additional
domain, approximately 300 amino acids long, containing a high
percentage (46%) of charged amino acids is specific to this plant
enzyme. Recombinant AtPI4K
expressed in baculovirus-infected insect
(Spodoptera frugiperda) cells phosphorylated phosphatidylinositol exclusively at the D4 position of the inositol ring. Recombinant protein was maximally activated by 0.6% Triton X-100
but was inhibited by adenosine with an IC50 of ~200
µM. Wortmannin at a concentration of 10 µM
inhibited AtPI4K
activity by ~90%. AtPI4K
transcript levels were similar in all tissues analyzed. Light or
treatment with hormones or salts did not change AtPI4K
transcript levels to a great extent, indicating constitutive expression
of the AtPI4K
gene.
INTRODUCTION
Top
Abstract
Introduction
References
-phosphatidyl-1-D-myo-inositol
(PtdIns)1 yields
phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2). PtdIns-4,5-P2
is hydrolyzed to inositol 1,4,5-trisphosphate, a stimulator of calcium
release from intracellular stores (1), and diacylglycerol, an activator of some protein kinase C isoforms (3), by phosphoinositide-specific phospholipase C (PI-PLC). In addition to their classical function as
precursors of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, phosphoinositides, including those phosphorylated at the D3 position of the inositol ring, have been shown to regulate cytoskeleton rearrangements through the association with a variety of
actin-binding proteins, including profilin, gelsolin, and villin (4,
5), and can also, for example, potentiate the activation of protein
kinase C (6) and PI-PLC (7, 8). They also constitute major regulators
of membrane-trafficking (9, 10). With the exception of
phosphatidylinositol 5-phosphate and phosphatidylinositol 3,4,5-trisphosphate, all of the inositol phospholipids found in animals
have also been identified in plants. Focusing on the role of inositol
lipids in plants, independent of their much questioned role in calcium
signaling, PtdIns-4-P and PtdIns-4,5-P2 are known to affect
the activity of several enzymes, including the plasma membrane
H+-ATPase (11) and phospholipase D (12) and can interact,
in vitro at least, with cytoskeletal components (13).
Profilin is believed to be involved in cytoskeleton dynamics in plant
cells as it is in animal cells, through interaction with
PtdIns-4,5-P2 and actin (14).
, which is a 97-kDa protein (21)).
The comparison of the primary structures of these proteins has enabled
the identification of several conserved domains. All PI 4-kinases
possess a catalytic domain of about 230 amino acid residues, which
constitute the C-terminal part of those proteins, and a so-called lipid
kinase unique (LKU) domain of about 100 residues, the location of which varies between the different isoforms. The larger isoforms as well as
PI4K
(21) all contain a PH domain that separates the LKU and
catalytic domains in these proteins. In addition, it was recently noted
that yeast PIK1 and a soluble PI 4-kinase from rat have a region in
common that could not be detected in any other PI 4-kinases
(22).
(28). This activation was
subsequently shown to be dependent on the phosphorylation status of
PIK-A49 (29). Recently, a partial cDNA from Arabidopsis thaliana encoding a putative PI 4-kinase, AtPI4K
, was isolated. The deduced amino acid sequence corresponding to this cDNA
comprised domains highly similar to the LKU, PH, and catalytic domains
of known PI 4-kinases and, thus, demonstrated that plants posses at
least one protein structurally related to known PI 4-kinases. The PH
domain present in this plant protein was expressed in bacteria and
shown to bind phosphatidic acid, PtdIns-4-P, and
PtdIns-4,5-P2 (30).
,
from A. thaliana.
EXPERIMENTAL PROCEDURES
-32P]dCTP
and [
-32P]ATP were obtained from ICN (Meckenheim,
Germany). PtdIns was obtained from Sigma. Unless otherwise indicated,
other chemicals were purchased from Boehringer Mannheim, Merck, or Sigma.
Bac (Life
Technologies, Inc.) was used for in vivo construction of
recombinant bacmids employed for protein expression in insect cells.
