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Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation.

ABSTRACT: Schizosaccharomyces pombe extracts synthesize InsP6 (myo-inositol hexaphosphate) from Ins(1,4,5)P3 plus ATP. An S. pombe soluble fraction converts Ins(1,4,5)P3 into Ins(1,4,5,6)P4 and Ins(1,3,4, 5)P4, in a constant ratio of approximately 5:1, and thence to Ins(1, 3,4,5,6)P5 and InsP6. We have purified a soluble Mg2+-dependent kinase of molecular mass approximately 41 kDa that makes Ins(1,4,5, 6)P4 and Ins(1,3,4,5)P4 in the same ratio and also converts Ins(1,4, 5,6)P4 or Ins(1,3,4,5)P4 into Ins(1,3,4,5,6)P5 and InsP6. Of InsP3 isomers other than Ins(1,4,5)P3, only the non-biological molecule Ins(1,4,6)P3 potently 'competed' with all steps in conversion of Ins(1,4,5)P3 into InsP6. Examination of molecular graphics representations allowed us to draw tentative conclusions about the environment needed for an hydroxyl group to be phosphorylated by this kinase and to predict successfully that the purified kinase would phosphorylate the 5-hydroxyl of Ins(1,4,6)P3. S. pombe that have been cultured with [3H]inositol contains a variety of 3H-labelled inositol polyphosphates, with Ins(1,4,5)P3 and InsP6 the most prominent, and the InsP6 concentration quickly increases in hyper-osmotically stressed S. pombe. This yeast therefore contains InsP6 and Ins(1,4,5)P3 as normal constituents, makes more InsP6 when hyper-osmotically stressed and contains a versatile inositol polyphosphate kinase that synthesizes InsP6 from Ins(1,4,5)P3.

SUBMITTER: Ongusaha PP

PROVIDER: S-EPMC1219831 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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Project description:The metabolism of biologically active inositol phosphates in developed ovarian follicles from Xenopus laevis was investigated. Techniques used were microinjection of tracer into the intact oocyte coupled by gap junctions to follicle cells, as well as addition of tracer to homogenates of ovarian follicles and to homogenates of oocytes stripped of outer follicle-cell layers. Metabolism was similar to that previously described for other types of cell and tissue, with several unusual features. Homogenates of ovarian follicles were shown to contain an apparent 3'-phosphomonoesterase capable of converting [3H]Ins(1,3,4,5)P4 predominantly into a substance with h.p.l.c. elution characteristics of Ins(1,4,5)P3. In intact ovarian follicles, little Ins(1,4,5)P3 was formed but the esterase was activated by the phorbol ester activator of protein kinase C, PMA (phorbol 12-myristate 13-acetate; 60 nM), as well as by acetylcholine (200 microM). In follicle homogenates, this enzyme also appeared to be active in converting [3H]Ins(1,3,4)P3 into a substance eluting as Ins(1,4)P2. The apparent 3'-phosphomonoesterase activity was not inhibited by intracellular (or higher) levels of Mg2+. Although PMA activated this enzyme in intact oocytes relative to 5'-phosphomonoesterase activation, it did not enhance overall metabolism, in contrast with reports on other tissues. Compared with the processing of inositol phosphates injected into the intact follicle, homogenization in simulated intracellular medium appeared to alter the activity and/or accessibility of several enzymes. The metabolism of inositol phosphates appears to occur predominantly in the follicle cells surrounding the oocyte, as collagenase treatment followed by defolliculation greatly diminished the rates of metabolism of several inositol phosphates. The presence in Xenopus ovarian follicles of a 3'-phosphomonoesterase activated by protein kinase C in addition to the well-known 3'-kinase suggests that, by forming a reversible interconversion between Ins(1,4,5)P3 and Ins(1,3,4,5)P4, this tissue may have the potential to prolong stimulatory signals on binding of appropriate agonists to receptors.

Project description:Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] originating in the vascular smooth-muscle cells (VSMCs) has been shown to modulate the Ca(2+) stores in endothelial cells (ECs). However, the reverse is not found, suggesting that Ins(1,4,5)P(3) movement might be unidirectional across gap junctions at the myoendothelial junction (MEJ), or that distribution of the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)-R] is different between the two cell types. To study trans-junctional communication at the MEJ, we used a vascular-cell co-culture model system and selectively modified the connexin composition in gap junctions in the two cell types. We found no correlation between modification of connexin expression and Ins(1,4,5)P(3) signaling between ECs and VSMCs. We next explored the distribution of Ins(1,4,5)P(3)-R isoforms in the two cell types and found that Ins(1,4,5)P(3)-R1 was selectively localized to the EC side of the MEJ. Using siRNA, selective knockdown of Ins(1,4,5)P(3)-R1 in ECs eliminated the secondary Ins(1,4,5)P(3)-induced response in these cells. By contrast, siRNA knockdown of Ins(1,4,5)P(3)-R2 or Ins(1,4,5)P(3)-R3 in ECs did not alter the EC response to VSMC stimulation. The addition of 5-phosphatase inhibitor (5-PI) to ECs that were transfected with Ins(1,4,5)P(3)-R1 siRNA rescued the Ins(1,4,5)P(3) response, indicating that metabolic degradation of Ins(1,4,5)P(3) is an important part of EC-VSMC coupling. To test this concept, VSMCs were loaded with 5-PI and BAPTA-loaded ECs were stimulated, inducing an Ins(1,4,5)P(3)-mediated response in VSMCs; this indicated that Ins(1,4,5)P(3) is bidirectional across the gap junction at the MEJ. Therefore, localization of Ins(1,4,5)P(3)-R1 on the EC side of the MEJ allows the ECs to respond to Ins(1,4,5)P(3) from VSMCs, whereas Ins(1,4,5)P(3) moving from ECs to VSMCs is probably metabolized before binding to a receptor. This data implicates the MEJ as being a unique cell-signaling domain in the vasculature.

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Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation.

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