(Received for publication, February 9, 1996; and in revised form, March 4, 1996)
From the
Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold
from calf brain using chromatography on heparin-agarose and affinity
elution with inositol hexakisphosphate. The final preparation contained
proteins of 48 and 36-38 kDa. All of these proteins had the same
amino-terminal sequence and were enzymatically active. The smaller
species represent proteolysis products with carboxyl-terminal
truncation. The Kof the enzyme for
inositol 1,3,4-trisphosphate was 80 nM with a V
of 60 nmol of product/min/mg of protein. The
amino acid sequence of the tryptic peptide
HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by
a human expressed sequence tag clone (GB T09063) at 16 of 22 residues.
The expressed sequence tag clone was used to screen a human fetal brain
cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that
predicts a protein of 46 kDa. The clone encodes the amino-terminal
amino acid sequence obtained from the purified calf brain preparation,
suggesting that it represents its human homologue. The cDNA was
expressed as a fusion protein in Escherichia coli and was
found to have inositol 1,3,4-trisphosphate 5/6-kinase activity.
Remarkably, both the purified calf brain and recombinant proteins
produced both inositol 1,3,4,6-tetrakisphosphate and inositol
1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This
finding proves that a single kinase phosphorylates inositol in both the
D5 and D6 positions. Northern blot analysis identified a transcript of
3.6 kilobases in all tissues with the highest levels in brain. The
composite cDNA isolated contains 3054 bp with a poly(A) tail,
suggesting that 500-600 bp of 5` sequence remains to be
identified.
The phosphatidylinositol signaling pathway involves a complex scheme in which cells use a series of kinases and phosphatases to interconvert the six known inositol lipids and the more than 20 inositol phosphates that exist in eukaryotic cells(1) . These molecules have been implicated in a number of intracellular events including calcium ion mobilization(2) , nuclear DNA synthesis(3) , trafficking of intracellular vesicles(4) , and cell proliferation in response to cytokines and growth factors(1, 2) . Many of the reactions in this pathway are catalyzed by several different isoenzymes, most notably phospholipase C (5) and inositol polyphosphate 5-phosphatase isoenzymes (6, 7) , where 8-10 isoforms of each have been discovered to date. We have now characterized an additional kinase of this pathway that utilizes inositol 1,3,4-trisphosphate as a substrate.
Inositol
1,3,4-trisphosphate (Ins(1,3,4)P) (
)is at a
branch point in inositol phosphate metabolism. It is dephosphorylated
by specific phosphatases to either inositol 3,4-bisphosphate or
inositol 1,3-bisphosphate. Alternatively, it is phosphorylated to
inositol 1,3,4,6-tetrakisphosphate
[Ins(1,3,4,6)P
] or inositol
1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P
] by
inositol trisphosphate 5/6-kinase (Ins(1,3,4)P
5/6-kinase)(8, 9) . Ins(1,3,4,6)P
is
the first intermediate in the pathway leading to the higher inositol
phosphates including other tetraphosphates, pentaphosphates, inositol
hexakisphosphate, and pyrophosphate forms of these(10) , all of
which are ubiquitously found in tissues. Because the Ins(1,3,4)P
5/6-kinase enzyme is at a branch point in metabolism leading to
multiple different end products, it is likely to be regulated by its
various end products. This enzyme has been partially purified from rat
liver(8, 9) , porcine brain(11) , and bovine
testes (11) and in each case was reported to phosphorylate
Ins(1,3,4)P
on either the 5 or 6 position yielding a
mixture of two products. This property seems remarkable since the 5 and
6 positions of myoinositol are on opposite faces of the ring. We
therefore isolated the enzyme from calf brain and used the amino acid
sequence that we determined to identify a human expressed sequence tag
(EST) and to clone a cDNA encoding the human Ins(1,3,4)P
5/6-kinase.
