Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase.

Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic beta-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic beta-cell line MIN6. Two novel cDNAs, designated PI4P5K-Ialpha and PI4P5K-Ibeta, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.

fusion of secretory granules with the plasma membrane requires membrane proteins, v-SNARE 1 and t-SNARE. Also required are cytosolic proteins, including N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins (reviewed in Ref. 1). Recently, two other cytosolic protein components required for ATP-dependent priming for Ca 2ϩ -activated secretion have been identified: phosphatidylinositol transfer protein (2) and the type I phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) (3). PI4P5K produces, from phosphatidylinositol 4-phosphate (PtdIns(4)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), which has been demonstrated to be important in various cellular processes. PtdIns(4,5)P 2 acts as a substrate for phospholipase C, generating the major second messenger molecules, inositol 1,4,5-triphosphate and diacylglycerol (4). In addition, PtdIns-(4,5)P 2 itself has also been demonstrated to function as a regulator molecule in several cellular processes, including actin filament reorganization (5,6) and exocytosis (7). More recently, it has been postulated that PtdIns(4,5)P 2 functions in the fusion of intracellular vesicles with target membranes (8). Despite these important roles played by the type I PI4P5K, molecular identification of the enzyme has yet to be carried out.
Insulin secretion from pancreatic ␤-cells is one example of regulated exocytosis. Extensive studies have been conducted, which suggest the importance of the polyphosphoinositide synthesis pathway and the breakdown of its products by phospholipase C (9 -11). We recently demonstrated that insulin secretion requires an ATP-dependent priming process (12), suggesting that the type I PI4P5K plays a significant role in insulin secretion from pancreatic ␤-cells. We have therefore made efforts to identify PI4P5K-like proteins in insulin-secreting MIN6 cells (13). Here, we have identified two type I isoforms of PI4P5K from a cDNA library of the murine pancreatic ␤-cell line MIN6.

EXPERIMENTAL PROCEDURES
Screening of MIN6 Cell cDNA Library-Two blocks of six amino acids, (Y/F)DLKGS and MDYSL(L/V), were chosen for the synthesis of degenerate oligonucleotide primers. These two sequences are highly conserved among human type II or type C PI4P5K (14,15) and its yeast homologues, Mss4p (16) and Fab1p (17). The sequences of the sense primers were 5Ј-T  15 and served as a PCR template. DNA amplification was achieved by a 40-cycle PCR protocol with a 30-s denaturing step at 95°C, a 30-s annealing step at 40 or 44°C, and a 60-s extension step at 72°C. Reactions were performed using a mixture of sense primers S1 and S3 (2:1) or S2 and S4 (2:1), and a mixture of antisense primers A1, A2, A3, and A4 (4:2:2:1). The concentration of each mixture was 4 M. The amplified DNA was separated on a 2% agarose gel. The purified * This work was supported in part by Grant-in-aid for Scientific Research 06557056 (to Y. O.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 GenBank TM /EBI Data Bank with accession number(s) D86176 and D86177.
Production of Isoform-specific Antibodies and Western Blotting-Two oligopeptides CLDLQDDASVLDVYL and PSFSRRSGPSGNSC were custom synthesized and conjugated to keyhole limpet hemocyanin (Research Genetics, AL) and injected into female New Zealand rabbits employing standard protocols (21). The COS7 cell lysates (20 g/lane) were subjected to SDS-PAGE (10%) and were probed with these antisera (1:100 dilution) or anti HA-tag monoclonal antibody (12CA5, 1 g/ml). Blots were developed using ECL reagents (Amersham, United Kingdom).
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) and was used for the PtdIns(4)P kinase assay. Phosphorylation of PtdIns (4) To determine whether lipid products were PtdIns(4,5)P 2 or PtdIns(3,4)P 2 , the TLC methods described by Pignataro and Ascoli were utilized (22). PtdIns(3,4)P 2 was generated, using PtdIns(4)P as a substrate, with p85/p110 phosphatidylinositol 3-kinase that had been immunoprecipitated with anti-p85 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) from murine brain lysates. TLC plates (Whatman LK6D) were immersed in a solution containing 1.3% potassium oxalate and 2 mM EDTA for 30 min and dried overnight and then activated by heating at 110°C for 30 min just prior to use. The plates were developed in chloroform:methanol:ammonium hydroxide (15 N Northern Blotting-A murine multiple tissue Northern blot (Clontech) was hybridized according to the manufacturer's instructions with an [␣-32 P]dCTP-labeled 0.5-kilobase BamHI-Aor51HI fragment from the 3Ј portion of I␣ isoform cDNA. The blot was de-probed and reprobed with 0.54-kilobase HindIII-XhoI fragment from the 3Ј portion of I␤ isoform cDNA.

