H. Banfic - Academia.edu (original) (raw)

Papers by H. Banfic

Research paper thumbnail of Unclear or nuclear: another role for the phosphatidylinositol cycle?

Biochemical Society Transactions, 1993

Research paper thumbnail of Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells

Biochemical Journal, 1995

Differentiation of murine erythroleukaemia cells with the chemical agent DMSO leads to a cessatio... more 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 s...

Research paper thumbnail of Croatian Institute for Brain Research: a model of priority research project

Research paper thumbnail of The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells

Leukemia, 2006

The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed... more The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, several studies demonstrated the activation of PI3K in the nuclei of all-trans-retinoic acid (ATRA)-differentiated HL-60 cells, raising the possibility that PI3K/Akt-inhibitors may block antitumor properties of retinoids. The aim of the present study was to investigate the possible activation of nuclear Akt in ATRA-treated cells and to test the effects of Akt-inhibitors on ATRA-mediated differentiation. The Akt-activity was found to be increased in the nuclei and lysates of ATRA-differentiated HL-60 and NB4 cells. The down-modulation of the expression of Akt protein in HL-60 cells using siRNA reduces the CD11b expression in ATRA-treated cells. The treatment of both cell lines with the commercially available Akt inhibitors inhibited the growth of both control and ATRA-treated cells. Aktinhibitors had no inhibitory effects on ATRA-mediated growth arrest and the expression of CD11b in HL-60 cells, but increased the percentage of control cells expressing CD11b. In contrast, the presence of Akt inhibitors reduced the expression of CD11b in ATRA-treated NB4 cells.

Research paper thumbnail of Biphasic Activation of PKBα/Akt In Platelets

Journal of Biological Chemistry, 1998

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggreg... more Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin ␣ IIb ␤ 3 activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2). After aggregation, a larger amount of PtdIns(3,4)P 2 is generated. We report that this latter PtdIns(3,4)P 2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca 2؉-dependent protease calpain. Elevation of cytosolic Ca 2؉ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KII␣." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the ␤␥ subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to G␤␥ and is, in addition, activated. Both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 have been shown to stimulate PKB␣/Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKB␣/Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P 3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P 2 generated after aggregation. There is thus potential for both pre-and postaggregation-dependent signaling by PKB␣/Akt.

Research paper thumbnail of Acetylcholine and cholecystokinin receptors functionally couple by different G‐proteins to phospholipase C in pancreatic acinar cells

FEBS Letters, 1988

We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C fro... more We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin‐octapeptide (CCK‐OP) and GTPγS but not carbachol (CCh)‐induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP‐analogue, 8‐bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist‐induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP‐ribosylated a 40 kDa protein, which was inhibited by CCK‐OP but not by CCh. We conclude from these data that both CCK‐ and muscarinic acetylcholine receptors functionally couple to phospholipase ...

Research paper thumbnail of Short-Term and Long-Term-Effects of Phorbol 12-Myristate 13-Acetate and Different Inhibitors on the Ability of Bone Marrow Cells to Form Colonies In Vitro

cclm, 1995

The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated i... more The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated increase in the colony-forming potential of murine (CBA) bone marrow cells was studied using known modulators of the mitogenic signal. Pretreatment of cells for 60 minutes with staurosporine (1 μηιοΙ/1), an inhibitor of protein kinase C, completely prevented colony formation in the control group of cells and significantly reduced the number of colonies formed in the phorbol ester-treated group. Brief exposure (60 min) of cells to the phospholipase A 2 inhibitors, mepacrine (500 μιηοΐ/ΐ) and heparin (1 g/1), reduced the number of colonies formed in the control group and completely abolished the increase in the number of colonies formed after treatment of the cells with phorbol ester. When inhibitors of protein kinase C or phospholipase A 2 were present during the entire period of the colony forming assay (7 days), no colonies could be scored in either the control or phorbol ester-treated groups of bone marrow cells. Long-term treatment or temporary exposure (60 min) of cells to indomethacin (50 μπιοΙ/1), an inhibitor of cyclooxygenase, or nordihydroguaiaretic acid (50 μπιοΐ/ΐ), an inhibitor of lipoxygenase, had no effect on colony formation in both groups. Pretreatment of cells for 45 min with calcium ionophore A23187 (10 μιηοΐ/ΐ) failed to increase the number of colonies, compared with the control group. Moreover, simultaneous treatment of cells for 45 min with phorbol ester (500 nmol/1) and A23187 (10 μιηοΐ/ΐ) did not produce any further increase in the number of colonies, compared with the phorbol ester-treated group, suggesting that elevation of intracellular calcium is unimportant in the phorbol ester-mediated response. Dibutyryl cyclic adenosine monophosphate (50 μιηοΐ/ΐ) in the presence or absence of phorbol ester, failed to stimulate colony formation, indicating that cyclic AMP-dependent protein kinases are not involved in the signalling process. Temporary exposure (75 min) of bone marrow cells to okadaic acid (1 μπιοΐ/l), a potent inhibitor of serine/ threonine phosphatases, or to tyrphostine AG-115 (20 μηιοΙ/1), a tyrosine kinase inhibitor, did not effect colony growth in the control or phorbol ester-treated group. The results indicate that phospholipase A 2 activation is involved in the phorbol ester-mediated increase in colony formation, since, of the different agents applied, only staurosporine, an inhibitor of protein kinase C, and mepacrine and heparin, putative inhibitors of phospholipase A 2 , were capable of abolishing phorbol ester-mediated effects.