The crude extract from 12
brains was filtered sequentially through 80-, 5-, and 1-µm filters
prior to being applied to a 3.5-liter heparin-agarose column (45
10 cm) equilibrated in 20 mM Bis-Tris, 1 mM ATP, 1 mM DTT, and 1 mM EGTA, pH 7.2 (heparin
buffer A). After washing with two column volumes of buffer A, the
column was step eluted with heparin buffer A containing 0.2 M NaCl. The fractions containing Ins(1,3,4)P
5/6-kinase
activity were pooled and precipitated with 50% ammonium sulfate. The
pellet was stored at -80 °C until 36 calf brains were
processed in the same manner. Between uses, the heparin-agarose column
was washed with two column volumes each of 2 M NaCl and 8 M urea to remove residual protein. The ammonium sulfate
pellets were combined, resuspended in homogenization buffer, and
dialyzed against heparin buffer A containing 3 mM MgCl
(heparin buffer B). The dialyzed samples were loaded onto a
500-ml heparin-agarose column (25
5 cm), equilibrated with
heparin buffer B, and eluted with 0.3 M NaCl in heparin buffer
B. The fractions containing Ins(1,3,4)P
5/6-kinase activity
were pooled, precipitated with 50% ammonium sulfate, and dialyzed
against heparin buffer B.
The sample was next applied to an 85-ml
heparin-agarose column (48 1.5 cm) equilibrated in heparin
buffer B. After washing with two column volumes of buffer B, the column
was eluted with a 1.6-liter linear gradient of 0-1 mM inositol hexakisphosphate (IP
) in heparin buffer B.
Two pools of Ins(1,3,4)P
5/6-kinase activity were collected
from this elution and were dialyzed against 55% ammonium sulfate in
heparin buffer B. After centrifugation, the pellets were dialyzed
against heparin buffer B and applied separately to a 1-ml Mono Q
column. Fractions were eluted using a 20-ml linear gradient of
0-0.3 M NaCl in heparin buffer B.
Clone 15 was
digested with EcoRI, and the resulting fragment was gel
purified and ligated into the Xpress(TM) System Protein Expression
TrcHis vector as per manufacturer's instructions (Invitrogen).
From a 25-ml culture having an A of 0.6 at the
time of induction with 1 mM isopropyl-1-thio-
-D-galactopyranoside, 1 ml was
removed at various time points, and the cells were pelleted by
centrifugation. Bacterial pellets were lysed by the addition of 50
µl of 20 mM MES, pH 6.1, 1 mM EDTA, 1 mM ATP, 10 mM benzamidine, 40 µM leupeptin, 1
mM phenylmethylsulfonyl fluoride, 40 µM iodoacetamide, 1 µM pepstatin A, 10 µM bestatin, 1 mM DTT, 17 µg of calpain inhibitor I/ml,
7 µg of calpain inhibitor II/ml, and 3 mM MgCl
, followed by freezing on dry ice and thawing. To
each pellet suspension, 5 µl of 10 mg of lysozyme/ml in the above
buffer was added, and samples were kept on ice until all time points
were collected. Assays for 5/6-kinase activity were done as described
above.
The purification of Ins(1,3,4)P 5/6-kinase exploited the finding that various conditions altered
the position of elution of enzyme from heparin-agarose columns. In the
absence of MgCl
, the Ins(1,3,4)P
5/6-kinase
activity elutes from heparin-agarose with 0.2 M NaCl (Fig. 1A). Upon rechromatography in the presence of 3 mM MgCl
, Ins(1,3,4)P
5/6-kinase activity
bound more tightly and was eluted with 0.3 M NaCl (Fig. 1B). Ins(1,3,4)P
5/6-kinase activity
was further purified on another heparin-agarose column eluted with a
linear gradient of IP
. In the absence of MgCl
,
Ins(1,3,4)P
5/6-kinase activity elutes very early in the
gradient (data not shown). In the presence of MgCl
,
Ins(1,3,4)P
5/6-kinase activity elutes midway through the
gradient at 0.4 mM IP
, as shown in Fig. 1C. At this point, the preparation was divided
into two parts based on the specific activity of the fractions. Pool 1
contained fractions with a specific activity of 5000-18,000
min
/mg, and pool 2 contained fractions with a
specific activity of 18,000-52,000 min
/mg (Fig. 1C).
