Type I Phosphatidylinositol-4-phosphate 5-Kinases
CLONING OF THE THIRD ISOFORM AND DELETION/SUBSTITUTION ANALYSIS OF MEMBERS OF THIS NOVEL LIPID KINASE FAMILY*

Hisamitsu IshiharaDagger §, Yoshikazu ShibasakiDagger , Nobuaki Kizuki, Takako Wada, Yoshio YazakiDagger , Tomoichiro AsanoDagger , and Yoshitomo Oka

From the Dagger  Third Department of Internal Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, and the  Third Department of Internal Medicine, Yamaguchi University School of Medicine, Kogushi, Ube, Yamaguchi 755, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma ) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha  and beta  isoforms. Northern blot analysis revealed that the gamma  isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta  isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma  isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta  and gamma  isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha  isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta  or the gamma  isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Recent advances in cell biology have revealed that phosphoinositide metabolism plays an essential role in various cellular processes. Synthesis and breakdown of certain phosphoinositides at appropriate times and intracellular sites appear to be required for complex regulation of these cellular processes. One of the phosphoinositides, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2),1 is located at an important branchpoint in phosphoinositide metabolism. PtdIns(4,5)P2 serves as a substrate for phosphoinositide-specific phospholipase C (EC 3.1.4.11), generating the second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (1). PtdIns(4,5)P2 can also be phosphorylated by phosphoinositide 3-kinase (EC 2.7.1.137), generating phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), the synthesis of which is activated in signaling pathways of several growth factor receptors (2). Furthermore, PtdIns(4,5)P2 modulates the activity of numerous enzymes, including actin-binding proteins (3), binds pleckstrin homology domains (4-6), and has been suggested to play a role in exocytosis (7-9). The major pathway of PtdIns(4,5)P2 synthesis is that mediated by type I phosphatidylinositol-4-phosphate 5-kinases (PIP5K; EC 2.7.1.68), phosphorylating the D-5 position of the inositol ring of phosphatidylinositol 4-phosphate (PtdIns(4)P). Despite these important functions of PtdIns(4,5)P2, direct investigations of the intracellular roles and of mechanisms regulating synthesis of this lipid molecule are limited. Until recently, a major factor hindering progress in this field was the absence of molecular tools.

Recently, cDNAs encoding two isoforms of type I PIP5K have been cloned (10, 11). Herein, we report molecular cloning of a third isoform of type I PIP5K (PIP5Kgamma ) from a cDNA library of the murine pancreatic beta -cell line MIN6 (12). This novel isoform has two alternative splicing forms of 87 and 90 kDa and is the most markedly stimulated by phosphatidic acid of the three isoforms. These molecular identifications revealed that PIP5K isoforms constitute a novel lipid kinase family, distinct from phosphoinositide 3-kinases, phosphatidylinositol 4-kinases, and diacylglycerol kinases. Demonstration of structural characteristics is essential for understanding the intracellular roles of these isoforms and the mechanisms by which they are regulated. Therefore, in this report, several aspects of the structural characteristics of these isoforms were also studied in vitro and in vivo. We found that a central region, consisting of approximately 380 amino acids, is sufficient for PtdIns(4)P kinase activity and that carboxyl-terminal regions are important for modulation of the kinase activities of these isoforms. We also found that expression of either the beta  or the gamma  isoform leads to actin rearrangement in COS7 cells, as was the case with the alpha  isoform (13), and that the central region is sufficient for this effect. Furthermore, surprisingly, the expression of a kinase-deficient substitution mutant generated a similar effect in COS7 cells.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of Murine Type I PIP5Kgamma -- A polymerase chain reaction (PCR) using degenerate oligonucleotide primers and screening of a MIN6 cell cDNA library were as described previously (10). To obtain a 5' sequence of PIP5Kgamma , a MarathonTM cDNA amplification kit (CLONTECH) was used according to the instructions of the manufacturer. The reverse transcription was performed using MIN6 cell poly(A)+ RNA and an antisense primer 5'-GGTGACGTAGAAGACAGAGCC-3'. The first PCR was performed using adapter primer 1 (CLONTECH) and an antisense primer, 5'-CTTCACTGGGGAAGAAGA TGC-3'. The second PCR was performed using adapter primer 2 (CLONTECH) and an antisense primer, 5'-GTGGCCCAGCTTCTTCCCATG-3'. The first and second PCR reactions were conducted with inclusion of dimethyl sulfoxide (5%), without which only shorter products were obtained. Individual clones were sequenced following subcloning into pGEM-T vector (Promega, WI) as described above. The consensus of three independent clones confirms the sequence of the 5' region of PIP5Kgamma .

Northern Blotting-- A murine multiple tissue Northern blot (CLONTECH) was hybridized according to the instructions of the manufacturer with an [alpha -32P]dCTP-labeled 0.4-kb Aor51HI-PstI fragment from the 3' portion of the gamma  isoform cDNA.

