Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP₇, is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP₇ regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP₇. Although the inhibitors have diverse targets, they all perturbed IP₇ levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP₇ levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K_m for ATP makes IP₇ synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP₇ concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP₇ indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP₇’s role in cellular signal transduction is likely to have been underestimated.     

Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates epithelial cell apoptosis in response to oxidative stress in Drosophila

Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP₃ 3-K, IP₃K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP₃Ks (IP₃K-1 and IP₃K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP₃K-1 directly increases H₂O₂ production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells–assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters–is significantly increased by IP₃K-1 under oxidative stress conditions, leading to apoptosis.The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP₃K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.     

Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells

Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F_2α (PGF_2α), resulted in a very rapid increase in the intracellular levels of [³H]-inositol triphosphate (IP₃), [³H]-inositol biphosphate (IP₂), and [³H]-inositol monophosphate (IP₁) in cells prelabeled with [³H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2?2 min and declined to control levels by 6–10 min. The stimulation was dose-dependent with maximal increases observed near 10^?6 M PGF_2α. The cholinergic agent, carbachol, also led to time and dose-independent increases in IP₃. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP₃ or on the accumulation of IP₁. In contrast, vanadate at 10^?6 or 10^?5 M did lead to a decrease in the breakdown of IP₁ and a concomitant increase in IP₁, IP₂, and IP₃. PGF_2α was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E₁ (PGE₁). PGE₁, in a dose-dependent manner, was found to inhibit the observed IP₃ increase by PGF_2α at 1 1/2 min of stimulation. PGF_2α treated and control cultures did not differ in cAMP or cGMP levels, cellular ⁴⁵Ca uptake, nor cellular ²²Na uptake. We propose that IP₃ may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF_2α and that it may play a crucial role with cAMP in growth regulation.     

Inositol phosphate signaling and gibberellic acid

To respond to physical signals and endogenous hormones, plants use specific signal transduction pathways. We and others have previously shown that second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] is used in abscisic acid (ABA) signaling, and that some mutants with altered Ins(1,4,5)P₃ have altered responses to ABA. Specifically, mutants defective in the myo-inositol polyphosphate 5-phosphatases (5PTases) 1 and 2 genes that hydrolyze 5-phosphates from Ins(1,4,5)P₃ and other PtdInsP and InsP substrates, have elevated Ins (1,4,5)P₃, and are ABA-hypersensitive. Given the antagonistic relationship between ABA and gibberellic acid (GA), we tested the response of these same mutants to a GA synthesis inhibitor, paclobutrazol (PAC). We report here that 5ptase1, 5ptase2 and 5ptase11 mutants are hypersensitive to PAC, suggesting a relationship between elevated Ins(1,4,5)P₃ and decreased GA signal transduction. These data provide insight into signaling cross-talk between ABA and GA pathways.Key words: inositol, phosphatidylinositol phosphate, paclobutrazol, gibberellic acid, inositol trisphosphate, paclobutrazol     

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated calcium (Ca²⁺) release channel and plays important roles in regulation of numerous Ca²⁺-dependent cellular responses. Many intracellular modulators and IP₃R-binding proteins regulate the IP₃R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP₃R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP₃R. The interaction was favored by the cytosolic Ca²⁺ concentration and it functionally inhibited IP₃R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP₃R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP₃R-mediated Ca²⁺ signal through a direct interaction with IP₃R in a cytosolic Ca²⁺-dependent manner.The inositol 1,4,5-trisphosphate (IP₃)³ receptor (IP₃R) consisting of three subtypes, IP₃R1, IP₃R2, and IP₃R3, is a tetrameric intracellular IP₃-gated calcium (Ca²⁺) release channel localized at the endoplasmic reticulum (ER) with its NH₂ terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP₃Rs are composed of five functional domains. The long NH₂-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP₃-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP₃R channel opening (2, 3). Ca²⁺ release activity of the IP₃R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca²⁺), protein kinases, and IP₃R-binding proteins (2, 4), and the tight regulation of IP₃R channel activity by these factors generates various spatial and temporal intracellular Ca²⁺ patterns such as Ca²⁺ spikes and Ca²⁺ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP₃R. A, schematic of ER residential IP₃R. The CTT of IP₃R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP₃R1 in vitro. GST and GST-IP₃R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP₃R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP₃R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP₃R1. F and G, both GIT1 and GIT2 bind to all three IP₃R subtypes. HeLa cells coexpressing GFP-fused IP₃R1, IP₃R2, or IP₃R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP₃R-mediated Ca²⁺ signaling is a pro-apoptotic regulator during apoptosis. Ca²⁺ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca²⁺ released from the IP₃Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-X_L (12), have been reported to interact with the internal coupling domain and/or the CTT of IP₃R and enhance the Ca²⁺-release activity of IP₃Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP₃R-binding proteins that bind to the CTT of IP₃R and inhibit apoptosis by regulation of IP₃R-mediated Ca²⁺ signal.     

