Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1)

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-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 Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.     

Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform,Pik1

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.     

Growth control of Golgi phosphoinositides by reciprocal localization of sac1 lipid phosphatase and pik1 4-kinase

Compartment-specific control of phosphoinositide lipids is essential for cell function. The Sac1 lipid phosphatase regulates endoplasmic reticulum (ER) and Golgi phosphatidylinositol-4-phosphate [PI(4)P] in response to nutrient levels and cell growth stages. During exponential growth, Sac1p interacts with Dpm1p at the ER but shuttles to the Golgi during starvation. Here, we report that a C-terminal region in Sac1p is required for retention in the perinuclear ER, whereas the N-terminal domain is responsible for Golgi localization. We also show that starvation-induced shuttling of Sac1p to the Golgi depends on the coat protein complex II and the Rer1 adaptor protein. Starvation-induced shuttling of Sac1p to the Golgi specifically eliminates a pool of PI(4)P generated by the lipid kinase Pik1p. In addition, absence of nutrients leads to a rapid dissociation of Pik1p, together with its non-catalytical subunit Frq1p, from Golgi membranes. Reciprocal rounds of association/dissociation of the Sac1p lipid phosphatase and the Pik1p/Frq1p lipid kinase complex are responsible for growth-dependent control of Golgi phosphoinositides. Sac1p and Pik1p/Frq1p are therefore elements of a unique machinery that synchronizes ER and Golgi function in response to different growth conditions.     

Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus

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.     

Conservation of regulatory function in calcium-binding proteins: human frequenin (neuronal calcium sensor-1) associates productively with yeast phosphatidylinositol 4-kinase isoform, Pik1

Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic alpha-helix and that frequenins bind to one side of this alpha-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.     

Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae

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.     

Multiple Roles for Frequenin/NCS-1 in Synaptic Function and Development

The calcium-binding protein frequenin (Frq), discovered in the fruit fly Drosophila, and its mammalian homologue neuronal calcium sensor 1 (NCS-1) have been reported to affect several aspects of synaptic transmission, including basal levels of neurotransmission and short- and long-term synaptic plasticities. However, discrepant reports leave doubts about the functional roles of these conserved proteins. In this review, we attempt to resolve some of these seemingly contradictory reports. We discuss how stimulation protocols, sources of calcium (voltage-gated channels versus internal stores), and expression patterns (presynaptic versus postsynaptic) of Frq may result in the activation of various protein targets, leading to different synaptic effects. In addition, the potential interactions of Frq's C-terminal and N-terminal domains with other proteins are discussed. Frq also has a role in regulating neurite outgrowth, axonal regeneration, and synaptic development. We examine whether the effects of Frq on neurotransmitter release and neurite outgrowth are distinct or interrelated through homeostatic mechanisms. Learning and memory are affected by manipulations of Frq probably through changes in synaptic transmission and neurite outgrowth, raising the possibility that Frq may be implicated in human pathological conditions, including schizophrenia, bipolar disorder, and X-linked mental retardation.     

The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication

? The rice-rice blast pathosystem is of great interest, not only because of the damaging potential of rice blast to the rice crop, but also because both the pathogen and its host are experimentally amenable. The rice blast resistance gene Pik, which is one of the five classical alleles located at the Pik locus on the long arm of chromosome 11, confers high and stable resistance to many Chinese rice blast isolates. ? The isolation and functional characterization of Pik were performed in the present study through genetic and genomic approaches. ? A combination of Pik-1 and Pik-2 is required for the expression of Pik resistance. Both Pik-1 and Pik-2 encode coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) proteins, and each shares a very high level of protein identity with corresponding proteins encoded by the Pik-m and Pik-p alleles. Pik could be distinguished from other Pik alleles, including Pik-m and Pik-p, by the allele-specific, single-nucleotide polymorphism T1-2944G. ? The coupled genes probably did not evolve as a result of a duplication event, and are far from any NBS-LRR R gene characterized. Pik is a younger allele at the locus that probably emerged after rice domestication.     

Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.     