Production of Antibody Specific to the gamma  Isoform and Western Blotting-- An oligopeptide, CASDEEDAPSTDIYF, was custom synthesized and conjugated to keyhole limpet hemocyanin (Research Genetics, AL) and injected into female New Zealand rabbits employing standard protocols (14). The COS7 cell lysates (10 µg/lane) and murine brain lysate (50 µg/lane) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (7.5%) and then probed with the antisera raised against the above peptide (1:100 dilution). Blots were developed using ECL reagents (Amersham, UK).

Epitope Tagging and Expression of PIP5K Isoforms by Recombinant Adenoviruses-- Epitope (influenza virus hemagglutinin (HA))-tagged cDNAs were generated as described previously (10). Recombinant adenoviruses bearing the cDNA of PIP5K isoforms with or without the HA tag were constructed as described previously (15-17). COS7 cells (1.5 × 106 cells) maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum were infected with recombinant adenoviruses (3-5 × 107 plaque-forming units) as described previously (17). Three days later, cells were lysed with 1 ml of lysis buffer (50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EGTA, 15 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) and used for Western blotting and immunoprecipitation.

Constructions of Mutant cDNAs-- Substitution mutant cDNAs were constructed by oligonucleotide-directed mutagenesis. For example, the PIP5Kalpha -K138A mutant cDNA was generated using primers 5'-ATGAATTCATCATCGCAACCGTTCAG-3' (underlined nucleotides encode a mutated alanine) and 5'-CTCCTGACTGCATGCAATACAGC-3'. The amino- and carboxyl-terminal deletion mutants were generated by using either endogenous restriction enzyme sites or a PCR-based strategy. Endonuclease BamHI, EcoRI, or NcoI digestion, followed by subcloning into the SwaI site of a cosmid vector (pAdex1CA) (16) generated mutants containing amino acids 1-456 with extra asparagine, 1-392, or 1-308 with extra lysine-leucine-isoleucine-lysine-leucine-valine, due to a poly-linker sequence of the vector, respectively. Amino-terminal deletion mutants were generated using an inner antisense primer, 5'-CTCCTGACTGCATGCAATACAGC-3', and an appropriate sense primer containing a SalI site for connection with a sequence for the HA epitope. Carboxyl-terminal mutants were generated using an inner sense primer, 5'-CTCTATTCCACAGCCATGGAATCC-3', and an appropriate antisense primer containing a stop codon and a BamHI site. The mutant cDNAs, confirmed by DNA sequencing, were subcloned into pBluescript containing a sequence coding the HA-epitope.

PtdIns(4)P Kinase Assay-- Immunoprecipitation was performed using a monoclonal antibody against the HA epitope (12CA5) and protein G-Sepharose 4 First Flow (Pharmacia Biotech Inc.). The immunoprecipitates were used for the PtdIns(4)P kinase assay. A standard assay for phosphorylation of PtdIns(4)P was carried out in an incubation medium containing a final concentration of 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 1 mM EGTA, 100 µM PtdIns(4)P, and 50 µM [gamma -32P]ATP (5 µCi/tube). For determination of the Km and the Vmax for PtdIns(4)P, concentrations of 10, 30, 100, and 200 µM were used with ATP at 25 µM. For determination of the Km for ATP, concentrations of 5, 10, 30, and 100 µM were used with PtdIns(4)P at 100 µM. To investigate the effects of phosphatidic acid, Triton X-100TM was added at a final concentration of 0.1%. The phosphorylation reaction was stopped by adding 20 µl of 8 M HCl and 160 µl of chloroform:methanol (1:1). Lipids were separated by developing thin layer chromatography plates (Silica gel 60, Merck) in chloroform:methanol:15 M ammonium hydroxide:water (90:90:7:22).

For comparison of kinase activities among various constructs (see Table I and Figs. 4, 5, and 6), half an aliquot of the immunoprecipitate was used for triplicate kinase activity assay and the other half for Western blotting with the rabbit anti-HA-epitope IgG (MBL, Nagoya) and 125I-labeled Protein A, by which the protein amount in the immunoprecipitate was estimated. Kinase activities were normalized with wild-type or mutant protein amounts. The signal intensities were measured with a BAS 2000 (Fuji Photo Film, Tokyo).

Immunofluorescence-- COS7 cells were plated on coverlips in Dulbecco's modified Eagle's medium with 10% fetal calf serum and infected with recombinant adenoviruses the next day. After 18 h, cells were fixed with 3% paraformaldehyde and incubated with anti-HA monoclonal antibody (12CA5) in phosphate-buffered saline with 0.2% gelatin at room temperature for 45 min. After washing three times with phosphate-buffered saline-gelatin, cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (DAKO) and rhodamine-conjugated phalloidin (Molecular Probes) for 30 min. Slides were observed under a Bio-Rad confocal microscope system (MRC 1024).