A. thaliana C24 plants were grown in a phytotron in soil
(GS90; Gebr. Patzer, Sinntal Jossa, Germany) with an 16-h light
(22 °C) and 8-h dark (15 °C) period. Plants grown in liquid culture medium were obtained as follows. Seed from A. thaliana Landsberg erecta were germinated on solid AM medium (1/2
MS, 1% sucrose) for 5 days. Subsequently, plantlets were transferred to liquid MS medium (31) containing 1% (w/v) sucrose and cultured for
2 weeks under gentle shaking conditions. For Northern analysis, plants
were treated for 5 days (see Fig. 6).
cDNA and Sequence
Analysis--
DNA manipulation was performed using standard protocols,
as e.g. described by Sambrook et al. (32). A
1.8-kb partial cDNA fragment with a corresponding amino acid
sequence showing high similarity to mammalian PI 4-kinases was
originally isolated from a potato leaf epidermal fragment cDNA
library.2 This fragment was
used to screen an A. thaliana genomic library in phage
EMBL 3 (kindly provided by U. Uwer, Max Planck Institute of Molecular
Plant Physiology, Golm, Germany). Hybridization was performed under low
stringency as described (33). Candidate phages hybridizing to the
potato cDNA were used as templates to amplify putative PI 4-kinase
encoding DNA fragments via PCR with degenerate forward
(5'-AARTCNGGNGAYGAYTGYMG-3') and reverse (5'-CCYTCNGCRTCNDARTCCAT-3') primers. These primers were designed according to the partial potato
sequence and a homologous rice EST (accession number D24320). A 1.2-kb
amplicon was found to represent a genomic fragment homologous to PI
4-kinase-coding regions. This fragment was subsequently used to screen
an A. thaliana hypocotyl
ZAP II cDNA library (CD4-16; obtained from the Arabidopsis Stock Center,
Columbus, OH) under stringent conditions as described previously (33). Plaque-purified phage clones were converted to pBluescript derivatives using helper phage ExAssist according to the supplier's (Stratagene) instructions. Clone pAtPI4K
, which contained the longest
cDNA insert, was used for further analyses.
protein were identified with the help of the SAPS program
(37).3
protein, fused to a polyhistidine
tag at its N terminus, was achieved with the help of the Bac-to-Bac
Expression System from Life Technologies, Inc. Transfer plasmid
pFB-His-PI4K was constructed as follows. A DNA fragment encompassing an
820-base pair-long 5' fragment of the AtPI4K
cDNA was
amplified via PCR using forward
(ATCGGGATCCATGCCGATGGGACGCTTTCTA-3' (added
BamHI site underlined; start ATG of the
AtPI4K
-coding region in italics) and reverse
(5'-ATCGGCGGCCGCCTCTGAGTTAGGTATTGGTTCA-3' (added
NotI site underlined) primers and cloned into plasmid
pFastBacHTb (Life Technologies, Inc.). The PCR-amplified fragment in
the resulting plasmid pFBH-PI4Kn was sequenced to confirm that no
nucleotide exchanges occurred during the PCR amplification.
Subsequently, a 2.6-kb-long fragment of the AtPI4K
cDNA was inserted via PvuII and NotI
restriction sites into plasmid pFBH-PI4Kn. This final cloning step
reconstituted the complete AtPI4K
-coding region. Recombinant baculoviruses obtained after transfection of insect cells
with bacmid DNA (i.e. E. coli/baculovirus shuttle
vector) were amplified twice to yield high titer virus stocks. For
expression of recombinant protein, 9 ml of virus stock was used to
infect cells (60-80% confluency) in 150-cm2 culture flasks.
--
Recombinant
AtPI4K
protein was purified from insect cells 3 days after
infection. Cells were harvested, washed once with ice-cold
phosphate-buffered saline buffer (10 mM
Na2HPO4, 1.8 mM
KH2PO4, 0.14 M NaCl, 2.7 mM KCl, pH 7.3), and lysed by sonification in lysis buffer
containing 50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 10% glycerol, 20 µg/ml aprotinin, and 50 µg/ml chymostatin.
Undisrupted cells were removed by centrifugation (14.000 rpm for 10 min, Centrifuge 5417, Eppendorf), and the supernatant was analyzed by
SDS-PAGE or used for protein purification. Recombinant protein was
batch-purified under native conditions using nickel nitrilotriacetic
acid resin according to manufacturer's (Qiagen) instructions. Yield of
recombinant AtPI4K
from a representative purification (24 ml of
insect cell culture) was 0.2 mg of protein. Purity of recombinant
AtPI4K
was above 95% as estimated from Coomassie-stained SDS-PAGE
protein gels. Protein concentrations were determined by the method of Bradford (1976) using bovine serum albumin as standard (41).