Melatonin Receptor-Mediated Stimulation of Phosphoinositide Breakdown in Chick Brain Slices

Abstract: The direct effect of melatonin and related agonists on Li⁺-amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo-[2-³H]-inositol. The melatonin receptor agonist 6-chloromelatonin (10–100 µM) increased, in a concentration-dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6-chloromelatonin (10 µM) was rapid as transient increases in IP₃/IP₄ (maximal increase, 29% at 20 s) and IP₂ levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP₁ level (30% at 5 min), when the amount of IP₃/IP₄ and IP₂ had already been decreased to the control level. The phosphoinositide response elicited by 6-chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6-chloromelatonin (10 µM) in isolated myo-[2-³H]inositol-prelabeled optic tectum membranes was dependent on the presence of guanosine-5′-O-(3-thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration-dependent manner the IP₁ accumulation stimulated by 6-chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5-hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5-hydroxytryptamine (10 µM)-, but not 6-chloromelatonin (10 µM)-, induced IP₁ accumulation. Melatonin receptor agonists increased IP₁ accumulation in a concentration-dependent manner reaching different maximal responses. N-Acetyl-5-hydroxytryptamine was more potent than melatonin in increasing IP₁ accumulation, suggesting activation of a melatonin receptor site other than the ML-1 melatonin receptor (i.e., N-acetyl-5-hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML-1 subtype leads to activation of the phospholipase C-mediated signal transduction pathway.     

Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F₁ mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F₁ mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F₁ fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F₁ mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.     

Vascular endothelial cells and smooth muscle cells mediate carbachol-induced hepatocyte proliferation via muscarinic receptors and IP3/PKC signaling cascades

An acetylcholine (ACh) agonist, carbachol (Cch), causes hepatocytes to proliferate in the presence of hepatic nonparenchymal cells (HNPCs). To identify the HNPCs and ACh receptor subtypes involved in carbachol-induced hepatocyte proliferation (CIHP), we examined two types of vascular cells as candidates for HNPCs mediating CIHP in cocultures of hepatocytes using the Transwell filter insert. In the coculture with vascular smooth muscle cells (VSMCs) or endothelial cells (VECs), but not in the monoculture, 72 h treatment with Cch significantly increased the numbers of hepatocytes. The results suggest that both VSMCs and VECs are involved in CIHP through soluble factors secreted from these cells. Interestingly, coculture with VECs, but not with VSMCs, markedly increased the number of hepatocytes, even in the absence of Cch. Cell proliferation assays using an analogue of thymidine, bromodeoxyuridine (BrdU), demonstrated that the hepatocytes in both cocultures transiently replicated their chromosomes 12 h after Cch administration. Blocking the muscarinic type 1 ACh receptor (M₁), M_3/5, intracellular inositol triphosphate (IP₃) receptor, or protein kinase C (PKC) pathways inhibited VSMC-mediated CIHP, whereas blocking the M_3/5, IP₃ receptor, or PKC pathways inhibited VEC-mediated CIHP. Co-culturing hepatocytes with both types of vascular cells markedly increased their albumin content, but addition of Cch had no effect. In conclusion, VSMCs among vascular cells mediate CIHP through M₁, M_3/5, and IP₃/PKC signal transduction pathways, whereas VECs do so through M_3/5, and IP₃/PKC pathways.     

Quantitative immunodetection of key elements of polyphosphoinositide signal transduction in osteoblasts from arthritic patients shows a direct correlation with cell proliferation

Phosphoinositides play an essential role in diverse cellular functions such as cell proliferation, cytoskeletal regulation, intracellular vesicle trafficking, motility, cell metabolism and death. Alteration of these pathways is common to many diseases. In this study, we show that osteoblasts from patients affected by osteoarthritis (OA) and by rheumatoid arthritis (RA) present a decreased cell proliferation and a reduced expression of the key elements of polyphosphoinositide signal transduction such as phosphatidylinositol-3-kinase (PI 3K), phospholipase C γ₁ (PLCγ₁), and protein kinase C ζ (PKCζ) compared to the post-traumatic (PT) patients. Our results suggest that a correlation may exist between the reduced osteoblast proliferation observed in OA and RA patients and the lowered expression of PI 3K, PLCγ₁, and PKCζ enzymes. The reduced proliferation rate of osteoblasts in response to these signal transduction effectors could counteract the evolution of arthritic disease.     

Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.     