Materials-- PtdIns(4)P from bovine brain and phosphatidic acid were purchased from Sigma. Oligonucleotides were custom synthesized and purchased from either Japan Bio-service Inc. (Saitama, Japan) or Becks Inc. (Itabashi, Tokyo).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of the Third Member of Type I PIP5K Family-- A polymerase chain reaction (PCR) using degenerated primers and subsequent screening of a MIN6 cell cDNA library, as described previously (10), identified a class of partial coding sequences with homology to type I PIP5K alpha  and beta  isoforms. The third cDNA sequence, designated PIP5Kgamma , contains four overlapping clones, one of which has an additional 78-base pair sequence at the 3' terminus and possibly arises by alternative splicing of the PIP5Kgamma gene. In-frame stop codons upstream from the first ATG codon of this cDNA could not be identified in the initial study. Therefore, to obtain an additional 5' sequence, an adapter ligation/PCR-based method (MarathonTM, CLONTECH) was employed. Although an additional 172 base pairs and another ATG codon were obtained, there were no in-frame stop codons in a 79-base pair sequence upstream from this ATG codon. Nonetheless, this ATG codon was concluded to be the initial translation codon for the following reasons. First, the ATG codon is in a favorable position for translation according to Kozak's rules (Fig. 1, Ref. 19). Second, as shown in Fig. 2A, recombinant proteins of the gamma  isoform with or without the 26 carboxyl-terminal amino acids expressed via adenoviral vectors migrated almost identically to either of the doublet bands (87 and 90 kDa) from brain tissue on SDS-PAGE. The gamma  isoform has two alternative splicing forms, consisting of 635 and 661 amino acids with calculated molecular masses of 69,563 and 72,469 Da, and isoelectric points of 5.40 and 5.27, respectively (Fig. 1). Because the 87-kDa protein was predominantly expressed in brain tissue (Fig. 2A) and MIN6 cells (data not shown), the gamma  isoform without the 26 carboxyl-terminal amino acids was used in subsequent analyses. As shown in Fig. 3, the central portions of the three type I isoforms were found to be very similar (approximately 80% identity) in amino acid sequence. In addition, the amino-terminal sequence of the type gamma  isoform shows partial homology with that of the beta  isoform (approximately 40% identity) whereas the carboxyl-terminal regions differed in length and amino acid sequence among the three isoforms. An entire coding sequence of murine cDNA homologous to human PIP5KIIalpha (20, 21), which was recently revealed to be phosphatidylinositol 5-phosphate (PtdIns(5)P) 4-kinase (22), was also cloned from a MIN6 cell cDNA library (data not shown). The murine PIP5KIIalpha consists of 405 amino acids, one residue less than its human counterpart, with only seven conserved amino acids differing between the two (Fig. 3).


View larger version (89K):
[in this window]
[in a new window]
 
Fig. 1.   Sequence of murine type I PIP5Kgamma . The single-letter codes for the deduced amino acid sequence are indicated below the nucleotide sequence. Underlined nucleotide sequence may result from alternative splicing of the PIP5Kgamma gene.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 2.   Novel cDNA encodes 90/87-kDa type I PtdIns(4)P kinase with abundant expression in brain tissue. A, COS7 cells were infected with recombinant adenoviruses containing cDNAs for PIP5K-Igamma (635) or PIP5K-Igamma (661) without the HA-epitope sequence. The lysates from murine brain (50 µg/lane) or infected COS7 cells (10 µg/lane) were subjected to SDS-PAGE (7.5%). Expressed proteins were probed with anti-PIP5Kgamma antisera. B, a mouse multiple tissue Northern blot (CLONTECH) was hybridized with specific probes for the gamma  isoform. Sk. mus., skeletal muscle. C, autoradiogram demonstrating the novel isoform to contain PtdIns(4)P kinase activity. Lysates of COS7 cells infected with a recombinant adenovirus encoding HA-tagged PIP5Kgamma or a control virus (lacZ) were subjected to immunoprecipitation with anti-HA monoclonal antibody. The immunocomplex was assayed for PtdIns(4)P kinase activity as described under "Experimental Procedures." D, PtdIns(4)P kinase activity of the gamma  isoform is markedly stimulated by phosphatidic acid (PA). Lipid kinase reaction was performed using PtdIns(4)P as a substrate (100 µM) in the presence and absence of PA (100 µM).


View larger version (91K):
[in this window]
[in a new window]
 
Fig. 3.   Alignment of three type I PIP5K isoforms and type II PIP5Kalpha . A, schematic representation of murine PIP5K isoforms. Kinase core domains were determined by analyses shown in Figs. 4 and 6. B, alignment of PIP5Ks. Identical residues among three or four enzymes are boxed in black. Gaps are represented by dashes.