RESULTS
Cloning of Novel PI4P5K Isoforms-With primers prepared on the basis of the highly conserved amino acid sequences among human type II/type C PI4P5K (14,15) and its yeast homologues, Mss4p (16) and Fab1p (17), polymerase chain reactions were performed and subsequent screening of a cDNA library of the pancreatic ␤-cell line MIN6 identified, in addition to the type II PI4P5K, two classes of complete coding sequences with homology to type II/type C PI4P5K. These two coding sequences, referred to herein as PI4P5K-I␣ and PI4P5K-I␤, contained 1617 and 1638 nucleotide open reading frames, respectively, preceded by in-frame stop codons. PI4P5K-I␣ encodes a 539-amino acid protein with a predicted M r of 60,722 and PI4P5K-I␤ a 546-amino acid protein with a M r of 60,466 (Fig. 1). The central portion of these isoforms were found to be very similar (approximately 75% identity, when calculated without gaps), while the amino-and carboxyl-terminal regions differed markedly in length and amino acid sequence (Fig. 1). These cDNAs were then tagged with an epitope derived from influenza virus hemagglutinin (HA-tag) and expressed in COS7 cells via an adenoviral vector. Lysates from COS7 cells infected with a control virus or recombinant adenoviruses bearing these cDNAs were subjected to SDS-PAGE. The 68-kDa products were identified by the anti-HA antibody or by isoformspecific antisera in cells infected with recombinant adenoviruses bearing these cDNAs (Fig. 2).
Novel cDNAs Encode Type I PI4P5K Proteins-To characterize the enzymatic activity of these isoforms, HA-tagged proteins of the two isoforms expressed in COS7 cells were immunoprecipitated using anti-HA antibody. The resulting immunocomplexes exhibited PtdIns(4)P kinase activity (Fig.  3A). To clarify whether the observed PtdIns(4)P kinase activity was due to PtdIns(4)P 3-kinase or PtdIns(4)P 5-kinase activity, a TLC was conducted that separates PtdIns(4,5)P 2 from PtdIns(3,4)P 2 (22). As shown in Fig. 3B, products of the I␣ or the I␤ isoform using PtdIns(4)P as a substrate were distinct from PtdIns(3,4)P 2 , a product of p85/p110 phosphatidylinositol 3-kinase. In addition, the PtdIns(4)P kinase activity was inhibited by 70 to 80% with the addition of an equimolar amount of PtdIns(4,5)P 2 (data not shown). Furthermore, the PtdIns(4)P kinase activities of the I␣ and the I␤ isoform were increased by 11.0 Ϯ 2.1-fold and 10.2 Ϯ 2.2-fold, respectively, when an equimolar amount of phosphatidic acid (PA) was added to the reaction solutions (Fig. 3C). Because PA sensitivity is a major characteristic of the type I isoform (23,24), we concluded that these novel murine cDNAs encode the type I PtdIns(4)P 5-kinase.
Tissue Distribution of PI4P5K Isoforms-Northern blotting analysis was performed using cDNA probes corresponding to the sequences close to termination codons. The nucleotide sequences of these regions differed significantly between the two isoforms, allowing the detection of isoform-specific expression. A 3.3-or 4.2-kilobase mRNA was detected utilizing the I␣ or the I␤ isoform probe, respectively, in murine poly(A) ϩ RNA from different tissues (Fig. 4). Tissue distributions differed between the two isoforms. The I␣ isoform was highly expressed in the brain and testis, but barely detectable in the liver and skeletal muscle, while the I␤ isoform was found to be expressed at high levels in skeletal muscle, testis, brain, and lung tissues.
Sequence Homology-As expected, a search of protein se- Molecular Cloning of Type I PtdIns(4)P 5-Kinase 23612 quence data bases with BLAST (25) revealed that these two isoforms of type I PI4P5K have approximately 30 -40% identity with human type II PI4P5K, or its putative yeast homologue Fab1p and Mss4p amino acid sequences. Surprisingly, PI4P5K-I␣ is 95% identical to the amino acid sequence of STM7, a gene identified in the critical region for the Friedreich's ataxia locus (26), but which was recently proven not to be responsible for the disease (27). The amino acid sequence of neither the I␣ nor the I␤ isoform contains regions homologous to known protein or lipid kinase domains. Both isoforms contain a sequence closely homologous to the PtdIns(4,5)P 2 -binding domain of chicken striated ␣-actinin (28): RLMKKLEH-SWK and RFVKKLEHSWK, in the I␣ and the I␤ isoforms, respectively. DISCUSSION We have identified two type I isoforms of PI4P5K, designated PI4P5K-I␣ and PI4P5K-I␤, in this study. PI4P5K catalyzes the formation of PtdIns(4,5)P 2 from PtdIns(4)P. PtdIns(4,5)P 2 has been demonstrated to be an important molecule in various cellular processes, including inositol 1,4,5-trisphosphate mediated Ca 2ϩ mobilization (4), actin filament reorganization (5, 6), and exocytosis (7). Co-existence of at least three isoforms of the PI4P5K (one previously cloned isoform (type II or C) and two type I) in an insulin-secreting cell line, MIN6, suggests that these isoforms have distinct roles in various cellular processes. These isoforms may reside in different subcellular compartments and that spatially segregated synthesis or metabolism of PtdIns(4,5)P 2 may be linked to different cellular functions. While these novel isoforms show approximately 75% identity in their central regions, the amino-terminal or carboxyl-terminal sequences are different. It is possible that the amino-and carboxyl-terminal regions have isoform-specific functions. Further studies of these isoforms are needed; of particular interest is their subcellular localization and possible association with other molecules, such as Rac1 (29) and Rho (30).
Proteins of both the I␣ and the I␤ isoform migrate at 68 kDa on SDS-PAGE, suggesting that these isoforms are identical or closely related to the type Ia isoform previously described (3,23). During the screening of the MIN6 cell cDNA library, we obtained another partial coding sequence closely homologous to the PI4P5K-I␣ and I␤ isoforms. Further studies are needed to characterize the third sequence.
Whether the type I PI4P5K plays an important roles in regulated exocytosis of insulin secretory granules remains to be determined. The type I but not the type II enzyme has been demonstrated to be activated by phosphatidic acid and appears to phosphorylate PtdIns(4)P on intact membrane (31). Recent studies on regulated exocytosis have postulated that a positive feedback loop catalyzed by phospholipase D and the type I FIG. 2. Cloned cDNAs encode 68-kDa proteins. Cloned cDNAs were tagged at the amino terminus with an epitope derived from influenza virus hemagglutinin (HA-tag). Recombinant adenoviruses containing these HA-tagged cDNAs were constructed as described previously (19,20). COS7 cells were infected with the recombinant adenoviruses or a control virus (Adex1CAlacZ), and the lysates (20 g/lane) were subjected to SDS-PAGE (10%). Expressed proteins were probed with anti-HA monoclonal antibody (left panel), anti-PI4P5K-I␣ antisera (central panel), or anti-PI4P5K-I␤ antisera (right panel), respectively, followed by detection with ECL reagents (Amersham).