Figure 1:
Purification of calf brain
Ins(1,3,4)P 5/6-kinase. A, heparin-agarose
chromatography minus MgCl
, 0.2 M NaCl was added at
fraction 1. The flow rate was 35 ml/min, and 20-ml fractions were
collected. B, heparin-agarose chromatography plus
MgCl
, 0.2 M NaCl was added at fraction 1 and 0.3 M NaCl at fraction 40. The column was allowed to flow by
gravity at varying flow rates, and the fraction size was 20 ml. C, affinity elution of heparin-agarose with IP
. A
1.6-liter gradient of 0-1 mM IP
in 20 mM Bis-tris, pH 7.2, 1 mM EGTA, 1 mM DTT, 1 mM ATP, and 3 mM MgCl
was developed at a flow
rate of 0.5-1.0 ml/min. The fraction size was 20 ml. D,
fast protein liquid chromatography ion exchange chromatography. A Mono
Q column (HR5/5) was run at a flow rate of 1 ml/min. Fraction size was
1.0 ml.
The two pools of enzyme activity were
applied separately to a Mono Q column. Elution of pool 2 with a linear
gradient of NaCl is shown in Fig. 1D. Ins(1,3,4)P 5/6-kinase activity elutes at 0.12 M NaCl with a small
peak of coincident protein. The bulk of protein elutes later in
fractions 13-15. The specific activity of the peak fraction was
3.7
10
min
/mg. Application of
pool 1 to the Mono Q column yielded an identical profile of protein and
Ins(1,3,4)P
5/6-kinase activity, but with lower specific
activities of the fractions (data not shown). SDS-gel analysis of the
Mono Q fractions from pool 2 indicated that the peak fraction contains
two discrete sets of protein bands. One protein has an apparent
molecular mass of 48 kDa, whereas the other is a triplet of proteins
ranging in size from 36-38 kDa (Fig. 2).
Figure 2:
SDS-polyacrylamide gel of purified
Ins(1,3,4)P 5/6-kinase. Fractions from elution of pool 2
from a Mono Q column were run on a 12% SDS gel and stained with
Coomassie Blue. Lane 1 contains 1 µg of molecular mass
markers; lanes 3-11 contain 15 µl each of Mono Q
fractions 6-14; lanes 12-15 contain 0.25, 0.5,
1.0, and 2 µg of molecular mass markers,
respectively.
A summary of
the purification is shown in Table 1. From 36 calf brains, a
total of 53 g of protein was obtained from the 48,000 g supernatant (crude extract). The final material (pool 2) had a
specific activity of 3.7
10
min
/mg protein. The overall yield for this
fraction was 3%, with a 12,900-fold purification.
Figure 3:
Gel assay of partially purified
Ins(1,3,4)P 5/6-kinase. A, Coomassie Blue stain of
a 12% SDS gel containing 2.0, 1.0, and 0.5 µg each of molecular
mass markers (lanes 1-3, respectively) and 20 µg of
partially purified Ins(1,3,4)P
5/6-kinase (lane
5). An additional lane containing 40 µg of Ins(1,3,4)P
5/6-kinase was sliced into 41 pieces (shown at the far right of the stained gel) and assayed for Ins(1,3,4)P
5/6-kinase activity. B, IP
produced in each
gel slice. The arrows indicate Coomassie-stained bands
corresponding to active Ins(1,3,4)P
5/6-kinase.
Amino-terminal amino acid sequence analysis,
obtained from sequencing excised bands from polyvinylidene difluoride
membranes from early preparations of lower specific activity
(350-550 min/mg protein), indicated that a
predominant 36-kDa band was aldolase type C (17) , which was a
major contaminant of the preparations (data not shown). Under
nonreducing conditions on a series of gel filtration columns, aldolase
elutes as a tetramer with an apparent molecular mass of 140 kDa,
whereas Ins(1,3,4)P
5/6-kinase activity was found only in
fractions of lower molecular weight (data not shown). It was recently
shown by Baron et al.(18) that aldolase type C binds
Ins(1,4,5)P
and that this binding is inhibited strongly by
Ins(1,3,4)P
, which may explain its copurification with
Ins(1,3,4)P
5/6-kinase.
A schematic diagram of the EST clone GB T09063 and the overlapping human fetal brain clone is shown in Fig. 4. The consensus clone contains 3054 bp with an open reading frame of 1242 bp and 1700 bp of 3`-untranslated region. A polyadenylation signal is located 23 bp upstream from a poly(A) tail. There is no in-frame stop codon in the sequence 5` of this, thus it is possible that initiation occurs in a 5` site not yet obtained. There is a weak Kozak consensus sequence (19) around bp 1 shown in Fig. 5. The open reading frame encodes a protein of 45.6 kDa with a pI of 6.1 (Fig. 5). The peptide sequences obtained from the calf brain preparation presumed to match the corresponding amino acids in the human protein are underlined. They were identical at 45 of 54 residues sequenced (83%).
