The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP 6 ) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P 3 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP 4 ) isomers, inositol pentakisphosphate (InsP 5 ) and InsP 6 , whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P 4 5-kinase does not increase the amount of InsP 5 and InsP 6 , although its depletion does block InsP 5 and InsP 6 production, showing that it is necessary for production of InsP 5 and InsP 6 . Expression of Ins(1,3,4,5,6)P 5 2-kinase increases the amount of InsP 6 by depleting the InsP 5 in the cell, and depletion of 2-kinase decreases the amount of InsP 6 and causes an increase in InsP 5 . These results are consistent with a pathway that produces InsP 6 through the sequential action of Ins(1,3,4)P 3 5/6-kinase, Ins(1,3,4,6)P 4 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P 3 to InsP 6 . Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.Ins(1,2,3,4,5,6)P 6 (InsP 6 ) 1 has been implicated in many cellular processes. It is required for mRNA export from the nucleus in yeast (1) and human cells (2). InsP 6 binds to the clathrin assembly proteins AP2 and AP180 (3, 4) and inhibits clathrin cage assembly in vitro (5, 6). InsP 6 inhibits serine and threonine protein phosphatases, which are thought to regulate L type Ca 2ϩ channels in pancreatic islet cells (7). Nonhomologous DNA end joining of double strand breaks is stimulated by InsP 6 through its binding to the Ku70/80 subunits of DNA-PK (8, 9). Most recently, InsP 6 has been suggested to stimulate endocytosis, possibly by the activation of protein kinase C and inhibition of synaptojanin (10). The many roles for InsP 6 necessitates an understanding of the pathway leading to its production.InsP 6 is synthesized ultimately from Ins(1,4,5)P 3 (Fig. 1). The action of phospholipase C on the lipid phosphatidylinositol (4,5)-bisphosphate yields Ins(1,4,5)P 3 and diacylglycerol. Ins(1,4,5)P 3 can then be phosphorylated by an Ins(1,4,5)P 3 3-kinase to Ins(1,3,4,5)P 4 or dephosphorylated by an inositol polyphosphate 5-phosphatase to Ins(1,4)P 2 (11); Ins(1,3,4,5)P 4 can be also be dephosphorylated by 5-phosphatases, yielding Ins(1,3,4)P 3 . When looking at the formation of the soluble inositol phosphates upon phospholipase C activation in rat pancreatoma cells, Menniti et al. (12) saw that in addition to the expected increase of the Ins(1,4,5)P 3 isomer, Ins(1,3,4)P 3 is also increased, as well as Ins(1,3,4,6)P 4 . They therefore argued that in vivo Ins(1,4,5)P 3 is phosphorylated to Ins(1,3,4,5)P 4 by an Ins(1,4,5)P 3 3-kinase, dephosphorylated to Ins(1,3,4)P 3 by a 5-phosphatase, and phosphorylated to Ins(1,3,4,6)P 4 (Fig. 1A). The increase of Ins(1,3,4)P 3 was also seen by Wong et al. (13) in WRK-1 cells st...
Many functions have been suggested for inositol 1,2,3,4,5,6-hexakisphosphate (InsP 6), including mRNA export, nonhomologous end-joining, endocytosis, and ion channel regulation. However, it remains to be demonstrated that InsP6 is necessary for in vivo survival. We previously isolated a cDNA encoding the mammalian inositol 1,3,4,5,6-pentakisphosphate (InsP 5) 2-kinase (2-kinase), the enzyme that converts InsP 5 to InsP6. We used the sequence to search the BayGenomics databases and identify an ES cell line (XA232) that has a gene trap construct embedded in the 2-kinase gene. We obtained a mouse from this line, produced heterozygotes, and confirmed that the heterozygotes contain the trapping construct and have diminished 2-kinase activity. Breeding the XA232 heterozygotes produced no homozygous offspring; thus, loss of 2-kinase is lethal in mice. Dissections of embryonic day-8.5 uteri yielded no homozygous embryos; thus, the mice die before day 8.5 postcoitum. The gene trap construct contains a -galactosidase͞neomycin reporter gene, allowing us to stain heterozygotes for -galactosidase to determine tissue-specific expression of 2-kinase protein. 2-kinase is expressed in the hippocampus, the cortex, the Purkinje layer of the cerebellum in the brain, in cardiomyocytes, and in the testes of adult mice. At day 9.5 postcoitum, 2-kinase was expressed in the notochord, the ventricular layer of the neural tube, and the myotome of the somites. Intense staining was also seen in the yolk sac, suggesting that InsP6 is necessary for yolk sac development or function. Furthermore, failure of yolk sac development or function is consistent with the early lethality of 2-kinase embryos.embryonic lethal ͉ gene deletion ͉ inositol signals
Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles. INTRODUCTIONDense-core secretory vesicles, also called secretory granules, are specialized vesicles with a condensed protein core (Halban and Irminger, 1994). The vesicle core can be described as a dynamic aggregate. First, it is assembled within the secretory pathway from previously soluble proteins. Conversion of a set of soluble proteins to a temporarily insoluble form can serve diverse functions, including their efficient storage in a state that imposes a reduced osmotic burden. Secondly, when an appropriate exocytic stimulus triggers the fusion of the vesicle membrane with the plasma membrane, the dissemination of the secreted contents involves re-or dis-assembly of the core. Granule core dynamics exemplify the potential for compartmentspecific regulation of protein interactions in the secretory pathway (Arvan and Castle, 1992), and offer the opportunity to investigate the underlying mechanisms.Interactions among proteins in transit may be influenced by their microenvironments, as for example gradients of pH and calcium concentration within and between secretory compartments. The potential importance of such modulation is not limited to granule assembly. For example, the assembly of T-cell receptor subunits may be controlled by their calcium-specific binding to BiP (Suzuki et al., 1991), and ligand-binding by the ER retrieval receptor may be regulated by the pH difference between the ER and the Golgi (Wilson et al., 1993). For proteins forming the dense cores of neuroendocrine granules, a combination of these factors in the TGN and newly formed secretory vesicles have been implicated in the induction of aggregation/ condensation (references in Natori and Hut...
