Inositol phosphates are a group of highly complex molecules for which cellular functions are being defined; however, this process has been difficult because of the large number of different inositol phosphates, with varying numbers of phosphates in different positions of the inositol ring. The synthetic pathway generating higher order inositol phosphates, inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P 5 ) 1 and inositol hexakisphosphate (InsP 6 ), in human cells is not completely defined. Current evidence supports inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) as the common precursor and Ins(1,3,4,5,6)P 5 and InsP 6 as the predominant higher inositol phosphates in human cells, and the synthetic pathway from Ins(1,4,5)P 3 to Ins(1,3,4,5,6)P 5 remains an area of active investigation (for general reviews, see Refs. 1 and 2). Hence, the goal of the current study is to define a catalytic step leading to Ins(1,3,4,5,6)P 5 synthesis.The best characterized InsP 6 synthetic pathway is in Saccharomyces cerevisiae (3). The common precursor Ins(1,4,5)P 3 is synthesized from phospholipase C and subsequently used by two distinct proteins, Ipk2p and Ipk1p, ultimately to generate InsP 6 . The IPK1 and IPK2 genes were isolated from a genetic screen for mutants that were synthetically lethal in combination with a gle1-2 mutant, which is defective in mRNA export. Sequential phosphorylation of Ins(1,4,5)P 3 at the D-6 and D-3 positions generates Ins(1,4,5,6)P 4 and Ins(1,3,4,5,6)P 5 , respectively, catalyzed by the IPK2 gene product (4). Next, phosphorylation at the D-2 position yields InsP 6 catalyzed by the IPK1 gene product (5).In human cells, the synthetic pathway to higher inositol phosphates shares the same initial step to produce the common precursor Ins(1,4,5)P 3 catalyzed by phospholipase C (2). However, the pathway from Ins(1,4,5)P 3 to Ins(1,3,4,5,6)P 5 is less clear. Two different synthetic pathways generating different isomers of InsP 4 have been described. First, the synthesis of Ins(1,3,4,5)P 4 from Ins(1,4,5)P 3 has been well documented to be catalyzed by inositol 1,4,5-trisphosphate 3-kinases (InsP 3 3-kinases) in response to stimulation by various ligands (6, 7). Genes encoding the InsP 3 3-kinase have been cloned (8 -10) and the enzymatic catalysis characterized (6). Second, the synthesis of Ins(1,3,4,6)P 4 from Ins(1,3,4)P 3 was described to be catalyzed by the inositol 1,3,4-trisphosphate 5/6-kinase (InsP 3 5/6-kinase), which has no activity toward the Ins(1,4,5)P 3 isomer (11). The Ins(1,3,4)P 3 isomer is synthesized from Ins(1,3,4,5)P 4 catalyzed by 5-phosphatase(s). The human enzymes responsible for the conversion of either Ins(1,3,4,5)P 4 or Ins(1,3,4,6)P 4 to Ins(1,3,4,5,6)P 5 have not been reported. The final step is the phosphorylation of Ins(1,3,4,5,6)P 5 at the D-2 position catalyzed by inositol 1,3,4,5,6-pentakisphosphate 2-kinase (12).To date, the synthesis of Ins(1,3,4,5,6)P 5 from InsP 4 has been characterized in S. cerevisiae, as noted above, and in rat. The rat inositol phosphate multikinas...
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...
We describe the SEM appearance of the rat endosteal bone lining cell ( BLC ) population, and the sequence of morphological changes of these cells as they self-incorporate into unmineralized bone matrix (osteoid), establish intercellular connections, and construct lacunae. The osteoblast/nascent osteocyte series was progressively unsheathed by gentle digestion of the osteoid with 0.25% collagenase. The osteoblasts which leave the polygonally packed BLC compartment rapidly develop numerous complexly branched processes that contact the processes elaborated by previous generations of maturing and mature osteocytes. As osteoblasts mature and approach the mineralization front, they appear to lose processes. The mature cells begin to form osteocyte lacunae by depositing an asymmetric perimeter of woven collagen fibrils, such that as the cells roof-over, the lacunae appear as pocketlike constructions. The collagen fibrils on the perilacunar matrix are oriented in a tangential or circular pattern, while those in the more distal matrix are arranged in a parallel pattern. With the completion of a lacuna, its wall appears to mineralize quickly, for lacunae could be recognized only when they are forming.
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