Tissue Distribution of Type I PIP5Kgamma -- Northern blotting analysis was performed using the gamma  isoform cDNA probe corresponding to the sequence close to the termination codon. A 4.8-kilobase mRNA was detected, as a major band, in murine poly(A)+ RNA from different tissues (Fig. 2B). The tissue distribution of the gamma  isoform differed from those of the alpha  and beta  isoforms, being essentially restricted to the brain, lung, and kidneys.

The Novel cDNA Encodes Type I PIP5K Protein-- To characterize the enzymatic activity of the third isoform, HA-tagged proteins of this isoform expressed in COS7 cells were immunoprecipitated using anti-HA-epitope monoclonal antibody 12CA5. The resulting immunocomplex exhibited PtdIns(4)P kinase activity (Fig. 2C). Although this thin layer chromatography separation did not provide information about whether the PtdInsP2 produced was PtdIns(4,5)P2 or PtdIns(3,4)P2, the close sequence similarity with the alpha  and beta  isoforms (Fig. 3) strongly suggests that the gamma  isoform is also a 5-kinase. Furthermore, the PtdIns(4)P kinase activities of the third isoform increased by more than 10-fold when an equimolar amount of phosphatidic acid was added to the reaction solutions (Fig. 2D), demonstrating the novel murine cDNA to encode the type I PtdIns(4)P 5-kinase (23, 24).

Comparison of Kinetic Activities of Type I PIP5K Isoforms-- For initial characterization of members of the PIP5K family, kinetic parameters for the PtdIns(4)P kinase activity of these murine isoforms were studied. For this purpose, recombinant proteins of isoforms with the HA epitope were expressed and immunoprecipitated with the anti-HA-epitope monoclonal antibody. One-half of each immunoprecipitate was used for kinase assay and the other half for Western blotting with rabbit anti-HA-epitope polyclonal IgG. Lipid kinase activity was normalized with the protein amount estimated by Western blotting (for example, see Fig. 5B). Kinetic parameters for these isoforms are summarized in Table I. While affinities for PtdIns(4)P and ATP were similar among the three isoforms, the beta  isoform had the greatest Vmax value, approximately 3.2-fold and 1.7-fold higher than those of the alpha  and gamma  isoforms, respectively. Study of phosphatidic acid sensitivity revealed the gamma  isoform to be most sensitive to phosphatidic acid.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetics of type I PIP5K isoenzymes
The relative Vmax for PtdIns(4)P kinase were obtained after normalization of the Vmax values with protein amounts estimated by Western blotting. The normalized Vmax value of the alpha  isoform was taken as 1.0. Km for PtdIns(4)P was measured at a ATP concentration of 25 µM and varying concentrations of PtdIns(4)P. Km for ATP was measured at a PtdIns(4)P concentration of 100 µM and varying concentrations of ATP. Phosphatidic acid (PA) sensitivity was measured in the presence of 100 µM of PA. Data are presented as means ± S.E. of five independent experiments each performed in triplicate.

Deletion Analysis of the Type I PIP5Kalpha Defines a Central Kinase Domain-- Because type I PIP5K isoforms have no sequence homology with other lipid kinases including phosphoinositide 3-kinases, phosphatidylinositol 4-kinases and diacylglycerol kinases (25, 26), it is of great importance to determine their structure and function relationships. To begin to address this issue, amino- or carboxyl-terminal deletion mutants of the type I PIP5Kalpha isoform were constructed using endogenous restriction enzyme sites and PCR-based methods. As summarized in Fig. 4, while all four mutants with stepwise deletions from the carboxyl terminus to glutamine residue 400 have activity almost equal to that of the wild-type alpha  isoform, the PIP5Kalpha -(1-392)/EcoRI mutant has little or no PtdIns(4) 5-kinase activity. In contrast to the long dispensable region in the carboxyl terminus, amino-terminal deletions had a pronounced effect. Although the first 17-amino acid deletion did not alter kinase activity, deletion of only 31 amino acids from the amino-terminal region resulted in significantly reduced kinase activity (34 ± 9% of the full-length alpha  isoform, mean ± S.E., n = 3), and a 46-amino acid deletion virtually abolished kinase activity.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4.   Deletion mutants of the type I PIP5Kalpha isoform. A, HA-tagged mutant proteins were immunoprecipitated from lysates of COS7 cells infected with recombinant adenoviruses, subjected to SDS-PAGE and blotted with anti-HA polyclonal antibody. B, deletion mutants are schematically represented with their relative kinase activities at 200 PtdIns(4)P and with their effects on actin reorganization. Effects on actin reorganization are represented as + when more than 70% cells expressing one of deletion mutants exhibited an increase in short actin fibers and a decrease in stress fibers, as ± when 10-70%, and - when less than 10% (for typical cells, see Fig. 7).

