Nuclear diacylglycerol, the cell cycle, the enzymes and a red herring (or how we came to love phosphatidylcholine).
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Animals↗
Biomedical subjects
N Divecha
Publications and source records attributed to N Divecha.
Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.
Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (PtdIns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp60(c-src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.
Blood Platelets↗
Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells.
Differentiation of murine erythroleukaemia cells with the chemical agent DMSO leads to a cessation of proliferation and the production of a number of erythrocyte markers such as haemoglobin. We have previously demonstrated that activation of proliferation leads to an increase in the production of nuclear diacylglycerol (DAG). Here we demonstrate that differentiation leads to a decrease in the levels of nuclear DAG and the activity of the nuclear-associated phosphoinositidase C (PIC). The change in activity appears to be due to a decrease in the mass levels of the beta 1 isoform, as demonstrated by the use of isoform-specific antibodies. Moreover, the changes correlate with the cessation of proliferation and an increase in the number of cells in G1 phase of the cell cycle, rather than with the number of cells which have differentiated. Indeed, although treatment of the cells with phorbol 12-myristate 13-acetate (PMA) inhibits the differentiation programme as assessed by haemoglobin staining, it does not inhibit the number of cells blocking in G1 of the cell cycle or the changes in nuclear DAG or PIC activity. The possible involvement of this nuclear inositide cycle during progression through the cell cycle is discussed.
Animals↗
DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product.
DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs. Here, we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays, however, indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p, Tor2p, and FRAP, S. pombe rad3, and the product of the ataxia telangiectasia gene, mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action.
Ataxia Telangiectasia↗
The cloning and sequence of the C isoform of PtdIns4P 5-kinase.
In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.
Amino Acid Sequence↗
Cloning and characterisation of two new cDNAs encoding murine triple LIM domains.
We have cloned and sequenced two new cDNAs that code for proteins carrying the related triple LIM domains (acronym of Lin-11, Isl-1, Mec-3) proteins. These LIM domains show good agreement to the LIM domain consensus sequence, but also exhibit some novel variations. The 1.36-and 2.8-kb cDNAs are probably splice variants of one gene and code for 42- and 50-kDa proteins, respectively. The larger transcript has a 900-nucleotide (nt) 3' untranslated region (UTR). High levels of the 2.8-kb transcript can be detected in many tissues, and all tissues show some level of expression of both transcripts, the larger transcript being more abundant. In adult testis there are very high levels of the 1.36-kb transcript and moderate levels of the 2.8-kb transcript. The wide tissue distribution and high levels of expression suggest an important role for these proteins in cellular function.
Amino Acid Sequence↗
The nuclear phosphoinositide cycle--does it play a role in nuclear Ca2+ homoeostasis?
The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca2+ changes changing in response to cytosolic Ca2+, with little evidence for an independently controlled nuclear Ca2+ homeostasis. There are InsP3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP2, which may have distinct intranuclear functions.
Animals↗
Inositol lipid signalling occurs in brush-border membranes during initiation of compensatory renal growth in the rat.
Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.
Animals↗
Inositides and the nucleus and inositides in the nucleus.
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3T3 Cells↗
Monoclonal antibodies to phosphatidylinositol 4-phosphate 5-kinase: distribution and intracellular localization of the C isoform.
We have raised a panel of monoclonal antibodies to PtdIns4P 5-kinase C purified from bovine brain [Divecha, Brooksbank and Irvine (1992) Biochem. J. 288, 637-642]. This panel includes antibodies which specifically recognize PtdIns4P 5-kinase C both in a native catalytically active condition, and/or when presented on Western blots. Some of the former antibodies will also inhibit PtdIns4P 5-kinase C activity. We have used the blotting antibodies to study the bovine tissue distribution of PtdIns4P 5-kinase C and its distribution in mammalian species. We have also studied its localization in Jurkat cells and found it to be predominantly bound to membranes, with only a minority localized to the cytoskeleton. Neither PtdIns4P 5-kinase activity nor PtdIns4P 5-kinase C, as detected by Western blotting, were increased in the cytoskeleton after stimulation of Jurkat cells with OKT3. These antibodies should prove to be extremely useful tools with which to study the regulation of PtdIns4P 5-kinase C.
Animals↗
Nuclear diacylglycerol is increased during cell proliferation in vivo.
Highly purified nuclei were prepared from livers and kidneys of rats undergoing compensatory hepatic or renal growth, the former being predominantly by cellular proliferation, and the latter mostly by cellular enlargement. In liver, an increase in nuclear diacylglycerol (DAG) concentration occurred between 16 and 30 h, peaking at around 20 h. At the peak of nuclear DAG production a specific translocation of protein kinase C to the nucleus could be detected; no such changes occurred in kidney. There was no detectable change in whole-cell DAG levels in liver, and the increase in DAG was only measurable in nuclei freed of their nuclear membrane. Overall, these results suggest that there is a stimulation of intranuclear DAG production, possibly through the activation of an inositide cycle [Divecha, Banfić and Irvine (1991) EMBO J. 10, 3207-3214] during cell proliferation in vivo.
Animals↗
Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus.
The presence of phosphoinositide-mobilizing enzymes has been investigated in purified rat liver nuclei by radiolabelling and by probing with antibodies. A Ca(2+)-activated phosphoinositidase C (PIC) is present and was shown immunologically to be the beta 1 isoform. No gamma- or delta-PIC was found. However, only 5% of the total beta 1-PIC isoform is nuclear, with the majority being cytosolic. G alpha q and G alpha 11, the suggested physiological activators of beta 1-PIC, were not present. A PtdIns4P 5-kinase is also present, which immunologically is shown to be the C isoform. All of these nuclear inositide enzymes still remained after the removal of the nuclear envelope with Triton X-100, consistent with the concept of an intranuclear inositide cycle [Divecha, Banfic and Irvine (1991) EMBO. J. 10, 3207-3214].
Animals↗
Unclear or nuclear: another role for the phosphatidylinositol cycle?
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Animals↗
Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases.
We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].
Animals↗
Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands.
Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with alpha-amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.
Animals↗
Phospholipids in the nucleus--metabolism and possible functions.
Most of the phospholipids in the nuclear envelope are contained in the double nuclear membrane, and this has an active lipid metabolism consistent with its origins as a component of the endoplasmic reticular system. However, even after removal of the nuclear membrane with detergents, some phospholipids, mostly of unknown location and function, remain. Amongst these are all of the components of what appears to be a nuclear polyphosphoinositide signalling system, distinct from the well-established inositide pathway found in the plasma membrane. The consequences for nuclear function of the activation of these two inositide pathways are discussed, with a detailed consideration of proposed intranuclear functions for protein kinase C, and the maintenance of nuclear Ca2+ homoeostasis.
Animals↗
Molecular species analysis of 1,2-diacylglycerols and phosphatidic acid formed during bombesin stimulation of Swiss 3T3 cells.
Swiss 3T3 cells were labelled with [3H]glycerol and stimulated with bombesin over a time course of 20 min. The individual 1,2-diacylglycerols produced were quantified by acetylation followed by analysis by HPLC and argentation chromatography. The major phospholipids and phosphatidic acid were acetolysed and then analysed in the same manner. The data show that even at an early time of stimulation (30 s), stimulated diacylglycerol formation comes from at least two sources--phosphoinositides and phosphatidylcholine.
Animals↗