Role of 1,4,5-inositol triphosphate-induced Ca2+ release in pollen tube orientation

 Pollen tube reorientation is a dynamic cellular event crucial for successful fertilization. Previously, it was shown that reorientation is preceded by an asymmetric increase of cytosolic free calcium ([Ca²⁺]_c) in the side of the apex to which the cell will bend. In order to find the targets for this signal transduction pathway, the effects of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in the reorientation process were analyzed. Ins(1,4,5)P₃ was artificially increased in different cell domains by localized photoactivation of caged Ins(1,4,5)P₃ and its effects on [Ca²⁺]_c monitored by ion confocal microscopy. It was found that photolysis of caged Ins(1,4,5)P₃ in the nuclear or subapical region resulted in a transient increase in [Ca²⁺]_c and reorientation of the growth axis, while photolysis in the apex frequently resulted in disturbed growth or tip bursting. Perfusion of the cells with the Ins(1,4,5)P₃ receptor blocker heparin prior to photoactivation inhibited the increase in [Ca²⁺]_c and no reorientation was observed. Ca²⁺ release from Ins(1,4,5)P₃-dependent stores localized in the shank of the tube thus seems to be part of the signal transduction pathway that controls tube guidance, although not the primary stimulus leading to reorientation. Received: 5 May 1998 / Accepted: 11 June 1998     

Structural Studies and Protein Engineering of Inositol Phosphate Multikinase

Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP₃ to IP₅, as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP₃ 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P₄ are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.     

Inositol 1,4,5‐trisphosphate transduction cascade in taste reception of the fleshfly,Boettcherisca peregrina

The role of an inositol 1,4,5‐trisphosphate (IP₃)‐mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP₃ production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP₃‐gated channel), IP₃, ruthenium red (a blocker of the IP₃‐gated channel), and 2‐aminoethoxydiphenylborate (2‐APB; an antagonist of the IP₃‐gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP₃ + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2‐APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP₃ transduction cascade, such as G protein α subunit (dGqα), phospholipase C (norpA), and IP₃ receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox‐neuro⁷⁰ mutant (poxn⁷⁰), which lacks taste receptor cells. The expressed levels of dGqα and itpr in the tarsus of poxn⁷⁰ mutant flies were reduced compared with those of wild‐type flies. These results suggest that the IP₃ transduction cascade is involved in the response of the sugar receptor cell of the fly. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 66–83, 2002     

hpc2研究进展

生物个体的胚胎发育以及细胞的增殖、分化,都同时受到多种基因的严格调控,PcG基因家族就是一类重要的发育相关基因.而hPc2基因是人PcG基因家族中的一个重要成员,其编码的HPC2蛋白,不仅可以和HPH、BMI-1以及RING1等其他人类PcG蛋白结合形成HPC/HPH PcG复合体,以蛋白复合体的形式参与对homeotic基因的表达抑制,以维持机体的正常发育以及细胞的增殖和定向分化,还发现它能与其他多种蛋白质相结合,提示HPC2可能具有多种功能.因此,对hPc2的深入研究不仅有助于进一步阐明PcG基因家族的作用机理,扩展人们对基因表达调控的认识,还有助于发现PcG基因家族与其他信号转导通路的联系,更好地理解细胞信号网络系统.     

Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth

The inositol 1,3,4,5,6-pentakisphosphate (IP₅) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP₆) in eukaryotic cells. Previous studies have shown that IP₆ is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1⁺ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1⁺ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP₆ production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1⁺ function, encoding an IP₆ kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1⁺ upstream of ipk1⁺. We propose that IP₆ production is a key signaling linchpin for regulating multiple essential cellular processes.     

Zebrafish inositol polyphosphate kinases: New effectors of cilia and developmental signaling

We described here our recent findings that Ipk1 catalyzed production of IP₆ regulates LR-axis specification (Sarmah et al., 2005) and that IP₆ is an essential effector of ciliary beating and length maintenance in zebrafish (Sarmah et al., 2007). We have also uncovered a novel role for the IP-kinase IP₆k2 in craniofacial development, neural crest cell migration, and hedgehog signal transduction (B.S. and S.R.W., unpublished). Together, these findings place IP production as a key mediator for cellular signaling mechanisms that regulate vital cellular and developmental processes. How these and other IPs are integrated with cell–cell signaling networks during complex processes, such as, tissue morphogenesis and maintenance of cell fate and function? We propose that with its enormous resource and unique set of structural, functional, and sensory attributes, cilium provides a platform for executing IP-based signaling functions. Given the evolutionary conservation of the IP repertoire and pathways, the developmental and molecular events uncovered in our studies in the zebrafish system could be applicable in other vertebrates including humans. This unbiased approach of systematic identification of IP functions in cilia and development will aid in understanding of multiple disease pathologies including ciliopathies and dysmorphic syndromes.