Phosphatidylinositol-4-phosphate 5-kinases in yeast

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).

Substrate specificities of phosphatidylinositol-4-phosphate 5-kinases

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).

Multiple genes code for phosphatidylinositol-4-phosphate 5-kinases

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).

Phosphatidylinositol-4-phosphate 5-kinases interact with Rac and Rho

Rho family GTPases appear to play an important role in the regulation of the actin cytoskeleton, but the mechanism of regulation is unknown. Since phosphoinositide 3-kinase and phosphatidylinositol 4,5-bisphosphate have also been implicated in actin reorganization, the possibility that Rho family members interact with phosphoinositide kinases was investigated. Both GTP- and GDP-bound Rac1 (see Drosophila Rac) associate with phosphatidylinositol-4-phosphate 5-kinase in vitro and in vivo. Phosphoinositide 3-kinase also bind to Rac1 and Cdc42Hs, and these interactions are GTP-dependent. Stimulation of Swiss 3T3 cells with platelet-derived growth factor induces the association of PI 3-kinase with Rac in immunoprecipitates. PI 3-kinase activity was also detected in Cdc42 immunoprecipitates from COS7 cells. These results suggest that phosphoinositide kinases are involved in Rho family signal transduction pathways and raise the possibility that the effects of Rho family members on the actin cytoskeleton are mediated in part by phosphoinositide kinases (Tolias, 1995).

Post-translationally modified Rho (see Drosophila Rho) in its GTP-bound state stimulates phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, immobilized Rho-GST was incubated with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, binds significant PIP5K activity. The binding of PIP5K is independent of whether Rho is in a GTP- or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognizes a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responds to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitates with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhances PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown (Ren, 1996).

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). Treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreases calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulates PIP 5-kinase activity, and this effect is blocked by botulinum C3 exoenzyme, suggesting that Rho is responsible. GTP-bound recombinant Rho stimulates PIP 5-kinase activity, whereas GDP-Rho is much less potent and GTP-bound Rac is ineffective. Microinjected botulinum C3 exoenzyme causes diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reverses the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis mediates the effects of Rho on the actin cytoskeleton (Chong, 1994).

The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Several isoforms of type I PI4P5K have been isolated. PI4P5K Ialpha induces massive actin polymerization resembling 'pine needles' in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity are lost. Although the dominant negative form of Rho, RhoN19, alone decreases actin fibers, those induced by PI4P5K are not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity (Shibasaki, 1997).

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. A diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases is enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways (Tolias, 1998a).

Other interactions of phosphatidylinositol-4-phosphate 5-kinases

Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms, and its mechanism of regulation is not yet clear. PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu fails to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations (Nishikawa, 1998).

Phosphatidylinositol-4-phosphate 5-kinases act downstream of surface receptors

skittles Evolutionary homologs part 2/2

skittles: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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