-32P]ATP (>600
Ci/mmol; ICN). The reaction was stopped after 30 min by adding 0.8 ml
of cold chloroform/methanol/H2O (1/2/0.8; v/v/v). To
extract lipids, 0.4 ml of chloroform/2.4 N HCl (1/1; v/v)
was added, and the reaction mixture was vortexed and spinned for
30 s at 12,000 rpm. The upper phase was removed, and an equal
volume of chloroform/methanol/HCl (5/245/235; v/v/v) was used to wash the chloroform phase a second time. An aliquot of the chloroform phase
(15 µl) was applied to thin-layer chromatography (TLC) plates (Silica
gel 60 plates; Merck). Chromatograms were developed with a
chloroform/methanol/concentrated NH3/H2O
(45/35/2/8, v/v/v/v) solvent mixture. Plates were exposed to Kodak
X-Omat AR films. TLC revealed that 32P in the chloroform
phase was incorporated into PtdIns-P only. To determine the effect of
Triton X-100, adenosine, and wortmannin (Calbiochem, Bad Soden,
Germany) on PI 4-kinase activity, liquid scintillation counting was
performed on the dried washed chloroform phase.
cDNA was used as
[
-32P]dCTP-labeled hybridization probe. Membranes were
hybridized at 65 °C in 250 mM sodium phosphate buffer,
pH 7.2, containing 7% SDS, 1% bovine serum albumin, and 1 mM EDTA. Washes were performed at 65 °C in 3× SSC (1×
SSC= 0.15 M NaCl and 0.015 M sodium citrate), 0.5% SDS for 15 min and in 0.2× SSC, 0.5% SDS for 20 min. Blots were
exposed to Kodak X-Omat AR films between intensifying screens for 2-3
days at
70 °C.
RESULTS
--
A partial cDNA from potato encoding
a polypeptide with high similarity to animal and yeast PI 4-kinases was
used to isolate via various intermediate cloning steps (for details see
"Experimental Procedures") a corresponding cDNA from A. thaliana. The longest hybridizing cDNA (4349 nucleotides)
obtained from this library, designated AtPI4K
to
differentiate it from the partial clone (AtPI4K
) isolated
by Stevenson et al. (30), contained an open reading frame of
3366 nucleotides, representing a 1121-amino acid polypeptide with a
calculated molecular mass of 126 kDa. The putative initiation codon was
preceded by an in-frame stop codon; therefore, this clone represented a
full-length cDNA (Fig.
1A). The calculated isoelectric point of 5.5 of AtPI4K
is similar to the isoelectric point of 5.8 determined for the partially purified PI 4-kinase from
Catharanthus roseus (44). A genomic fragment harboring the
entire AtPI4K
-coding region was present in a P1 clone
(MHJ24; GenBankTM accession number AB008266) sequenced by
the Kazusa DNA Research Institute. A comparison between the two DNA
sequences revealed the presence of 16 exons (varying in size between 49 and 2163 base pairs) and 15 introns (sizes between 95 and 511 base
pairs) in the genomic fragment (Fig. 1B). The
computer-predicted exon/intron structure was mostly correct with the
exception of the 3' region of the gene, where three instead of two
exons were found (marked in Fig. 1B); an additional exon was
detected at the 3' end of the gene (Fig. 1B). The complete
AtPI4K
gene had a size of approximately 8 kb, including
5'- and 3'-untranslated regions and putative 5' regulatory elements.
Mapping analysis indicated that the AtPI4K
gene is
located at the bottom of chromosome V, between markers CIC9B5L and
T04492.
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Fig. 1.
Sequence analysis and gene structure of
AtPI4K . A, nucleotide and
predicted amino acid sequences. Numbers on the right
indicate amino acid positions. Note the presence of 11 repeated motifs
(underlined) within the N-terminal half of the protein (see
also text and Fig. 3). B, exon/intron structure of the
AtPI4K
gene. Exons are presented as boxes. The
four marked exons in the 3' region of the gene were
identified via comparison with the AtPI4K
cDNA and
were found to differ from the original computer prediction (see
accession number AB008266). Numbers indicate exon sizes
(nucleotides).
protein sequence possessed two
regions highly similar to the LKU and catalytic domains of known PI
4-kinases (Fig. 2A). The LKU
domain in AtPI4K
was located close to the N terminus and was
20-37% identical to LKU domains from previously identified PI
4-kinases (Fig. 2B). The C terminus of AtPI4K
contained the putative catalytic domain, which was 43-57% identical to the corresponding domains from other PI 4-kinases (Fig. 2C). In
addition, the A. thaliana protein described here, like all
the members of subfamily 1.1, lacks a PH domain, a domain that
characterizes members of subfamily 1.2. These observations indicated
that AtPI4K
represented a new member of the subfamily 1.1 of PI
4-kinases, with a structure resembling most closely that of the yeast
PIK1 protein. Overall, AtPI4K
was more similar to subfamily 1.1 PI 4-kinases than to the other A. thaliana partial PI 4-kinase,
AtPI4K
, and was most closely related to a putative PI 4-kinase from
D. discoideum, with 30% identical and 52% similar amino
acid residues.