Arf1 directly recruits the Pik1-Frq1 PI4K complex to regulate the final stages of Golgi maturation

Proper Golgi complex function depends on the activity of Arf1, a GTPase whose effectors assemble and transport outgoing vesicles. Phosphatidylinositol 4-phosphate (PI4P) generated at the Golgi by the conserved PI 4-kinase Pik1 (PI4KIIIβ) is also essential for Golgi function, although its precise roles in vesicle formation are less clear. Arf1 has been reported to regulate PI4P production, but whether Pik1 is a direct Arf1 effector is not established. Using a combination of live-cell time-lapse imaging analyses, acute PI4P depletion experiments, and in vitro protein–protein interaction assays on Golgi-mimetic membranes, we present evidence for a model in which Arf1 initiates the final stages of Golgi maturation by tightly controlling PI4P production through direct recruitment of the Pik1-Frq1 PI4-kinase complex. This PI4P serves as a critical signal for AP-1 and secretory vesicle formation, the final events at maturing Golgi compartments. This work therefore establishes the regulatory and temporal context surrounding Golgi PI4P production and its precise roles in Golgi maturation.     

Pik3ip1 Modulates Cardiac Hypertrophy by Inhibiting PI3K Pathway

Cardiac hypertrophy is an adaptive response to various physiological and pathological stimuli. Phosphoinositide-3 kinase (PI3K) is a highly conserved lipid kinase involved in physiological cardiac hypertrophy (PHH). PI3K interacting protein1 (Pik3ip1) shares homology with the p85 regulatory subunit of PI3K and is known to interact with the p110 catalytic subunit of PI3K, leading to attenuation of PI3K activity in liver and immune cells. However, the role of Pik3ip1 in the heart remains unknown. In the present study, the effects of Pik3ip1 on cardiac hypertrophy were examined. We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts. The interaction of Pik3ip1 with the p110a subunit of PI3K in the heart was identified by immunoprecipitation using neonatal rat cardiomyocytes (NRCM). Approximately 35% knockdown of Pik3ip1 was sufficient to induce myocardial hypertrophy. Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size. However, adenovirus-mediated overexpression of Pik3ip1 attenuated PI3K-mediated cardiac hypertrophy. Pik3ip1 was upregulated by PHH due to swimming training, but not by pathological cardiac hypertrophy (PAH) due to pressure-overload, suggesting that Pik3ip1 plays a compensatory negative role for PHH. Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.     

Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.     

Two Frequenins in <Emphasis Type="Italic">Drosophila</Emphasis>: unveiling the evolutionary history of an unusual Neuronal Calcium Sensor (NCS) duplication

Background  

Drosophila Frequenin (Frq), the homolog of the mammalian Neuronal Calcium Sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, Frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species.     

The mammalian class 3 PI3K (PIK3C3) is required for early embryogenesis and cell proliferation

The Pik3c3 gene encodes an 887 amino acid lipid kinase, phosphoinositide-3-kinase class 3 (PIK3C3). PIK3C3 is known to regulate various intracellular membrane trafficking events. However, little is known about its functions during early embryogenesis in mammals. To investigate the function of PIK3C3 in vivo, we generated Pik3c3 null mice. We show here that Pik3c3 heterozygous are normal and fertile. In contrast, Pik3c3 homozygous mutants are embryonic lethal and die between E7.5 and E8.5 of embryogenesis. Mutant embryos are poorly developed with no evidence of mesoderm formation, and suffer from severely reduced cell proliferations. Cell proliferation defect is also evident in vitro, where mutant blastocysts in culture fail to give rise to typical colonies formed by inner cell mass. Electron microscopic analysis revealed that epiblast cells in mutant embryos appear normal, whereas the visceral endoderm cells contain larger vesicles inside the lipid droplets. Finally, we provide evidence that mTOR signaling is drastically reduced in Pik3c3 null embryos, which could be a major contributor to the observed proliferation and embryogenesis defects.     

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreER^T2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreER^T2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1–S6K1–rpS6 signaling pathway.     

The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast

We report the isolation of Pi1, a gene conferring broad-spectrum resistance to rice blast (Magnaporthe oryzae). Using loss- and gain-of-function approaches, we demonstrate that Pi1 is an allele at the Pik locus. Like other alleles at this locus, Pi1 consists of two genes. A functional nucleotide polymorphism (FNP) was identified that allows differentiation of Pi1 from other Pik alleles and other non-Pik genes. A extensive germplasm survey using this FNP reveals that Pi1 is a rare allele in germplasm collections and one that has conferred durable resistance to a broad spectrum of pathogen isolates.     