These data suggest that an approximately 380 amino acid central portion of the PIP5Kalpha isoform (amino acid residues 18 to 399) constitutes the kinase core domain. Indeed, as described below, this central portion alone retains kinase activity (see Fig. 6B). This region of the PIP5Kalpha isoform has about 80% amino acid identity with the corresponding regions of both the beta  and the gamma  isoform. The amino-terminal half of the kinase core domain is especially conserved among the three isoforms (more than 90%) (Fig. 3). In addition, sequence alignment between type I PIP5Ks and PIP5KIIalpha suggests that the latter enzyme (PtdIns(5)P 4-kinase) consists essentially of the kinase region of type I PIP5Ks with approximately 40% identity (Fig. 3).

Mutations in the Putative Nucleotide Binding Region-- In several protein and lipid kinases, a glycine-rich sequence followed by a lysine residue 10-30 residues downstream constitutes an important region for phosphate-transfer reactions (25, 27). Although there is no typical region for such a glycine-rich sequence, the region of amino acid residues 121 to 138 in the alpha  isoform is similar to the ATP binding domain of cyclic AMP-dependent protein kinase (PKA, Fig. 5A). To examine the role of this region, two alpha  isoform mutants were constructed in which glycine 124 was substituted with valine (PIP5Kalpha -G124V mutant) or lysine 138 with alanine (PIP5Kalpha -K138A mutant). A lipid kinase assay revealed type I PIP5Kalpha -G124V to have 67 ± 11% of the PtdIns(4)P kinase activity of the wild-type protein, while type I PIP5Kalpha -K138A had virtually no kinase activity (Fig. 5B).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   Role of the putative ATP binding domain of type I PIP5Kalpha . A, lipid and protein kinase sequences are aligned around lysine 72 of PKA. PI3K p110alpha , catalytic subunit of bovine phosphoinositide 3-kinase (35); PI4K 230kDa, rat 230-kDa phosphatidylinositol 4-kinase (36); DGK, human diacylglycerol kinase zeta  (37); INS-R, human insulin receptor (38); amino acid numbers of (putative) ATP binding lysine residues (bold letters) are indicated. B, the effects of mutations in the putative ATP binding domain of PIP5Kalpha . Kinase activities were normalized with the amounts of immunoprecipitated enzymes estimated by Western blotting. PtdIns(4)P kinase activity of the wild-type alpha  isoform was taken as 100%. Data are presented as means ± S.E. of three independent experiments, each performed in triplicate.

Role of Amino- and Carboxyl-terminal Variable Regions of Type I PIP5Ks-- As indicated above, these type I PIP5K isoforms consist of a similar central domain and variable amino- and carboxyl-terminal regions. To characterize these domains, amino- and/or carboxyl-terminal deletion mutants of the three isoforms, carrying the HA epitope, were constructed based on the results of deletion analysis of the alpha  isoform (Fig. 6A). As summarized in Fig. 6B, the central regions of the three isoforms showed essentially equivalent lipid kinase activities. There were no marked changes when amino- and/or carboxyl-terminal regions were deleted from the alpha  isoform. As shown in Table I, the beta  isoform had approximately three-fold higher activity than the alpha  isoform. When the carboxyl-terminal region was deleted from the beta  isoform, the activity was reduced to a level approaching that of the alpha  isoform. In addition, a deletion of the carboxyl-terminal region of the gamma  isoform also resulted in a reduction in its kinase activity. The phosphatidic acid sensitivities of these deletion mutants were also examined (Fig. 6C). The central regions alone of the three isoforms can be stimulated by phosphatidic acid. Although the gamma  isoform showed the highest sensitivity to phosphatidic acid, its carboxyl-terminal deletion mutant exhibited a magnitude of phosphatidic acid stimulation similar to that of the alpha  isoform.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Roles of amino- and/or carboxyl-terminal regions of the three type I PIP5Ks. A, schematic representation of amino- and/or carboxyl-terminal deletion mutants of the type I PIP5K isoforms. B, PtdIns(4)P kinase activities of deletion mutants. Assays were performed using immunoprecipitated wild-type and mutant enzymes expressed in COS7 cells at a PtdIns(4)P concentration of 200 µM and ATP at 50 µM. Kinase activities were normalized with the amounts of immunoprecipitated enzymes estimated by Western blotting. PtdIns(4)P kinase activity of the wild-type alpha  isoform was taken as 1.0. Data are presented as means ± S.E. of at least five independent experiments, each performed in triplicate. *, Difference from the relevant wild-type isoform at p < 0.05. C, stimulation of the PtdIns(4)P kinase activity of deletion mutants by phosphatidic acid. Assays were performed using immunoprecipitated wild-type and mutant enzymes at a PtdIns(4)P concentration of 100 µM and 50 µM ATP in the absence or presence of 100 µM phosphatidic acid. Data are presented as means ± S.E. of five independent experiments, each performed in triplicate. *, difference from the relevant wild-type isoform at p < 0.05.

