Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells.

Previous data suggest the existence of discrete pools of inositol lipids, which are components of a nuclear phosphoinositide (PI) cycle. However, it is not known whether the contents of these pools are regulated during cell proliferation. In the present study we demonstrate that the mass levels of three important constituents of the nuclear PI cycle are regulated during the cell cycle. Radioactive label incorporation into PtdIns(4,5)P(2) was seen to increase dramatically as synchronized cells entered S-phase. This did not coincide with any significant changes in the nuclear mass levels of this lipid, suggesting that the rate of turnover of this molecule was increased. Levels of PtdIns4P, the major substrate for PtdIns(4,5)P(2) production by Type I PtdInsP kinases (PIPkins), were regulated during the cell cycle and indicated a complex relationship between these two lipids. An alternative substrate for PtdIns(4,5)P(2), PtdIns5P, phosphorylated by Type II PIPkins, was present in nuclei at much smaller amounts than the PtdIns4P, and thus is unlikely to contribute significantly to PtdIns(4,5)P(2) turnover. However, a large increase in nuclear PtdIns5P mass was observed when murine erythroleukaemia cells are in G(1), and this could represent a potential pool of nuclear inositol lipid that has a specific signalling role. Analysis of extracted lipid fractions indicated the absence of any PtdIns3P in these nuclei.

A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress.

BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a key second messenger found ubiquitously in higher eukaryotic cells. The activation of Class I phosphoinositide 3-kinases and the subsequent production of PtdIns(3,4,5)P(3) is an important cell signaling event that has been causally linked to the activation of a variety of downstream cellular processes, such as cell migration and proliferation. Although numerous proteins regulating a variety of biological pathways have been shown to bind PtdIns(3,4,5)P(3), there are no data to demonstrate multiple mechanisms for PtdIns(3,4,5)P(3) synthesis in vivo. RESULTS: In this study, we demonstrate an alternative pathway for the in vivo production of PtdIns(3,4,5)P(3) mediated by the action of murine Type Ialpha phosphatidylinositol 4-phosphate 5-kinase (Type Ialpha PIPkinase), an enzyme best characterized as regulating cellular PtdIns(4,5)P(2) levels. Analysis of this novel pathway of PtdIns(3,4,5)P(3) synthesis in cellular membranes leads us to conclude that in vivo, Type Ialpha PIPkinase also acts as a PtdIns(3,4)P(2) 5-kinase. We demonstrate for the first time that cells actually contain an endogenous PtdIns(3,4)P(2) 5-kinase, and that during oxidative stress, this enzyme is responsible for PtdIns(3,4,5)P(3) synthesis. Furthermore, we demonstrate that by upregulating the H(2)O(2)-induced PtdIns(3,4,5)P(3) levels using overexpression studies, the endogenous PtdIns(3,4)P(2) 5-kinase is likely to be Type Ialpha PIPkinase. CONCLUSIONS: We describe for the first time a novel in vivo activity for Type Ialpha PIPkinase, and a novel pathway for the in vivo synthesis of functional PtdIns(3,4,5)P(3), a key lipid second messenger regulating a number of diverse cellular processes.

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity.

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.

Nuclear inositides: inconsistent consistencies.

It is now clear that phosphoinositides, which play a major role in the regulation of a variety of cellular processes in the cytoplasm, are found within the nucleus. Their role in this subcellular compartment is still contentious: however, data has suggested that nuclear inositides generate substrates, such as PtdIns(4,5)P2, utilised by a number of nuclear signalling pathways: for example, nuclear phospholipase C and the PtdIns 3-kinase cascade. There is also evidence that PtdIns(4,5)P2 may play a role in the localisation and regulation of a number of nuclear proteins such as the BAF complex, which is involved in the regulation of chromatin structure. Although the presence of nuclear inositides has been demonstrated in a number of different cell types, suggesting that it is ubiquitous, there are many inconsistencies within the literature concerning the locations and isotypes of enzymes that are involved in their regulation and in the potential second messengers which are generated by them. This review aims to highlight some of these inconsistencies in order to focus on areas that need further characterisation.

Nuclei contain two differentially regulated pools of diacylglycerol.

A number of recent studies have highlighted the presence of a nuclear pool of inositol lipids [1] [2] that is regulated during progression through the cell cycle [1] [3], differentiation [1] [2] and after DNA damage [2], suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG) [1] [3] [4], which is important in the activation of nuclear protein kinase C (PKC) [5] [6] [7]. Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase [4] and through G2 to mitosis [3] [8] [9] [10] [11]. Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cbeta1 (PIC-beta1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs).

