(Received for publication, December 6, 1994)
From the
Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P)
occupies an essential position in the phosphoinositide signal
transduction cascades as the precursor to second messengers and is
thought to regulate many cellular proteins directly. The final step in
the synthesis of PtdIns(4,5)P
is the phosphorylation of
PtdIns(4)P by PtdIns(4)P 5-kinase (PIP5K). Using peptide sequences from
a purified PIP5K, a cDNA for a human placental PIP5K was isolated and
sequenced. Expression of this cDNA in Escherichia coli produced an active PIP5K. Surprisingly, the sequence of this PIP5K
has no homology to known PtdIns kinases or protein kinases. However,
the PIP5K is homologous to the Saccharomyces cerevisiae proteins Fab1p and Mss4p.
Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P) (
)directly modulates the in vitro activity of a
number of enzymes, including protein kinase C, casein kinase I, and
phospholipase D (1, 2, 3) . The activities of
cytoskeletal proteins, profilin, gelsolin, protein 4.1,
-actinin,
and others are also regulated by
PtdIns(4,5)P
(4, 5, 6) . Recently,
pleckstrin homology domains were found to bind
PtdIns(4,5)P
, and several proteins that contain these
domains are involved in signal transduction(7) .
PtdIns(4,5)P
is also a bifurcation point in the PtdIns
signal transduction
cascade(8, 9, 10, 11, 12) .
PtdIns(4,5)P
can be hydrolyzed by receptor-activated
phospholipase C enzymes(9) , generating inositol
1,4,5-trisphosphate, which stimulates intracellular Ca
release(8) , and diacylglycerol, which activates some
protein kinase C isoforms(10) . Alternatively,
PtdIns(4,5)P
can be a substrate for receptor-stimulated
PtdIns 3-kinases, which synthesize PtdIns(3,4,5)P
, a second
messenger of unknown function(11, 12) . The candidate
functions of PtdIns(4,5)P
are thought to include regulation
of secretion, cell proliferation, differentiation, and
motility(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
Given the roles of PtdIns(4,5)P
, its synthesis is likely to
be stringently regulated.
The first step in PtdIns(4,5)P synthesis is the phosphorylation of PtdIns on the fourth hydroxyl
of the myo-inositol ring by PtdIns 4-kinases(11) .
Multiple PtdIns 4-kinases have been identified biochemically, and two
PtdIns 4-kinase genes, PIK1 and STT4 have been
characterized from Saccharomyces
cerevisiae(11, 12, 13, 14, 15) .
Pik1p is an essential nuclear associated
enzyme(13, 14) . STT4 was identified as a
staurosporine-sensitive mutant in the protein kinase C
pathway(15) . Although STT4 is not essential, mutants
grow slowly and have cell cycle defects similar to protein kinase C
mutations(15) . The distinct phenotypes of these PtdIns
4-kinases suggest that they have different cellular roles.
PtdIns(4,5)P synthesis is catalyzed by the
phosphorylation of PtdIns(4)P on the fifth hydroxyl of the myo-inositol ring (11) . Two isoforms of PtdIns(4)P
5-kinase (PIP5K) have been characterized and are denoted type I and II
PIP5Ks (PIP5KI and PIP5KII) (16, 17) . PIP5KI and
PIP5KII are distinguished by their lack of immunocross-reactivity, by
phosphatidic acid stimulation of PIP5KI, and by the low activity of
purified PIP5KII toward native membranes(16, 17) .
Both of these PIP5Ks use PtdIns(4)P as a substrate, and their product
is PtdIns(4,5)P
(16, 17) .
Immunological evidence suggests that multiple PIP5KI and PIP5KII isoforms exist in different cell types and may have different functions (16, 17, 18, 19) . For example, the G-protein Rho stimulated a PIP5K, and this appeared to be required for integrin-dependent, platelet-derived growth factor-stimulated phosphoinositide turnover(20) . This suggests that regulation of PIP5K activity is more crucial for phosphoinositide signaling than was previously thought.
The phosphoinositides have been implicated
directly in Ca-regulated
secretion(3, 21) . (
)One of the cytosolic
factors required for ATP-dependent priming of
Ca
-regulated secretion corresponds to a PIP5KI.
Purified erythroid 68-kDa PIP5KI stimulated priming; however, a
brain 90-kDa PIP5KIb (17) appeared to have greater priming
activity.
Surprisingly, PIP5KII had no detectable activity
in ATP-dependent priming of regulated secretion.
To assign regulatory mechanisms and cellular roles to individual PIP5Ks, isolation of the cDNAs and determination of the sequences of the PIP5Ks are critical. Here we report the isolation and characterization of the first cDNA encoding a PIP5K.
Figure 1: Nucleotide and predicted amino acid sequences for the coding region of the human 53-kDa PIP5KII cDNA. The first ATG start codon in the open reading frame is preceded by an in-frame termination codon and is within a Kozak consensus translation initiation sequence(26) . The existence of at least 60 additional nucleotides 5` to the cDNA sequence displayed is suggested by a 5`-rapid amplification of cDNA ends procedure on human placental 5`-rapid amplification of cDNA ends-ready cDNA (CLONTECH). The alignments of peptide sequences from 53-kDa PIP5KII are underlined. The putative SH3 domain-binding sites are boxed. The termination codon is denoted by an asterisk.