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Fig. 2.
Structural organization of A. thaliana PI 4-kinase. A, protein domains present
in AtPI4K , in comparison with selected yeast and animal PI
4-kinases. Numbers indicate amino acid positions. Accession
numbers are as follows: 92kPI4K (rat), D84667; ScPIK1 (yeast), P39104;
PI4K
(human), L36151; 230kPI4K (human), AF012872. B,
multiple amino acid sequence alignment of the LKU domain of selected PI
4-kinases. Accession numbers: AtPI4K
, AF035936; DdPI4K (D. discoideum), U23479; PI4K
(human), U81802. C,
multiple amino acid sequence alignment of the catalytic
(Cat) domains. In B and C, amino acid
residues identical in at least four sequences (conserved exchanges
included) are indicated by gray and black
shading. D, multiple amino acid sequence alignment of
the novel homology (NH) domain present in A. thaliana AtPI4K
, yeast ScPIK1, and rat 92kPI4K.
also possessed a region
similar to part of a sequence previously thought to be shared only by
yeast PIK1 and a soluble 92-kDa PI 4-kinase from rat brain (Fig.
2D). The region common to these three proteins, designated
NH, is approximately 90 amino acid residues in length.
, whereas the rest of the protein had a
more amphipathic character. Part of the hydrophilic segment, covering
amino acids 239-560, contained a high percentage (46%) of charged
amino acid residues, with 25% acidic (glutamate and aspartate) and
21% basic (lysine, arginine, and histidine) amino acids. This domain
was not present in any of the previously identified PI 4-kinases. A
more careful analysis of this region revealed the presence of a
repeated motif. Fig. 3 shows that the repeated sequence, which occurs 11 times between amino acid position 212 and 508 (see Fig. 1A), has a length of 19-20 amino
acids with a highly conserved inner core sequence of
hydrophobic/positively charged/hydrophobic amino acids. This core
sequence was N-terminal-flanked by acidic amino acids (aspartate or
glutamate), and C-terminal-flanked by alternating positively and
negatively charged amino acids.
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Fig. 3.
Unique repetitive motif of
AtPI4K . Alignment of the 11 repeated
motifs present between amino acid positions 212 and 508 (see also Fig.
1A). Red, acidic amino acids; blue,
basic amino acids; green, hydrophobic amino acids.
--
To investigate whether AtPI4K
encodes a
functional PI 4-kinase, we expressed it with a polyhistidine tag at its
N terminus in baculovirus-infected insect (S. frugiperda)
cells. As shown in Fig. 4A,
affinity-purified recombinant AtPI4K
appeared as a single band on a
protein gel (Fig. 4A, left panel, lane
4). The purity of the recombinant protein was estimated to be
higher than 95%, because almost no additional bands were present on
the gel. Although it was not possible to detect recombinant AtPI4K
in the corresponding crude extract after Coomassie staining, an antibody directed against the N-terminal polyhistidine tag showed that
recombinant protein was present in the crude extract (Fig. 4A, right panel, lane 2). No
cross-reacting protein was detected in cells expressing an unrelated
protein without a polyhistidine tag. The molecular mass of the
recombinant AtPI4K
protein was estimated to be 125-130 kDa, which
closely agrees with the calculated mass of 126 kDa.
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Fig. 4.
Expression and biochemical analysis of
recombinant AtPI4K expressed in insect cells.
A, SDS-PAGE (left panel) and Western blot
analysis (right panel). Lanes 1-3, crude protein
extract from insect cells expressing the following proteins: lane
1, a His-tagged plant protein unrelated to AtPI4K
; lane
2, AtPI4K
fused to a His tag; lane 3, a plant
protein unrelated to AtPI4K
(without a His tag). Lane 4,
affinity-purified AtPI4K
(with His tag). Immunodetection of protein
was performed using an antiserum directed against the polyhistidine
tag. B, TLC analysis of the reaction product of recombinant
AtPI4K
. Activity of recombinant AtPI4K
was analyzed as described
under "Experimental Procedures." Left panel,
autoradiogram; right panel, iodine staining. Lane
1, control protein, purified from insect cells; Lane 2,
purified His-tagged AtPI4K
; lane 3, unlabeled PtdIns-4-P.
