The Saccharomyces cerevisiae protein MSS4 is essential and homologous to mammalian phosphatidylinositol-4-phosphate (PI(4)P) 5-kinases. MSS4 is shown to be a lipid kinase. MSS4 has dual substrate specificity in vitro, converting PI(4)P to PI(4, 5)P2 and to a lesser extent PI(3)P to PI(3,4)P2; no activity was detected with PI or PI(5)P as a substrate. Cells overexpressing MSS4 contain an elevated level specifically of PI(4,5)P2, whereas mss4 mutant cells have only approximately 10% of the normal amount of this phosphorylated phosphoinositide. Furthermore, cells lacking MSS4 are unable to form actin cables and to properly localize their actin cytoskeleton during polarized cell growth. Overexpression of RHO2, encoding a Rho-type GTPase involved in regulation of the actin cytoskeleton, restores growth and polarized distribution of actin in an mss4 mutant. These results suggest that MSS4 is the major PI(4)P 5-kinase in yeast and provide a link between phosphoinositide metabolism and organization of the actin cytoskeleton in vivo (Desrivieres, 1998).

Phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2], an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase [PtdIns(4)P 5K] from phosphatidylinositol 4-phosphate [PtdIns(4)P] or by phosphatidylinositol-5-phosphate 4-kinase [PtdIns(5)P 4K] from phosphatidylinositol 5-phosphate [PtdIns(5)P]. Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. A hemagglutinin epitope-tagged form of Mss4p was constructed and Mss4p was found to have PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated; its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicate that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization (Homma, 1998).

Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Wortmannin is toxic to the yeast Saccharomyces cerevisiae and genetic and biochemical evidence is presented that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, is potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 confers wortmannin resistance, as does deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate (Cutler, 1999).

Phosphatidylinositol (4)P 5-kinase [PtdIns(4)P 5-kinase] catalyzes the last step in the synthesis of phosphatidylinositol 4, 5-bisphosphate [PtdIns(4,5)P2]. PtdIns(4,5)P2 is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2 must be stringently regulated. PtdIns(4)P 5-kinase has been extracted, purified, and characterized from the plasma membranes of Schizosaccharomyces pombe. Evidence is provided that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombe homolog of casein kinase I. Phosphorylation by Cki1 in vitro decreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pombe results in a reduced synthesis of PtdIns(4,5)P2 and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombe and that Cki1 is involved in regulation of PtdIns(4, 5)P2 synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase (Vancurova, 1999).

Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2), a key molecule in the phosphoinositide signalling pathway, was thought to be synthesized exclusively by phosphorylation of PtdIns-4-P at the D-5 position of the inositol ring. The enzymes that produce PtdIns-4,5-P2 in vitro fall into two related subfamilies [type I and type II PtdInsP-5-OH kinases, or PIP(5)Ks] based on their enzymatic properties and sequence similarities. The substrate specificities of these enzymes has been reinvestigated. As expected, the type I enzyme phosphorylates PtdIns-4-P at the D-5 position of the inositol ring. Surprisingly, the type II enzyme, which is abundant in some tissues, phosphorylates PtdIns-5-P at the D-4 position, and thus should be considered as a 4-OH kinase, or PIP(4)K. The earlier error in characterizing the activity of the type II enzyme is due to the presence of contaminating PtdIns-5-P in commercial preparations of PtdIns-4-P. Although PtdIns-5-P was previously thought not to exist in vivo, evidence has been found for the presence of this lipid in mammalian fibroblasts, establishing a new pathway for PtdIns-4,5-P2 synthesis (Rameh, 1997).

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3, 4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules (Zhang, 1997).

Inositol phospholipids regulate a variety of cellular processes including proliferation, survival, vesicular trafficking, and cytoskeletal organization. Recently, two novel phosphoinositides, phosphatidylinositol-3,5-bisphosphate (PtdIns-3,5-P2) and phosphatidylinositol- 5-phosphate (PtdIns-5-P), have been shown to exist in cells. PtdIns-3,5-P2, which is regulated by osmotic stress, appears to be synthesized by phosphorylation of PtdIns-3-P at the D-5 position. No evidence yet exists for how PtdIns-5-P is produced in cells. Understanding the regulation of synthesis of these molecules will be important for identifying their function in cellular signaling. To determine the pathway by which PtdIns-3,5-P2 and Ptd-Ins-5-P might be synthesized, the ability of the recently cloned type I PtdIns-4-P 5-kinases (PIP5Ks) alpha and beta to phosphorylate PtdIns-3-P and PtdIns at the D-5 position of the inositol ring was examined. Type I PIP5Ks phosphorylate PtdIns-3-P to form PtdIns-3,5-P2. The identity of the PtdIns-3,5-P2 product was determined by anion exchange high performance liquid chromatography analysis and periodate treatment. PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are also produced from PtdIns-3-P phosphorylation by both isoforms. When expressed in mammalian cells, PIP5K Ialpha and PIP5K Ibeta differ in their ability to synthesize PtdIns-3,5-P2 relative to PtdIns-3,4-P2. The type I PIP5Ks phosphorylate PtdIns to produce PtdIns-5-P and phosphorylate PtdIns-3,4-P2 to produce PtdIns-3,4,5-P3. These findings suggest that type I PIP5Ks synthesize the novel phospholipids PtdIns-3,5-P2 and PtdIns-5-P. The ability of PIP5Ks to produce multiple signaling molecules indicates that they may participate in a variety of cellular processes (Tolias, 1998b).

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] 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)P2 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 has been isolated and sequenced. Expression of this cDNA in Escherichia coli produces 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 (Boronenkov, 1995).

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, the polymerase chain reaction was employed 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 migrate at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibit phosphatidylinositol-4-phosphate 5-kinase activity, which is 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 depicts that tissue distributions differ 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 (Ishihara, 1996).

Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) synthesize phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling that also regulates some proteins and cellular processes directly. Two distinct PIP5Ks have been characterized in erythrocytes, the 68-kDa type I (PIP5KI) and 53-kDa type II (PIP5KII) isoforms. Using peptide sequences from the erythroid 68-kDa PIP5KI, cDNAs encoding PIP5KIalpha have been isolated from human brain. Partial cDNAs obtained for a second isoform, PIP5KIbeta, establish that the human STM7 gene encodes a previously unrecognized PIP5KI. However, the peptide sequences demonstrates that erythroid PIP5KI corresponded to PIP5KIalpha. Recombinant, bacterially expressed PIP5KIalpha possesses PIP5K activity and is immunoreactive with erythroid PIP5KI antibodies. By Northern analysis, PIP5KIalpha and PIP5KIbeta have wide tissue distributions, but their expression levels differ greatly. PIP5KIs had homology to the kinase domains of PIP5KIIalpha, yeast Mss4p and Fab1p, and a new Caenorhabditis elegans Fab1-like protein identified in the data base. These new isoforms have refined the sequence requirements for PIP5K activity and, potentially, regulation of these enzymes. Furthermore, the limited homology between PIP5KIs and PIP5KIIalpha, which was almost exclusively within the kinase domain core, provided a molecular basis for distinction between type I and II PIP5Ks (Loijens, 1996a).

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. The cloning of the third member (PIP5Kgamma) is reported 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. 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, overexpression of either the beta or the gamma isoform induces an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induces an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules (Ishihara, 1998).