Lysophosphatidic acid activates the 70-kDa S6 kinase via the lipoxygenase pathway.

Many hormones are known to activate the 70-kDa S6 kinase (p70(S6K)). The signalling pathways mediating p70(S6K) activation are only partially characterized. We investigate, in this report, the mechanisms by which lysophosphatidic acid (LPA) activates p70(S6K). We observed that p70(S6K) activation was conventional, in that it was sensitive to both rapamycin and PI3 kinase inhibition. p70(S6K) activation appeared to be caused by the activation of several phospholipase pathways. LPA was an effective stimulus of phospholipase C induced intracellular calcium mobilization, which appeared to participate in p70(S6K) activation. Similarly, the effect of LPA on p70(S6K) activity was antagonized by butan-1-ol but not butan-2-ol suggesting the involvement of agonist stimulated phospholipase D activity. Further, antagonism of the phospholipase A(2) and lipoxygenase pathways attenuated p70(S6K) activation indicating a novel mechanism of p70(S6K) regulation. We conclude that in Swiss 3T3 cells LPA coordinates activation of several phospholipases to regulate p70(S6K).

Activation of endogenous thrombin receptors causes clustering and sensitization of epidermal growth factor receptors of swiss 3T3 cells without transactivation.

The G protein-coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin-containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cgamma1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.

Insulin induces epidermal growth factor (EGF) receptor clustering and potentiates EGF-stimulated DNA synthesis in swiss 3T3 cells: a mechanism for costimulation in mitogenic synergy.

In many cellular systems, activation with more than one ligand can produce a cellular response that is greater than the sum of the individual responses to the ligands. This synergy is sometimes referred to as coactivation. In Swiss 3T3 fibroblasts, activation of the epidermal growth factor (EGF) receptor produces a weak induction of DNA synthesis. Insulin has no stimulatory effect on this response. However, in combination, EGF and insulin synergize to cause a large induction of S phase. The underlying cellular biochemistry of this effect has been examined. The data indicate that phospholipase C activation is a major component of agonist-induced DNA synthesis. In contrast, activation of p70 S6 kinase by single agonists was inversely related to their ability to stimulate DNA synthesis. Therefore, it was examined whether stimulation of Swiss 3T3 cells with insulin causes changes in the subcellular distribution of EGF receptors and phospholipase Cgamma1 that could potentially explain the observed synergy or costimulation. It was found that insulin effectively induced the accumulation of EGF receptors on the actin arc of cells without activation of the EGF receptor. In contrast, EGF, when added for several hours, did not cause accumulation of the EGF receptor at this site. However, both EGF and insulin stimulated the accumulation of phospholipase Cgamma1 at the actin arc, which was coincident with the EGF receptor in the case of insulin- stimulated cells. Therefore, it is suggested that the insulin-induced coclustering of the EGF receptor with phospholipase Cgamma1 at the actin arc may allow for greater efficiency of signal transduction, resulting in the synergy observed for these two hormones in stimulation of DNA synthesis.

Cellular function of p70S6K: a role in regulating cell motility.

The 70 kDa ribosomal S6 kinase (p70S6K) is activated by numerous mitogens, growth factors and hormones. Activation of p70S6K occurs through phosphorylation at a number of sites and the primary target of the activated kinase is the 40S ribosomal protein S6, a major component of the machinery involved in protein synthesis in mammalian cells. In addition to its involvement in regulating translation, p70S6K activation has been implicated in cell cycle control and neuronal cell differentiation. Recent data obtained in this laboratory suggests that p70S6K may also function in regulating cell motility, a cellular response that is important in tumour metastases, the immune response and tissue repair. The present paper reviews the regulation and cellular function of p70S6K and proposes a novel function of p70S6K in regulating cell motility.

Nitric oxide donors selectively potentiate thrombin-stimulated p70(S6k) activity and morphological changes in Swiss 3T3 cells.

Thrombin stimulates both DNA synthesis and cell morphological changes in Swiss 3T3 cells, although the mechanism of signal coordination leading to these responses is unknown. We report here that nitric oxide (NO) donors selectively enhance thrombin-stimulated p70(S6k) activity by 40-60%, an effect that was sustained for 24 h. Potentiation of p70(S6k) also was observed with cGMP analogues indicating that this effect is mediated by cGMP-activated protein kinase. NO donors also induced morphological changes characterized by spindle-shaped cells in confluent, nondividing cells or by extended protrusions from the trailing edge in subconfluent, polarized cells. NO donors had no significant effects on intracellular Ca(2+) mobilization, DNA synthesis, proliferation, or ERKs 1 and 2 and p90RSK activities, indicating that mitogenic responses and cell division are not altered by NO donors. We conclude that NO donors modulate the morphological changes associated with cellular motility in response to thrombin stimulation through selective enhancement of p70(S6k) activity.

Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes.

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P3 induces the activation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P3 receptors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concentration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caused near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2+ release. Similar results were obtained using another Ins(1,4,5)P3-receptor agonist, 2-hydroxyethyl-alpha-d-glucopyranoside 2,3',4'-trisphosphate. Anti-type-1 Ins(1,4,5)P3-receptor monoclonal antibody 18A10 inhibited vasopressin-stimulated Ca2+ inflow but had no observable effect on vasopressin-induced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. However, it did not substantially affect Ca2+ inflow and ER Ca2+ release induced by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release induced by ionomycin or Ins(1,4, 5)P3P4(5)-1-(2-nitrophenyl)ethyl ester ['caged' Ins(1,4,5)P3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)homopiperazine, HCl (ML-9), an inhibitor of myosin light-chain kinase, also inhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow and ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-induced Ins(1,4,5)P3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P3 requires the participation of a small region of the ER, which is distinguished from other regions of the ER by a different apparent affinity for Ins(1,4,5)P3 analogues and is associated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation of SOCs.