The background activity seen in lane 1 is because of
residual PI 4-kinase activity endogenous to insect cells. C,
HPLC analysis of the reaction product. The lipid products obtained as
in B were purified, deacylated, and analyzed by HPLC as described (42).
The migration of 3H standards glycerophosphoinositol
3-phosphate (GroPIns3P) and glycerophosphoinositol
4-phosphate (GroPIns4P) is indicated with open
symbols; 32P product is indicated with closed
symbols.
yielded a
phospholipid that on TLC, co-chromatographed with authentic PtdIns-4-P (Fig. 4B). The reaction product was deacylated and analyzed
by HPLC on a Partisphere SAX column. The deacylated product co-eluted exactly with [3H]glycerophosphoinositol 4-phosphate after
glycerophosphoinositol 3-phosphate (Fig. 4C). We also
analyzed the levels of PtdIns-4-P in insect cells. Cells expressing the
AtPI4K
consistently showed 25-30% increase in PtdIns-4-P levels as
compared with mock-infected cells (data not shown). These results
confirm that AtPI4K
is a functional PI 4-kinase.
was almost unaffected by Triton X-100 concentrations below
0.3% (w/v) but increased 4- to 5-fold when the detergent concentration
was increased to 0.6% (Fig.
5A).
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Fig. 5.
Effect of Triton X-100 (A),
adenosine (B), and wortmannin (C) on
enzyme activity of recombinant AtPI4K .
Activities are given as the percentage of maximal activity detected.
Each experiment was performed at least three times. Data indicate the
mean ±S.D.
, a
moderate inhibitory effect was observed, i.e. 50%
inhibition was reached at 200 µM adenosine (Fig.
5B). Maximal activity of recombinant AtPI4K
protein was
approximately 12 nmol/min/mg of protein (in the presence of 0.6%
Triton X-100).
Gene--
The full-length AtPI4K
cDNA
was used as hybridization probe to study transcript levels. A single
transcript of approximately 4.5 kb was detected in leaves, roots,
flowers, and stems of A. thaliana (Fig.
6, top left).
AtPI4K
transcript levels were similar in the various
tissues analyzed. An almost identical result was obtained using
quantitative reverse transcription PCR (data not shown). These
experiments also indicated the presence of AtPI4K
transcript in epidermal fragments, a tissue preparation highly enriched
for stomatal guard cells (data not shown). AtPI4K
transcript levels in leaves were closely similar in the light and dark
(Fig. 6, top right). Treatment of small plantlets with
hormones, CaCl2, or NaCl had no effect on
AtPI4K
mRNA levels (Fig. 6, bottom). The
AtPI4K
gene appeared therefore to be constitutively
expressed in A. thaliana.
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Fig. 6.
RNA blot analysis of steady state
AtPI4K transcript levels.
AtPI4K
transcript levels were tested in various tissues
of intact Arabidopsis plants grown in soil (top
left). The effects of dark or light treatment (top
right), salts, or hormones (bottom) were tested in
leaves harvested from plantlets cultivated in liquid culture medium for
5 days. Medium was supplemented with one of the following compounds,
giving the final concentrations as indicated: CaCl2 (5 mM); NaCl (200 mM); gibberellic acid
(GA3; 100 µM);
1-aminocyclopropane-1-carboxylic acid (ACC; 25 µM); abscisic acid (ABA; 10 µM);
indole acetic acid (IAA; 10 µM). No additional
compounds were added in the case of control experiments.
DISCUSSION
or
AtPI4K
.
possesses the two conserved
domains present in all PI 4-kinases, namely the LKU and the kinase
catalytic domains, but lacked a PH domain. These structural features
indicate that AtPI4K
is a new member of subfamily 1.1 of PI
4-kinases. The molecular structure of AtPI4K
most resembles that of
the yeast PIK1 and the Dictyostelium DdPI4K proteins. We
also identified a novel domain, NH, that corresponds to part of the
sequence that was previously identified only in the yeast PIK1 protein
and a rat soluble PI 4-kinase (22). Surprisingly, AtPI4K
is the only
known PI 4-kinase to possess a unique repetitive motif constituted of
11 repeats of a charged core unit. Interestingly, three putative
PtdIns-P kinases from Arabidopsis (Refs. 50 and 51 and
accession number U95973) also contain a repeated motif, whereas none of
the animal enzymes possess such a repeated motif. No clear function can
be assigned to the LKU, the NH, or the domain with the repetitive
motif; they may play a role in the interaction of PI 4-kinases with
other proteins and/or membrane structures.