Effects on Actin Polymerization of Overexpressing PIP5K Isoforms and Their Mutants in COS7 Cells-- As reported previously, overexpression of type I PIP5Kalpha via an adenoviral vector led to a decrease in typical stress fibers and an increase in disarrayed short actin fibers (13). In this study, we examined the effects of overexpressing three type I PIP5K isoforms, type II PIP5Kalpha , and their mutants on actin polymerization (Fig. 7). Abnormal reorganization of actin fibers was also observed in COS7 cells overexpressing either the beta  or the gamma  isoform (Fig. 7, B and C). These in vivo analyses using deletion mutants of the alpha  isoform are summarized in Fig. 4. All deletion mutants of the alpha  isoform with the complete kinase core domain induced abnormal actin reorganization in COS7 cells. In addition, COS7 cells expressing Ialpha -dNdC, Ibeta -dNdC, or Igamma -dNdC, mutants in which both amino- and carboxyl-terminals are deleted from the three isoforms (Fig. 6A), had enormous amounts of short actin fibers and relatively few stress fibers (data not shown). In contrast, PIP5Kalpha -(1-392)/EcoRI and PIP5Kalpha -(47-539), mutants with small deletions at the carboxyl- and amino-terminals, respectively, of the kinase core domain failed to induce abnormal actin rearrangement (Fig. 7E). All other deletion mutants with the incomplete kinase core domain (Fig. 4) and type II PIP5Kalpha (data not shown) exhibited behaviors similar to those of PIP5Kalpha -(1-392)/EcoRI and PIP5Kalpha -(47-539).


View larger version (96K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of expression of type I PIP5Ks and their mutants on actin polymerization. COS7 cells infected with recombinant adenoviruses were maintained in the presence of 10% fetal calf serum, then fixed and stained with anti-HA antibody followed by fluorescein isothiocyanate-conjugated anti-mouse IgG, to confirm expression of infected constructs (data not shown), and rhodamine-phalloidin to visualize polymerized actin. A, control (lacZ expressing); B, PIP5Kbeta (wild); C, PIP5Kgamma (wild); D, PIP5Kalpha (wild); E, PIP5Kalpha -(1-392)/EcoRI; F, PIP5Kalpha -K138A. Bar, 10 µm.

These data appeared to indicate that kinase activity of PIP5K would be necessary for abnormal actin polymerization. However, surprisingly, expression of a kinase-deficient substitution mutant, PIP5Kalpha -K138A, also led to a decrease in typical stress fibers and an increase in short actin fibers (Fig. 7F). In addition, as was the case with the wild-type alpha  isoform (13), COS7 cells expressing the kinase defective substitution mutant also exhibited decreased adhesion activity. They became rounded and readily detached from the bottoms of culture dishes (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A novel isoform of PIP5K was identified in this study. This novel isoform (PIP5Kgamma ) migrated at 90/87 kDa on SDS-PAGE, suggesting this isoform to be identical or closely related to the type Ib isoform (90 kDa) previously purified (23). The co-existence of three isoforms of the type I PIP5K in insulin-secreting clonal cells suggests that these isoforms have specific functions in vivo. Since the 90-kDa isoform has been reported to have higher activity than the 68-kDa isoform in restoring Ca2+-regulated catecholamine release from cytosol-depleted neuroendocrine cells (7), it appears likely that the gamma  isoform plays an important role in regulated secretion. Type I PIP5K and phospholipase D are postulated to be involved in exocytotic processes (8). Stimulation of PtdIns(4,5)P2 synthesis by phosphatidic acid, which may be generated by phospholipase D, was considered to be important in this process (8). The highest sensitivity to phosphatidic acid of the PIP5Kgamma isoform might be a reason for the 90-kDa isoform playing a more active role in Ca2+-regulated secretion (7). More specific expression of the gamma isoform in the brain may reflect an important role of this isoform in neurotransmitter release.

Molecular cloning of type I PIP5K isoforms allows study of the structure-function relationships of these important enzymes. Recent studies revealed that type I PIP5K isoforms can phosphorylate several lipid substrates other than PtdIns(4)P (28). In this study, we studied the structure-function relationships with regard to PtdIns(4)P 5-kinase activity since PtdIns(4)P is the preferred substrate of type I PIP5K isoforms (28). The minimal kinase core domain of the type I PIP5K isoforms was determined to be an approximately 380-amino acid region. The amino acid sequence in this region does not, however, contain typical sequences homologous to known protein or lipid kinase domains, the exception being one which exhibits weak homology with a phosphate binding loop of PKA. Almost complete loss of PtdIns(4)P kinase activity by substitution of lysine 138 with alanine suggests that this lysine residue plays a role similar to lysine 72 in PKA, which was proposed to interact with the alpha - and beta -phosphate groups of ATP (29). It was also found that the amino-terminal half of the kinase core domain is highly conserved among type I PIP5K isoforms. Especially, in the region spanning residue 80 to 161 of the alpha  isoform, 98% of 82 residues are identical or conserved among three isoforms. In addition to the putative nucleotide binding domain, there may be domains essential for lipid kinase activities in this region.