Injection of rat hepatocyte poly(A)+ RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels.

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP3F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin- and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less thari 5 microM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 microM Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with "mock"-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

Evidence that the pertussis toxin-sensitive trimeric GTP-binding protein Gi2 is required for agonist- and store-activated Ca2+ inflow in hepatocytes.

The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca2+ inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein alpha subunits, and carboxyl-terminal alpha-subunit synthetic peptides. An anti-Gi1-2 alpha antibody and a Gi2 alpha peptide (Gi2 alpha) Ile345-Phe355), but not a Gi3 alpha peptide (Gi3 alpha Ile344-Phe354), inhibited vasopressin- and thapsigargin-stimulated Ca2+ inflow, had no effect on vasopressin-stimulated release of Ca2+ from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca2+. An anti-Gq alpha antibody also inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. Immunofluorescence measurements showed that Gi2 alpha is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, Gq/11 alpha was found principally at the cell periphery. It is concluded that the trimeric G-protein, Gi2, is required for store-activated Ca2+ inflow in hepatocytes and acts between the release of Ca2+ from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca2+ channel protein(s) in the plasma membrane.

Evidence obtained using single hepatocytes for inhibition by the phospholipase C inhibitor U73122 of store-operated Ca2+ inflow.

The ability of 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipase C (Smith et al., J Pharmacol Exp Ther 253:688-697, 1992), to inhibit agonist-stimulated and store-operated Ca2+ inflow in single hepatocytes was investigated with the aim of testing whether the activation of phospholipase C is a necessary step in the process of agonist-stimulated Ca2+ inflow in this cell type. U73122 inhibited the release of Ca2+ from intracellular stores and plasma membrane Ca2+ inflow induced by vasopressin. An inactive analogue of U73122, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 2,5-pyrrolidone-dione (U73433), did not inhibit vasopressin-induced release of Ca2+ from intracellular stores, but did partially inhibit Ca2+ inflow. Neither U73122 nor 'inactive' analogue U73433 inhibited the release of Ca2+ from intracellular stores when this was initiated by the photolysis of 'caged' guanosine (5'-[gamma-thio]triphosphate (GTP gamma S) introduced to the cytoplasmic space by microinjection. However, both compounds inhibited GTP gamma S-stimulated Ca2+ inflow. U73122 also inhibited the actions of glycerophosphoryl-myo-inositol-4,5-diphosphate (GPIP2), a slowly-hydrolysed analogue of inositol 1,4,5-triphosphate (InsP3) which is released by photolysis of 'caged' 1-(alpha-glycerophosphoryl)-myo-inositol-4,5-diphosphate, P4(5)-1-(2-nitrophenyl)ethyl ester, and thapsigargin in stimulating Ca2+ inflow. U73122 did not inhibit GPIP2-stimulated release of Ca2+ from intracellular stores, but did partially inhibit the ability of thapsigargin to induce Ca2+ release. It is concluded that, while U73122 does inhibit phospholipase C beta in hepatocytes, complete inhibition of this enzyme in situ requires an intracellular concentration of U73122 higher than that achieved in the present experiments. Moreover, both U73122 and 'inactive' analogue U73433 have one or possibly two additional sites of action. These are likely to be the hepatocyte plasma membrane Ca2+ inflow channel protein (or a protein involved in the activation of this channel by the InsP3-sensitive intracellular Ca2+ store), and a protein involved in thapsigargin action.

A role for a pertussis toxin-sensitive trimeric G-protein in store-operated Ca2+ inflow in hepatocytes.

The mechanism of store-operated Ca2+ inflow in hepatocytes was investigated using fluo-3 and fura-2 to monitor changes in the concentration of intracellular free Ca2+ in single cells, and 1-(alpha-glycerophosphoryl)-myo-inositol 4,5-diphosphate, P4(5)-1-(2-nitrophenyl)ethyl ester ('caged' GPIP2) and 'caged' guanosine 5'-[gamma thio]triphosphate (GTP gamma S) (introduced into the cytoplasmic space by microinjection), thapsigargin and 2,5-di-tert- butylhydroquinone (DBHQ) to stimulate Ca2+ inflow. Photolysis of 'caged' GPIP2 or 'caged' GTP gamma S stimulated Ca2+ inflow. The abilities of GPIP2, thapsigargin and DBHQ to stimulate Ca2+ inflow were inhibited by the pre-treatment of hepatocytes with pertussis toxin in vivo for 36 h. Thapsigargin-stimulated Ca2+ inflow was also inhibited by guanosine 5'-[beta-thio]diphosphate (GDP beta S) (introduced by microinjection). It is concluded that, in hepatocytes, store-operated Ca2+ inflow induced by the actions of either inositol 1,4,5-trisphosphate, thapsigargin or DBHQ requires a pertussis toxin-sensitive trimeric G-protein.

A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes.

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or guanosine 5'-[beta gamma-imido]triphosphate to the cells, but not adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex.

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.