It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono-and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast.

Elk-1 responds to stress-induced, as well as mitogenic, signals by stimulating c-fos transcription through the serum response element. Phosphorylation of Elk-1 by SAPKs and the ensuing expression of Fos protein thus constitutes an additional mechanism by which cells can upregulate AP-1 activity in response to stress.

Phosphatidylinositol 4-kinase, Pik1, is essential for viability. GFP-Pik1 localized to cytoplasmic puncta and the nucleus. The puncta colocalized with Sec7-DsRed, a marker of trans-Golgi cisternae. Kap95 (importin-β) was necessary for nuclear entry, but not Kap60 (importin-α), and exportin Msn5 was required for nuclear exit. Frq1 (frequenin orthologue) also is essential for viability and binds near the NH2 terminus of Pik1. Frq1-GFP localized to Golgi puncta, and Pik1 lacking its Frq1-binding site (or Pik1 overexpressed in frq1Δ cells) did not decorate the Golgi, but nuclear localization was unperturbed. Pik1(Δ10-192), which lacks its nuclear export sequence, displayed prominent nuclear accumulation and did not rescue inviability of pik1Δ cells. A Pik1-CCAAX chimera was excluded from the nucleus and also did not rescue inviability of pik1Δ cells. However, coexpression of Pik1(Δ10-192) and Pik1-CCAAX in pik1Δ cells restored viability. Catalytically inactive derivatives of these compartment-restricted Pik1 constructs indicated that PtdIns4P must be generated both in the nucleus and at the Golgi for normal cell function.

The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca 2؉ -bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca 2؉ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices ( In animal cells and yeast (1, 2), phosphoinositides mediate selective recruitment of proteins to membranes (3-6) and serve as precursors for intracellular second messengers (7-9). Phosphoinositide biosynthesis begins with phosphorylation of the myo-inositol headgroup of phosphatidylinositol (PtdIns) 3 at the D-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC 2.7.1.67) (10 -12). The first PtdIns 4-kinase to be purified (13), and the corresponding gene cloned (14), was Pik1 from the yeast Saccharomyces cerevisiae. PIK1 is an essential gene required for vesicular trafficking in the late secretory pathway (15, 16), for nuclear functions (17), and possibly cytokinesis (18). Pik1-like isoforms are conserved in metazoans (10,11,19).Yeast frequenin (Frq1), a 22-kDa Ca 2ϩ -binding protein, copurifies with Pik1 and is essential for its optimal activity (20). The site where Frq1 docks on Pik1 was localized to a region (residues 121-174) that lies far upstream of the catalytic domain (residues 792-1066) (21). Mammalian frequenin also interacts with Pik1 (22), and frequenin may regulate PtdIns 4-kinase activity in animal cells (23-25). Ca 2ϩ -dependent activation of PtdIns 4-kinase by frequenin may be especially important in neurons because modulation of phosphoinositide synthesis by intracellular Ca 2ϩ controls exocytosis (26) and is involved in synaptic plasticity (27).Frq1 and other frequenins belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, which includes recoverin and neurocalcin (28 -31). These proteins are small (Յ25 kDa) and characterized by a consensus signal for N-terminal myristoylation and four EF-hand Ca 2ϩ -binding sites (Fig. 1). We have shown previously that, at saturation, Frq1 binds only three Ca 2ϩ (32). Frq1, which is itself essential for the viability of yeast cells (20), associates with membranes in a manner that depends on both the N-myristoyl group and conformational changes induced upon Ca 2ϩ binding, suggesting that Frq1, like other NCS proteins, may possess a Ca 2ϩ -myristoyl switch (32). Indeed, prior work indicated that N-myristoylation of Frq1 is important (but not essential) for stimulating both the catalytic activity (20) and the membrane recruitment of Pik1 (17).Three-dimensional structures for Frq1 and other NCS proteins have been determined by x-ray crystallography (23,(33)(34)(35)(36)(37) and NMR spectroscopy (32, 38 -40). The structure of * This work was su...