Among the three type I isoforms, the beta  isoform has the greatest Vmax for PtdIns(4)P kinase activity while the gamma  isoform is most markedly stimulated by phosphatidic acid. Our results using deletion mutants indicate an important role of the carboxyl-terminal regions for these characteristics. Since the type I PIP5K isoforms have recently been reported to phosphorylate PtdIns(3)P and PtdIns(3,4)P2 (28), it would be intriguing to examine whether amino- and/or carboxyl-terminal sequences are involved in recognition of these different substrates. In addition, these regions might be important for possible associations with other unknown molecules. Further studies are needed to elucidate roles of these amino- and carboxyl-terminal variable regions.

We also found that overexpression of any one of the three isoforms led to the production of massive amounts of short actin fibers while disrupting actin stress fibers in COS7 cells. A surprising result was that a kinase defective mutant, PIP5Kalpha -K138A, induces similar effects. The mechanism by which PIP5K isoforms and the kinase-deficient substitution mutant induce such effects remains to be determined. The causal relationship between short actin fiber formation and disruption of actin stress fibers is also unclear. It has been reported that expression of PtdIns(4,5)P2 5-phosphatases in COS7 cells decreased the number of actin stress fibers via the hydrolysis of PtdIns(4,5)P2 bound to actin regulatory proteins (13, 30). An opposite mechanism (i.e. via an increase in PtdIns(4,5)P2) is unlikely to lead an increase in short actin fibers in cells overexpressing 5-kinases since the kinase-deficient mutant induced a similar effect, although the possibility of endogenous 5-kinase activity playing some part in the effect cannot be ruled out. Indeed, it was reported that overexpression of type I PIP5K isoforms in COS7 cells did not increase cellular levels of PtdInsP2 (31), providing evidence that effects on actin reorganization of overexpressing PIP5Ks were not mediated by the kinase activity of overexpressed proteins. The fact that the kinase inactive mutant induces actin reorganization similar to that seen with the wild-type enzyme suggests that structural interactions with other as yet unknown molecules mediate this effect. Small GTP binding proteins, Rac and Rho, are possible candidates (32-34). In this regard, it should be noted that the structure of the kinase core domain was found to be sufficient for inducing abnormal actin polymerization. There may be a binding site for such an interacting molecule within the kinase core domain. Future studies should be designed to identify the molecules interacting with PIP5K isoforms.

Recent findings suggest that PIP5Ks play various roles in signaling pathways, by participating in the synthesis of a number of phosphoinositides (28). The present results suggest that structural interactions are also important in PIP5Ks functions. Much research remains to be done in order to elucidate the complex signaling pathways in which these lipid kinases are involved.

    FOOTNOTES

* This work was supported in part by Grant-in Aid for Scientific Research (A) 09357009 (to Y. O.) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from Uehara Memorial Foundation (to Y. O.).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 DDBJ, EBI, and GenBankTM Data Bank with accession number(s) AB006916 and AB009615.

§ To whom correspondence should be addressed: Third Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3815-5411 (ext. 3121); Fax: 81-3-5803-1874; E-mail: ishihara-tky{at}umin.u-tokyo.ac.jp.