(30), possesses a PH domain, and its structure resembles that of
members of subfamily 1.2 of PI 4-kinases. It was shown that the PH
domain of AtPI4K
can bind PtdIns-4-P and PtdIns-4,5-P2 but not PtdIns. It was suggested that AtPI4K
, by binding
phosphoinositides at its PH domain, could be involved in the regulation
of actin polymerization. It appears, thus, likely that the PH domain of type 1.2 PI 4-kinases is not responsible for binding their substrate. Consequently, it is possible that substrate binding in PI 4-kinases is
controlled by the LKU domain or a domain conserved structurally but not
at the sequence level.
is inhibited by wortmannin at concentrations that are significantly higher than
those required to inhibit PI 3-kinases. The residue corresponding to
the Lys residue of the PI 3-kinase PI3K
at which wortmannin binds
covalently (52) is conserved in AtPI4K
as it is in all other cloned
PI 4-kinases, including AtPI4K
. Recently, wortmannin at high
concentrations was shown to inhibit PI 3-kinase and PI 4-kinase
activities in tobacco cells in vivo as well as protein sorting to the plant vacuole (53). These results suggest that the
sorting of proteins to the plant vacuole is controlled by at least two
different mechanisms, one of which is wortmannin-sensitive and may
involve phosphoinositides (53). It has been shown in animal cells that
a wortmannin-sensitive PI 4-kinase enzyme was involved in the formation
of agonist-stimulated inositol trisphosphate (54). Because plants
appear to express similar PI 4-kinase isoforms as animals, it will be
of interest to test whether any of the factors that affect
phosphoinositide levels in plants, such as light, mastoparan, and
fungal elicitors (55), involve the response of a wortmannin-sensitive
PI 4-kinase enzyme.
-type of mammalian
PI-PLCs, but all lack a PH domain. Because phosphoinositides are now
known to be important in processes not involving the canonical second
messengers inositol trisphosphate and diacylglycerol, it will be of
great interest to compare the regulation and function of PI 4-kinases
between animal and plants. In addition, whereas the plant PI-PLC
isoforms are expressed in tissue- and/or environmental-specific manner
(56, 59), the plant PI 4-kinase reported here is ubiquitously expressed
in the plant. Future experiments will tell whether plants possess
additional subfamily 1.1 PI 4-kinase genes whose expression patterns
are developmentally and/or environmentally regulated.
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ACKNOWLEDGEMENTS |
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We thank Thomas Ehrhardt, Steffanie Hartje, and Sabine Zimmermann for help with the insect cell culture and Maryse Laloi for providing some of the RNA blots. We thank Gunnar Plesch, Nicola Weigmann, and Irina Staxen for critical comments on the manuscript. Charles Brearley thanks The British Council (British-German, ARC program) for support.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ002685.
§ Recipient of a fellowship from the Max Planck Society/Chinese Academy of Sciences. Present address: National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, 300 Fenglin Rd., 200032 Shanghai P.R. China.
Supported by the European Community Grant BIO-CT96-0775.
A BBSRC Advanced Research Fellow.
§§ Supported by the Max Planck Society. To whom correspondence should be addressed: Tel.: ++49-331-977-2787; Fax: ++49-331-977-2301; E-mail: mueller{at}mpimp-golm.mpg.de.
2 C. Pical, J. Kopka, F. Aitken, J.E. Gray, and B. Müller-Röber, unpublished information.
3 http://ulrec3.unil.ch/software/SAPS_form.html.
4 T. Westergren, L. Ekblad, B. Jergil, and M. Sommarin, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
PtdIns, L--phosphatidyl-1-D-myo-inositol;
PtdIns-4-P, PtdIns 4-phosphate;
PtdIns-4, 5-P2, PtdIns
4,5-bisphosphate;
PI-PLC, phosphoinositide-specific phospholipase C;
LKU, lipid kinase unique;
PH, Pleckstrin homology;
PCR, polymerase
chain reaction;
kb, kilobase pair(s);
PAGE, polyacrylamide gel
electrophoresis;
HPLC, high performance liquid chromatography;
bp, base pair(s).
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REFERENCES |
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