Neuronal calcium sensor (NCS) proteins transduce Ca2؉ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an ␣-NH 2 -linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca 2؉ , assembled into a compact structure. In Ca 2؉ -free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca 2؉ -bound Ncs1, the myristoyl group is extruded (Ca 2؉ -myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca 2؉ -free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca 2؉ -dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins. Neuronal calcium sensor (NCS)2 proteins (1-3) are a conserved subclass of the calmodulin (CaM) superfamily that regulate signal transduction in the brain and retina. All members of the NCS family includes ϳ200 residues, are N-myristoylated on their ␣-NH 2 group, and possess four EF-hand motifs, but the first contains a diagnostic CPXG sequence that disables its ability to bind Ca 2ϩ (4,5). Recoverin, the most intensively studied NCS protein, is a Ca 2ϩ sensor in rod and cone cells, where it controls desensitization of rhodopsin (6 -9). The guanylate cyclase-activating proteins, GCAP1 (10) and GCAP2 (11), are NCS proteins also found in rods and cones, which regulate the recovery phase of visual excitation and are genetically linked to retinal diseases (12, 13). Brain NCS family members include neurocalcin (14), hippocalcin (15), visinin, and visinin-like proteins (16), KChIPs (17), and NCS-1 (also called frequenin) (18). Brain NCS proteins have diverse functions. Neurocalcins and visinin-like proteins regulate guanylate cyclase and nicotinamide acetylcholine receptors implicated in synaptic plasticity (16). KChIPs (17), hippocalcin (19), and NCS-1 (20) bind to various ion channels and thus control neuronal excitability.Remarkably, even the genomes of yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) encode a protein that is more than 60% identical to mammalian NCS-1 (Fig. 1A). The budding yeast (S. cerevisiae) homolog (Frq1) is essential for cell growth (21) and activates a PtdIns 4-kinase (Pik1) (22, 23). The fission yeast (S. pombe) homolog (Ncs1) regulates sporulation (24) and confers Ca 2ϩ tolerance (25). Sporulation defects in Ncs1 knock-out fission yeast are rescued by overexpressing S. cerevisiae Frq1 or Pik1, suggesting that Ncs1 activates the homologous S. pombe PtdIns 4-kinase. Indeed, the Frq1-binding site in Pik1 (26) is conserved in its ...

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P 2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P 2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P 2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P 2 synthesis.

Recognition that phosphoinositides and inositol phosphates are key regulators of many processes in eukaryotic cells has brought increased attention to the enzymes that regulate the synthesis and turnover of these molecules (reviewed in Refs. 1-3). Of particular interest are the enzymes responsible for producing the various polyphosphoinositides situated on the cytosolic face of cellular membranes, which initiate several different signaling pathways by serving as highly specific recognition determinants for the selective recruitment of proteins to membranes (reviewed in Refs. 4 -7) and as the precursors for several intracellular second messengers (reviewed in Refs. 8 -10). The first committed step in the synthesis of the polyphosphoinositide, phosphatidylinositol 4,5-bisphosphate, is considered to be ATP-dependent phosphorylation of the hydrophilic myo-inositol head group of phosphatidylinositol (PtdIns) 1 at the D-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC 2.7.1.67) (reviewed in Refs. 11-13) . The resulting product, PtdIns(4)P, can be phosphorylated on the D-5 position by PtdIns(4)P 5-kinase to generate PtdIns(4,5)P 2 , PtdIns(4,5)P 2 can be phosphorylated on the D-3 position by yet other lipid kinases, and the phosphoinositides so generated can be converted to other species by specific phosphatases and phospholipases (reviewed in Refs. 14 -17).The first PtdIns 4-kinase to be purified to homogeneity from any organism (18), and to have the corresponding gene cloned (19,20), was Pik1 from the yeast Saccharomyces cerevisiae. Thereafter, a second isoform, Stt4, which is the product of a discrete gene, was described (21). Absence of either Pik1 or Stt4 is lethal, and overproduction of each protein cannot compensate for absence of the other, indicating that these enzymes participate in distinct cellular processes and generate discrete pools of PtdIns(4)P that are essential for yeast cell viability. Indeed, subsequent work has shown that, together, Pik1 and Stt4 account for all of the PtdIns(4)P generated in the yeast cell (22) and that Pik1 is required for vesicular trafficking in the late secretory pathway (23, 24) and perhaps for cytokinesis (20), whereas Stt4 plays roles in cell wall integrity, maintenance of vacuole morphology, and aminophospholipid transport from the endoplasmic reticulum to the Golgi (25-27). The presence of Pik1-and Stt4-like isoforms is also conserved in metazoans (11,12,28).We have shown previously that Frq1, a small calcium-bind-