1 The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HA, hemagglutinin; PKA, cyclic AMP-dependent protein kinase.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-204[CrossRef][Medline] [Order article via Infotrieve]
  2. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
  3. Janmey, P. A. (1994) Annu. Rev. Physiol. 56, 169-191[CrossRef][Medline] [Order article via Infotrieve]
  4. Harlan, J. H., Hajuk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Nature 371, 168-170[CrossRef][Medline] [Order article via Infotrieve]
  5. Salim, K., Bottomley, M., J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. L., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250[Abstract]
  6. Rameh, L. E., Arvidsson, A., Carraway III, K. L., Couvillon, A. D., Rathbun, G., Crompton, A., VanRenterghem, B., Czech, M. P., Ravichandran, K. S., Burakoff, S. J., Wang, D.-S., Chen, C.-S., and Cantley, L. C. (1997) J. Biol. Chem. 272, 22059-22066[Abstract/Free Full Text]
  7. Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takenawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173-177[CrossRef][Medline] [Order article via Infotrieve]
  8. Liscovitch, M., and Cantley, L. C. (1995) Cell 81, 659-662[Medline] [Order article via Infotrieve]
  9. Fensome, A., Cunnungham, E., Prosser, S., Tan, S. K., Swigart, P., Thomas, G., Hsuan, J., and Cockcroft, S. (1996) Curr. Biol. 6, 730-738[Medline] [Order article via Infotrieve]
  10. Ishihara, H., Shibasaki, Y., Kizuki, N., Katagiri, H., Yazaki, Y., Asano, T., and Oka, Y. (1996) J. Biol. Chem. 271, 23611-23614[Abstract/Free Full Text]
  11. Loijens, J. C., and Anderson, R. A. (1996) J. Biol. Chem. 271, 32937-32943[Abstract/Free Full Text]
  12. Ishihara, H., Asano, T., Tsukuda, K., Katagiri, H., Inukai, K., Anai, M., Kikuchi, M., Yazaki, Y., Miyazaki, J.-I., and Oka, Y. (1993) Diabetologia 36, 1139-1145[Medline] [Order article via Infotrieve]
  13. Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., and Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581[Abstract/Free Full Text]
  14. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  15. Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai, M., Sakaki, T., Sugano, S., and Saito, I. (1995) Nucleic Acids Res. 23, 3816-3821[Abstract]
  16. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1320-1324[Abstract/Free Full Text]
  17. Niwa, H., Yamamura, K., and Miyazaki, J.-I. (1991) Gene 108, 193-200[CrossRef][Medline] [Order article via Infotrieve]
  18. Ishihara, H., Nakazaki, M., Kanegae, Y., Inukai, K., Asano, T., Katagiri, H., Yazaki, Y., Kikuchi, M., Miyazaki, J.-I., Saito, I., and Oka, Y. (1996) Diabetes 45, 1238-1244[Abstract]
  19. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148[Abstract]
  20. Boronenkov, I. V., and Anderson, R. A. (1995) J. Biol. Chem. 270, 2881-2884[Abstract/Free Full Text]
  21. Divecha, N., Truong, O., Hsuan, J. J., Hinchliffe, A. K., and Irvine, R. F. (1995) Biochem. J. 309, 715-719[Medline] [Order article via Infotrieve]
  22. Rameh, L. E., Tolias, K. F., Duckworth, B. C., and Cantley, L. C. (1997) Nature 390, 192-196[CrossRef][Medline] [Order article via Infotrieve]
  23. Jenkins, G. H., Fisette, P. L., and Anderson, R. A. (1994) J. Biol. Chem. 269, 11547-11554[Abstract/Free Full Text]
  24. Moritz, A., De Graan, P. N. E., Gispen, W. H., and Wirtz, K. W. A. (1992) J. Biol. Chem. 267, 7207-7210[Abstract/Free Full Text]
  25. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52[Medline] [Order article via Infotrieve]
  26. Hunter, T. (1995) Cell 83, 1-4[Medline] [Order article via Infotrieve]
  27. Saraeste, M., Sibbald, P. R., and Wittinghofer, A. (1990) Trends Biochem. Sci. 15, 430-434[CrossRef][Medline] [Order article via Infotrieve]
  28. Zhang, X., Loijens, J. C., Boronenkov, I. V., Parker, G. J., Norris, F. A., Chen, J., Thum, O., Prestwich, G. D., Majerus, P. W., and Anderson, R. A. (1997) J. Biol. Chem. 272, 17756-17761[Abstract/Free Full Text]
  29. Madhusudan, E., Trafny, A., Xuong, N. H., Adams, J. A., Ten Eyck, L. F., Taylor, S. S., and Sowadski, J. M. (1994) Protein Sci. 3, 176-187[Abstract/Free Full Text]
  30. Sakisaka, T., Itoh, T., Miura, K., and Takenawa, T. (1997) Mol. Cell. Biol. 17, 3841-3849[Abstract]
  31. Nathan Davis, J., Rock, C. O., Cheng, M., Watson, J. B., Ashmun, R. A., Kirk, H., Kay, R. J., and Roussel, M. F. (1997) Mol. Cell. Biol. 17, 7398-7406[Abstract]
  32. Chong, L. D., Traynor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507-513[Medline] [Order article via Infotrieve]
  33. Tolias, K. F., Cantley, L. C., and Carpenter, C. L. (1995) J. Biol. Chem. 270, 17656-17659[Abstract/Free Full Text]
  34. Ren, X-D., Bokoch, G. M., Traynor-Kaplan, A., Jenkins, G. H., Anderson, R. A., and Schwartz, M. A. (1996) Mol. Biol. Cell 7, 435-442[Abstract]
  35. Hiles, I. D., Otsu, M., Valinia, S., Fry, M. J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., Totty, N. F., Hsuan, J. J., Courtneudge, S. A., Parker, P. J., and Waterfield, M. D. (1992) Cell 70, 419-429[Medline] [Order article via Infotrieve]
  36. Nakagawa, T., Goto, K., and Kondo, K. (1996) J. Biol. Chem. 271, 12088-12094[Abstract/Free Full Text]
  37. Bunting, M., Tang, W., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1996) J. Biol. Chem. 271, 10230-10236[Abstract/Free Full Text]
  38. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzeli, L. M., Dull, T. J., Gray, A., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.