FIG. 3. Cloned cDNAs encode type I PtdIns(4)P 5-kinase.
A, autoradiogram demonstrating that the immunocomplex contained PtdIns(4)P kinase activity. Lysates of COS7 cells infected with adenoviruses encoding HA-tagged novel proteins were immunoprecipitated with anti-HA monoclonal antibody. The immunocomplex was assayed for PtdIns(4)P kinase activity as described under "Experimental Procedures." B, autoradiogram demonstrating that the novel protein products were distinct from PtdIns(3,4)P 2 . PtdIns(3,4)P 2 was generated, using PtdIns(4)P as a substrate, with p85/p110 phosphatidylinositol 3-kinase from murine brain lysates. C, effect of phosphatidic acid on PtdIns(4)P kinase activity. The immunocomplex was assayed using PtdIns Molecular Cloning of Type I PtdIns(4)P 5-Kinase 23613 PI4P5K is involved in exocytotic processes (8), which may form a microdomain enriched in negatively charged phospholipids on the vesicle membrane. Our demonstration of several isoforms of the type I PI4P5K in insulin-secreting cells (MIN6), together with a previous demonstration of phospholipase D in pancreatic islets (32), raises the possibility that such a positive feedback loop may operate in insulin-secreting cells. Furthermore, it was previously reported that glucose could cause activation of phosphatidylinositol metabolism, which was partly ascribed to increased de novo synthesis of PA and polyphosphoinositide in pancreatic islets (10,11). These observations suggest that activation of phosphatidylinositol metabolism, in concert with membrane depolarization, may serve as a link between glucose metabolism and exocytosis of insulin secretory granules. Molecular identification of the type I PI4P5K isoforms will provide essential information for the precise role of phosphatidylinositol metabolism in exocytosis of insulin-containing dense core vesicles.