Figure 4: Schematic diagram of clones. The representation of the composite clone is shown at the top of the diagram, and the relationship between the EST clone and clone 15 isolated from a human fetal brain library is shown at the bottom. The position of the 200-bp HindIII fragment used to screen the library is indicated by arrows at the top of the diagram.
Figure 5:
Nucleotide and predicted amino acid
sequence of human Ins(1,3,4)P 5/6-kinase. The numbering of
the nucleotides (left side) and the amino acids (right
side) begin at the start methionine. Amino-terminal amino acid
sequence obtained from purified calf brain Ins(1,3,4)P
5/6-kinase is underlined with a dashed line.
Peptide sequences from lysyl endopeptidase and trypsin digestion of the
purified calf brain Ins(1,3,4)P
5/6-kinase are underlined with solid
lines.
The amino acid sequence of
Ins(1,3,4)P 5/6-kinase shares small regions of similarity
to the epsilon isoform of protein kinase C (PKC
) from both rabbit
and human sources, as determined from a BLAST search(20) . The
three regions are spaced throughout both the Ins(1,3,4)P
5/6-kinase sequence and the PKC
sequences (Fig. 6A). In the first conserved region, comprised of
12 amino acids, there is 50% identity and 75% similarity between the
two PKC
isoforms and Ins(1,3,4)P
5/6-kinase. The
second region contains 32% identity and 56% similarity, and the third
region contains 72% identity and 81% similarity. In addition, the
sequence obtained for Ins(1,3,4)P
5/6-kinase has similarity
to the predicted amino acid sequences of two other ESTs in the Genbank. Fig. 6B shows a comparison between the predicted amino
acid sequence of human Ins(1,3,4)P
5/6-kinase and the
predicted amino acid sequences available for the EST clones GB Z25963
from Arabidopsis thaliana and GB D46351 from rice. The
predicted amino acid sequence of the Arabidopsis partial cDNA
is 44% identical to and 79% similar to Ins(1,3,4)P
5/6-kinase over 54 amino acids and contains a methionine near the
putative initation methionine. The predicted amino acid sequence of the
rice partial cDNA is 32% identical and 76% similar to Ins(1,3,4)P
5/6-kinase over 139 amino acids.
Figure 6:
Amino acid alignments of human
Ins(1,3,4)P 5/6-kinase (h 5/6-kinase) with human (h) and rabbit (r) PKC
isoforms (A) and
with the predicted amino acid sequences from putative homologs from Arabidopsis and from rice (B). The identities are
indicated by boxes.
Figure 7: Northern blot analysis of human mRNA. A, a Northern blot containing mRNA from human tissues probed with a 200-bp HindIII fragment of GB clone T09063. B, a Northern blot containing mRNA from various regions of the human brain probed with a 200-bp HindIII fragment of GB clone T09063.
Figure 8:
Phosphorylation of Ins(1,3,4)P
by recombinant human Ins(1,3,4)P
5/6-kinase. Bacterial
extracts were used 5 h after induction with
isopropyl-1-thio-
-D-galactopyranoside. Over a 20-min
incubation period with 14 (
) and 28 (
) ng of crude
bacterial lysate from E. coli expressing histidine-tagged
recombinant 5/6-kinase, samples were assayed for 5/6-kinase
activity.
Figure 9:
HPLC analysis of products of
phosphorylation of Ins(1,3,4)P by Ins(1,3,4)P
5/6-kinase purified from calf brain (A) and from the
histidine-tagged recombinant enzyme produced in E. coli (B).
H-products (
) and a
P internal standard of Ins(1,3,4,5)P
(
)
are shown.