Fatty acid synthase (FAS) promotes energy storage through de novo lipogenesis and participates in signaling by the nuclear receptor PPAR␣ in noncardiac tissues. To determine if de novo lipogenesis is relevant to cardiac physiology, we generated and characterized FAS knockout in the myocardium (FASKard) mice. FASKard mice develop normally, manifest normal resting heart function, and have normal cardiac PPAR␣ signaling as well as fatty acid oxidation. However, they decompensate with stress. Most die within 1 h of transverse aortic constriction, probably due to arrhythmia. Voltage clamp measurements of FASKard cardiomyocytes show hyperactivation of L-type calcium channel current that could not be reversed with palmitate supplementation. Of the classic regulators of this current, Ca 2؉ / calmodulin-dependent protein kinase II (CaMKII) but not protein kinase A signaling is activated in FASKard hearts, and knockdown of FAS in cultured cells activates CaMKII. In addition to being intolerant of the stress of acute pressure, FASKard hearts were also intolerant of the stress of aging, reflected as persistent CaMKII hyperactivation, progression to dilatation, and premature death by ϳ1 year of age. CaMKII signaling appears to be pathogenic in FASKard hearts because inhibition of its signaling in vivo rescues mice from early mortality after transverse aortic constriction. FAS was also increased in two mechanistically distinct mouse models of heart failure and in the hearts of humans with end stage cardiomyopathy. These data implicate a novel relationship between FAS and calcium signaling in the heart and suggest that FAS induction in stressed myocardium represents a compensatory response to protect cardiomyocytes from pathological calcium flux.Storing energy preserves the function of many mammalian tissues during physiological and pathological stress. One of the ways that tissues store energy is through de novo lipogenesis, the synthesis of saturated fatty acids from carbohydrate precursors (1). This process, accomplished by iterative two-carbon additions to a fatty acid chain, is mediated by the multifunctional enzyme FAS 2 (2, 3). Initially relegated to cellular housekeeping, FAS was recently also shown to participate in intracellular signaling. Whole body FAS-deficient embryos die before uterine implantation (4), but liver-specific FAS knock-out mice (5) have decreased expression of fatty acid oxidation genes (classic targets of PPAR␣ signaling) and a phenotype resembling PPAR␣-deficient mice (6). Pharmacological activation of PPAR␣ reverses these features, suggesting that FAS generates an endogenous ligand for PPAR␣, and this FAS-dependent molecule was recently identified as a discrete phosphatidylcholine species (7). FAS also triggers PPAR␣ signaling in the hypothalamus (8) and in macrophages (9), raising the possibility that molecules generated by this enzyme are necessary for activating fatty acid metabolism, even in fatty acid-dependent tissues, such as the heart.Myocardial reliance on fatty acids as an energy source...
Cells amplify and regulate signals through the generation of a variety of second messengers. The inositol polyphosphate family of second messengers has grown in complexity with the discovery of new functions for the soluble, more highly phosphorylated inositols. The common precursor of all soluble inositol phosphates in mammalian cells is Ins(1,4,5)P 3 , which is produced when phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate yielding InsP 3 and diacylglycerol. InsP 3 is then metabolized to a number of more highly phosphorylated inositol species through the actions of several phosphatases and kinases; the cellular functions of these inositol polyphosphates are beginning to be elucidated (1-4). An understanding of the enzymes responsible for the production of soluble inositol polyphosphates will be critical to establishing their roles in cellular physiology.The major inositol pentakisphosphate isomer in eukaryotic cells, inositol 1,3,4,5,6-pentakisphosphate (InsP 5 ), 1 is converted to inositol hexakisphosphate (InsP 6 ) by phosphorylation at the D2 position of the inositol ring.
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