GRP94 promotes muscle differentiation by inhibiting the PI3K/AKT/mTOR signaling pathway

The glucose-regulated endoplasmic reticulum chaperone protein 94 (GRP94) is required for many biological processes, such as secretion of immune factors and mesoderm induction. Here, we demonstrated that GRP94 promotes muscle differentiation in vitro and in vivo. Moreover, GRP94 inhibited the PI3K/AKT/mTOR signaling pathway. Using both in vitro and in vivo approaches, in myoblasts, we found that this inhibition resulted in reduced proliferation and increased differentiation. To further investigate the mechanism of GRP94-induced muscle differentiation, we used co-immunoprecipitation and proximity ligation assays and found that GRP94 interacted with PI3K-interacting protein 1 (Pik3ip1). The latter protein promoted muscle differentiation by inhibiting the PI3K/AKT/mTOR pathway. Furthermore, GRP94 was found to regulate Pik3ip1 expression. Finally, when Pik3ip1 expression was inhibited, GRP94-induced promotion of muscle differentiation was diminished. Taken together, our data demonstrated that GRP94 promoted muscle differentiation, mediated by Pik3ip1-dependent inhibition of the PI3K/AKT/mTOR signaling pathway.     

Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase

Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane.     

Proliferative defect and embryonic lethality in mice homozygous for a deletion in the p110alpha subunit of phosphoinositide 3-kinase

Phosphatidylinositol 3,4,5-trisphosphate is a phospholipid signaling molecule involved in many cellular functions including growth factor receptor signaling, cytoskeletal organization, chemotaxis, apoptosis, and protein trafficking. Phosphorylation at the 3 position of the inositol ring is catalyzed by many different 3-kinases (classified as types IA, IB, II, and III), but the physiological roles played by each of the different 3-kinase isozymes during embryonic development and in homeostasis in animals is incompletely understood. Mammalian type IA kinase isozymes are heterodimers that are active at 37 degrees C when the catalytic 110-kDa subunit interacts through an amino-terminal binding domain with a regulatory 85- or 55-kDa subunit. Using gene targeting in embryonic stem cells, we deleted this binding domain in the gene encoding the alpha isoform of the 110-kDa catalytic subunit (Pik3ca) of the alpha isozyme of the type IA kinases, leading to loss of expression of the p110 catalytic subunit. We show that Pik3cadel/del embryos are developmentally delayed at embryonic day (E) 9.5 and die between E9.5 and E10.5. E9. 5 Pik3cadel/del embryos have a profound proliferative defect but no increase in apoptosis. A proliferative defect is supported by the observation that fibroblasts from Pik3cadel/del embryos fail to replicate in Dulbecco's modified Eagle's medium and fetal calf serum, even with supplemental growth factors.     

Characterization of the rice blast resistance gene Pik cloned from Kanto51

To study similar, but distinct, plant disease resistance (R) specificities exhibited by allelic genes at the rice blast resistance locus Pik/Pikm, we cloned the Pik gene from rice cultivar Kanto51 and compared its molecular features with those of Pikm and of another Pik gene cloned from cv. Kusabue. Like Pikm, Pik is composed of two adjacent NBS-LRR (nucleotide-binding site, leucine-rich repeat) genes: the first gene, Pik1-KA, and the second gene, Pik2-KA. Pik from Kanto51 and Pik from Kusabue were not identical; although the predicted protein sequences of the second genes were identical, the sequences differed by three amino acids within the NBS domain of the first genes. The Pik proteins from Kanto51 and Kusabue differed from Pikm in eight and seven amino acids, respectively. Most of these substituted amino acids were within the coiled-coil (CC) and NBS domains encoded by the first gene. Of these substitutions, all within the CC domain were conserved between the two Pik proteins, whereas all within the NBS domain differed between them. Comparison of the two Pik proteins and Pikm suggests the importance of the CC domain in determining the resistance specificities of Pik and Pikm. This feature contrasts with that of most allelic or homologous NBS-LRR genes characterized to date, in which the major specificity determinant is believed to lie in the highly diverged LRR domain. In addition, our study revealed high evolutionary flexibility in the genome at the Pik locus, which may be relevant to the generation of new R specificities at this locus.