We have isolated an Ins(1,3,4)P 5/6-kinase from
calf brain that phosphorylates Ins(1,3,4)P
. In bovine
tissues the Ins(1,3,4)P
5/6-kinase activity is most
abundant in calf brain, constituting 0.13% of total soluble protein,
and is 20- and 200-fold less prevalent in liver and in skeletal muscle,
respectively. In the case of skeletal muscle, the lower amounts of
Ins(1,3,4)P
5/6-kinase activity correlate with the low
levels of Ins(1,3,4,6)P
in skeletal muscle reported by Mayr
and Thieleczek(25) . By contrast, inositol polyphosphate
4-phosphatase, which also utilizes Ins(1,3,4)P
as a
substrate, is present in relatively high amounts in skeletal muscle (26) , as is inositol 1,3-bisphosphate(25) , the
product of Ins(1,3,4)P
dephosphorylation by inositol
polyphosphate 4-phosphatase(27) . The relative abundance of
these enzymes may contribute to the low level of Ins(1,3,4,6)P
found in this tissue.
Our purification of Ins(1,3,4)P 5/6-kinase employed chromatography in the presence and the
absence of MgCl
and affinity elution with IP
.
The final preparation had a peak specific activity of 3.7
10
min
/mg protein, with two sets of
protein bands of apparent molecular masses of 48 and 36-38 kDa.
Identification of these protein bands as Ins(1,3,4)P
5/6-kinase was accomplished by gel renaturation assays, in which
bands excised from an SDS gel were shown to phosphorylate
Ins(1,3,4)P
.
The specific activity of the preparation
reported here is greater than 200 times higher than that reported for
the enzyme purified from rat liver(9) . There is evidence of
carboxyl-terminal proteolysis in the calf brain preparation, and the
protein purified from rat liver has a molecular mass of 36 kDa, which
is consistent with proteolysis in that preparation. A similar situation
occurred in the isolation of inositol 1,4,5-trisphosphate 3-kinase
where early reports of purification found proteolyzed and relatively
inactive enzyme compared with the full-length protein(28) . A
major contaminant of the calf brain Ins(1,3,4)P 5/6-kinase
at early stages of the purification is aldolase type C. Because
aldolase C is an inositol polyphosphate binding protein, it may also
have contaminated the rat liver preparation.
Using protein sequence
obtained from the purified calf brain protein, we have cloned and
expressed the human homolog of Ins(1,3,4)P 5/6-kinase. The
cDNA obtained encodes a 46-kDa protein with regions of similarity to
human and rabbit PKC
isoforms. Only one of the three conserved
regions, residues 237-273 of Ins(1,3,4)P
5/6-kinase,
lies in a defined domain of PKC
(reviewed by Hug and
Sarre(29) ). This region is in a loop that is very sensitive to
proteolysis by calpain. The predicted amino acid sequence is also
similar to the predicted amino acid sequence of two plant cDNA EST
clones in GenBank. Although limited sequence identities are described
here, complete sequencing of these genes may yield further regions of
similarity. These proteins may therefore represent the plant homologs
of human Ins(1,3,4)P
5/6-kinase. There is no sequence
similarity to other inositol polyphosphate kinases or phosphatases
including the phosphatases that utilize the same substrate as
Ins(1,3,4)P
5/6-kinase.
The cloning of Ins(1,3,4)P 5/6-kinase allowed definitive identification of the products of
this enzyme. In enzyme preparations from tissues it was conceivable
that two kinases were copurified that phosphorylated either the 5 or 6
position of Ins(1,3,4)P
. The demonstration of both 5- and
6-kinase activities toward Ins(1,3,4)P
by the recombinant
kinase rules out contamination of the protein preparation. This dual
product formation from a single substrate makes Ins(1,3,4)P
5/6-kinase unique among the inositol polyphosphate kinases and
phosphatases. There is a constant ratio of the two tetrakisphosphate
products formed by the purified calf brain enzyme and the recombinant
human enzyme, with a preference for the 6 position, which might result
from the formation and ultimate hydrolysis of a cyclic phosphate
intermediate. If the two products result from hydrolysis of a cyclic
intermediate, it is possible that the product ratio is different in
cells. Ins(1,3,4)P
5/6-kinase phosphorylates one of the two
possible positions (i.e., IP
is not a product).
This finding would also be consistent with a cyclic 5/6 phosphate
intermediate. Alternatively, the production of dual products may serve
some as yet undiscovered cellular function. With Ins(1,3,4)P
5/6-kinase available as a recombinant enzyme, the mechanism of
its dual phosphorylation and studies of formation of higher
phosphorylated inositol polyphosphates may be carried out.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U51336[GenBank].