Figure 2:
Purification and kinase activity of
PIP5KII expressed in E. coli. A: left panel,
bacterially expressed PIP5KII is recognized by polyclonal antibodies
raised against human erythroid 53-kDa PIP5KII(16) . Shown is a
Western blot with PIP5KII antibodies of purified human erythrocyte
PIP5KII; E. coli lysates (14 µg) with vector
alone(-) or vector containing the cDNA (+); and
affinity-purified, polyhistidine-tagged recombinant PIP5KII
(His-PIP5KII). Right panel, Coomassie blue-stained
SDS-polyacrylamide gel of a total lysate of E. coli expressing
His-PIP5KII and purified His-PIP5KII. B, PtdIns(4)P kinase
activity of native erythrocyte PIP5KII and recombinant His-PIP5KII.
Shown is the PtdIns(4,5)P synthesized by purified erythroid
PIP5KII (6 ng); clarified E. coli lysates (350 µg) from
induced cells harboring the vector alone(-) or expressing PIP5KII
(+); and affinity-purified, recombinant His-PIP5KII (6 ng). PIP
,
PtdIns(4,5)P
.
Figure 3:
Northern blot analysis of human tissues. A
human multiple tissue Northern blot (CLONTECH) was hybridized (25) with an -
P-labeled 497-bp EcoRI
fragment derived from the coding region of the PIP5KII
cDNA.
Searching
the sequence data bases with BLAST(24) , we found a significant
similarity to two S. cerevisiae gene products, Fab1p ()and Mss4p(32) , at their C-terminal ends (Fig. 4A). Within this region, the identity between
PIP5KII and these proteins is 27 and 34%, respectively. If conservative
amino acid replacements are taken into account, the similarity among
the three proteins is close to 60% in this region. This sequence
identity is reminiscent of that among diverse protein kinases and
between PtdIns 3- and
4-kinases(12, 13, 14, 15, 28, 29, 30, 31) .
This is illustrated by the clustering of conserved residues into
several domains (Fig. 4B). These conserved domains may
represent parts of the catalytic core.
Figure 4: Comparison of the amino acid sequence of human placental PIP5KII with those of S. cerevisiae Fab1p and Mss4p. A, schematic representation of the proteins. Homologies among PIP5KII, Mss4p, and Fab1p are shown by shading. B, comparison of the amino acid sequences within the PtdIns(4)P kinase homology domain in the full-length human placental isoform of PIP5KII, Mss4p (residues 330-779), and Fab1p (residues 1900-2278). The alignment was performed using the PileUp program (University of Wisconsin Genetics Computer Group, Madison, WI). Conserved residues are in boldface.
The glycine-rich motif of the homologues at residues 149-155 (Fig. 4B) resembles the phosphate-binding loop of protein kinases, heterotrimeric G-proteins, RecA, elongation factor Tu, adenylate kinase, and H-ras(30) . It is also similar to the GTP-binding sequences in dynamin, Mx1, and Vps1p(31) . Since PIP5Ks can use ATP and GTP as phosphate donors(16) , this homology is consistent. Other conserved regions have no clear homology to kinases.
The S. cerevisiaeFAB1 gene was isolated based on the fab1 mutant phenotype, in which cells have profoundly enlarged
vacuoles that are not acidified. These mutants also have
defective chromosomal segregation and abnormal spindle morphology. A
chromosomal segregation defect is also observed upon mutation of the S. cerevisiaePLC1 gene, which encodes a
phosphoinositide-specific phospholipase
C(36, 37, 38) . However, there is no reported
phenotypic changes of the vacuoles in PLC1 mutants. The
presence of the PLC1 gene in S. cerevisiae suggests
that MSS4 and FAB1 may be required for second
messenger production (36, 37, 38) . MSS4 and FAB1 could also regulate other events, such as
cytoskeletal assembly, by an interaction of PtdIns(4,5)P
with the Pfy1p profilin homologue (22) or other
proteins or enzymes that have not yet been described.
The mammalian
PIP5KI and PIP5KII isoforms are structurally and apparently
functionally
distinct(16, 17, 18, 19, 20) . The different phenotypes associated with mutations of the yeast
PIP5K homologues and the fact that MSS4 is essential, whereas FAB1 is not, suggest that these proteins in S. cerevisiae have diverse cellular roles, similar to the PtdIns 3- and
4-kinases. If this hypothesis is correct, then the PIP5K family of
enzymes will likely be as large and diverse as the PtdIns 3- and
4-kinase families. The most striking feature of this PIP5K is its lack
of sequence homology to known protein or PtdIns kinases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14957[GenBank].
Note Added in Proof-The product of PIP kinase reaction with recombinant enzyme was unambiguously shown to be PtdIns(4,5)Pz by alkaline hydrolysis, followed by HPLC analysis.