Phospholipid signalling in the nucleus. Een DAG uit het leven van de inositide signalering in de nucleus.

Diverse methodologies, ranging from activity measurements in various nuclear subfractions to electron microscopy, have been used to demonstrate and establish that many of the key lipids and enzymes responsible for the metabolism of inositol lipids are resident in nuclei. PtdIns(4)P, PtdIns(4,5)P2 and PtdOH are all present in nuclei, as well as the corresponding enzyme activities required to synthesise and metabolise these compounds. In addition other non-inositol containing phospholipids such as phosphatidylcholine constitute a significant percentage of the total nuclear phospholipid content. We feel that it is pertinent to include this lipid in our discussion as it provides an alternative source of 1, 2-diacylglycerol (DAG) in addition to the hydrolysis of PtdIns(4, 5)P2. We discuss at length data related to the sources and possible consequences of nuclear DAG production as this lipid appears to be increasingly central to a number of general physiological functions. Data relating to the existence of alternative pathways of inositol phospholipid synthesis, the role of 3-phosphorylated inositol lipids and lipid compartmentalisation and transport are reviewed. The field has also expanded to a point where we can now also begin to address what role these lipids play in cellular proliferation and differentiation and hopefully provide avenues for further research.

Membrane association, localization and topology of rat inositol 1,4,5-trisphosphate 3-kinase B: implications for membrane traffic and Ca2+ homoeostasis.

We previously reported the isolation of a rat cDNA clone encoding a protein with significant sequence homology to the B isoform of human myo-inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase B); this protein was thus designated rat IP3 3-kinase B [Thomas, Brake, Luzio, Stanley and Banting (1994) Biochim. Biophys. Acta 1220, 219-222]. However, no IP3 kinase isoform had been shown to generate the physiologically important isoform of inositol tetrakisphosphate, i.e. inositol 1,3,4,5-tetrakisphosphate. We now present direct evidence that the putative rat IP3 3-kinase B is genuinely an IP3 3-kinase. We also show that the enzyme exists both as a peripheral membrane protein tightly associated with the cytosolic face of the extended endoplasmic reticulum network, and as a cytosolic protein. Association of the IP3 3-kinase with membranes is not affected by treatment with brefeldin A, Na2CO3 (pH 11.5), 2 M NaCl, or alteration of [Ca2+]. However, treatment of isolated membranes with 4 M urea leads to dissociation of the kinase from the membrane, implying that membrane association involves specific, conformation-dependent protein-protein interactions. The fact that IP3 3-kinase B is localized exclusively to membranes of Ca2+ stores, is consistent with a model where this kinase plays a role in IP3-dependent Ca2+ release.

Phospholipases in the nucleus.

There is a well established role for various phospholipases involved in the production of intracellular signals at the plasma membrane. In contrast much less is known of their role in other intracellular compartments, however, emerging evidence would suggest that some of these enzymes are also involved in the production of signals within the nucleus. Translocation to and activation of protein kinase C (PKC) within the nucleus has been suggested to be important in a number of diverse cellular processes suggesting the requirement for the intranuclear production of diacylglycerol (DAG), a known physiological activator of this enzyme. As the activation of a number of phospholipases leads to the production of DAG this review will consider the notion that these enzymes are present within the nucleus and that their activities can be stimulated to produce this important regulator of PKC.

Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus.

We have expressed rat myo-inositol 1,4,5-trisphosphate (IP3) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli. The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE). However, soluble, calmodulin-binding non-fusion IP3 3-kinase B is enzymically inactive. The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.

Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes.

The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.

Identification of high molecular weight forms of inositol 1,4,5-trisphosphate 3-kinase in rat thymus and human lymphocytes.

A soluble inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) has been characterized from extracts of rat thymus. The enzyme was shown to have a molecular weight within the range 98,000-114,000 M(r) as determined by regeneration of enzyme activity from sodium dodecyl sulphate polyacrylamide gels. The enzyme phosphorylates inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) with an apparent Km of 3.1 +/- 0.4 microM. The enzyme is stimulated 4-6-fold by Ca2+/calmodulin and is not recognised by polyclonal antisera raised against rat brain InsP3 3-kinase A. High levels of InsP3 3-kinase activity were also detected in soluble extracts of human lymphocyte preparations. The human lymphocyte enzyme was shown to have a molecular weight between 61,000 and 70,000 M(r) as judged by SDS-PAGE, and was stimulated approximately 10-fold in the presence of Ca2+/calmodulin. These results establish that InsP3 3-kinase from rat thymus and human lymphocyte preparations represent high molecular weight isoenzymes of the InsP3 3-kinase family.

Stimulation of adenylate cyclase by amylin in CHO-K1 cells.

The CHO-K1 cell line responds to the peptide amylin by a rapid elevation of cAMP. The related peptide calcitonin gene-related peptide (CGRP) is 100 times less potent at stimulating adenylate cyclase than is amylin. The actions of amylin at this receptor are concentration dependent and not antagonized by the CGRP antagonist CGRP-(8-37). Although these cells have receptors for calcitonin, amylin is unable to take part in any high affinity interaction with these receptors, as assessed by radioligand binding. The CHO-K1 cell line has receptors for amylin that are distinct from those for calcitonin and CGRP.

Reversible calcitonin binding to solubilized sheep brain binding sites.

In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of 125I-labelled salmon CT (125I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with beta-octyl glucoside, 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +/- 0.5 nM and a maximum binding site concentration of 6.2 +/- 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems, Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mM. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 125I-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40% and 46% dissociation after 2 h at 4 degree C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11,18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr approximately solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified. one of Mr approximately 71,000 and another of Mr approximately 88,000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues.

Biologically active, derivatizable salmon calcitonin analog: design, synthesis, and applications.

An analog of salmon calcitonin (sCT) has been synthesized, substituting Arg at positions 11 and 18 and Lys at position 14 to provide a free amino group for derivatization. The potency of [Arg11,18,Lys14]sCT was equivalent to that of sCT in activating adenylate cyclase in UMR 106-06 cells. The analog was derivatized with biotin, fluorescein, or 4-azidobenzoate without loss of activity. The derivatized analog was not degraded by lysine-specific endoprotease, whereas the underivatized [Arg11,18,Lys14]sCT was cleaved at Lys-14. The derivatized analogs were purified by HPLC and subsequently shown to possess full biological activity. The photoactive analog was used to photolabel 88,000 and 71,000 mol wt components of the calcitonin receptor in rat osteoclasts, but only an 88,000 mol wt component was photolabeled in the UMR 106-06 cells.

Solubilization of functional calcitonin receptors.

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.

Human placental calcitonin receptors.

Receptors for the hypocalcaemic hormone, calcitonin (CT), have been identified in a membrane fraction prepared from term human placentae. Binding of 125I-labelled salmon CT (125I-sCT) to the membranes was time- and temperature-dependent, saturable (Bmax. 58 +/- 11 fmol/mg of protein), of high affinity (Kd 80 +/- 21 pM) and poorly reversible. Species-specific CTs and CT analogues competed for 125I-sCT binding with potencies proportional to their known biological potencies. Various unrelated peptide hormones did not compete, indicating that receptor binding was specific for CT. Photoaffinity labelling using a derivatized biologically active sCT analogue, [Arg11,18,3-nitrophenylazide-Lys14]sCT, identified a receptor component of Mr approximately 85,000, comparable with findings in osteoclasts and other target cells. The presence of CT receptors in the human placenta supports other evidence that CT may have a role in the regulation of placental function.

Calcitonin receptors of human osteoclastoma.

Osteoclast-rich cultures were prepared by disaggregation of osteoclastomas (giant cell tumour of bone) and settlement onto glass or plastic surfaces. Autoradiography using [125I]-salmon calcitonin ([125I]-sCT) revealed specific binding only to multinucleate giant cells (osteoclasts) and a minor population of mononuclear cells. [125I]-sCT competitive binding studies indicated a Kd of 5 x 10(-10) M and receptor number of approximately 1 million sites/osteoclast. sCT treatment resulted in a dose-dependent rise in cAMP (EC50 10(-10) M). Homogenates of an osteoclastoma also demonstrated specific binding of [125I]-sCT. Chemical cross-linking of a labelled synthetic sCT derivative. [125I]-[Arg11,18,Lys14]-sCT, using disuccinimidyl suberate, resulted in labelling of a receptor component of approximate Mr 85-90,000. The multinucleate giant cells (osteoclasts) of human osteoclastomas possess large number of CT receptors which exhibit the same binding kinetics and apparent Mr as those of other CT target cells.