Research paper thumbnail of Inositides and the nucleus and inositides in the nucleus

Cell, 1993

Although there are many forms of evidence linking phosphoinositides to nuclear function, the subs... more Although there are many forms of evidence linking phosphoinositides to nuclear function, the substance of the links remains largely undefined. One link between inositide metabolism and the nucleus is suggested by the implication of inositol trisphosphate (IP3) in the process of nuclear envelope reassembly (Sullivan et al., 1993). That paper will be discussed below in its context, but this review will principally focus on another nuclear-inositide connect ion--a potential inositide cycle in the nucleus. It comes as something of a shock to see data that point to a phosphoinositide cycle entirely separate from the familiar one in the plasma membrane. Again contrary to expectation, the data suggest that the cycle is not in the nuclear membrane but appears to be within the nucleus. This aspect of inositide function has profound implications for the role of inositides in cell division and growth. For example, it makes us rethink the tumor-promoting actions of phorbol esters and the teratogenic effects of Li ÷ that have been associated with inositide homeostasis. In this article the evidence of a nuclear inositide cycle and what is known about its control are reviewed, and the role it may play in eukaryotic cell function is discussed. For a discussion of proposed nuclear functions for protein kinase C and what little is known about nuclear Ca 2÷, the reader is referred to a more comprehensive recent review (Irvine and Divecha, 1992). Evidence for a Nuclear Inositide Cycle When incubated with [y-32P]ATP, nuclei purified from rat liver (Smith and Wells, 1984) or Friend cells (Cocco et al., 1987) incorporate radioactivity into PtdlP, PtdlP2, and PtdOH. These findings suggest that all the enzymes necessary for making polyphosphoinositides are present in nuclei. The pattern of incorporation of radioactivity into nuclear lipids differs between differentiated and undifferentiated Friend cells (Cocco et al., 1987); the lack of any difference in the patterns in whole-cell homogenates argues against simple contamination of the nuclei with other cellular membranes. However, the best case for a distinct inositide cycle in the nucleus may come from data on Swiss 3T3 cells (Divecha et al., 1991). Stimulation of quiescent Swiss 3T3 cells with insulin-like growth factor 1 (IGF-1) has no detectable effect on the levels of PtdlP2 or diacylglycerol in the plasma membrane, but leads to a decrease in PtdlP2 and an increase in diacylglycerol in the

Research paper thumbnail of Hepatocyte growth factor activates phosphoinositide 3-kinase C2β in renal brush-border plasma membranes

Biochemical Journal, 2002

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid met... more Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH3 and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2β activity, which is sensitive to wortmannin (10nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices wi...

Research paper thumbnail of Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2β to the nucleus

Biochemical Journal, 2009

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2α and PI3K-C2β act acutely downstr... more Although the class II phosphoinositide 3-kinase enzymes PI3K-C2α and PI3K-C2β act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2β translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2β resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2β co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2β levels and lipid kinase activity following epidermal growth factor s...

Research paper thumbnail of Activation of phosphoinositide 3-kinase C2β in the nuclear matrix during compensatory liver growth

Advances in Enzyme Regulation, 2006

In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable... more In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable PI3K-C2b activity is observed, which is sensitive to wortmannin (10 Mm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On western blots PI3K-C2b revealed a single immunoreactive band of 180 kD, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed in the nuclear matrix, suggesting that observed activation of enzyme is achieved by proteolysis. As it is know that PI3K-C2a is associated with nuclear speckles [Didichenko SA, Thelen M. Phosphatidylinositol 3-kinase C2a contains a nuclear localization sequence and associates with nuclear speckles. J Biol Chem 2001;276:48135-42.], the data presented in this report show that in the nuclear matrix PI3K-C2b is activated during the compensatory liver growth, which clearly demonstrates that different class II PI3K enzymes have different subnuclear localization and therefore might have different intranuclear functions.

Research paper thumbnail of Nuclear diacylglycerol, the cell cycle, the enzymes and a red herring (or how we came to love phosphatidylcholine)

Biochemical Society Transactions, 1997

Research paper thumbnail of Stimulation of T-cell proliferation by pancreastatin and its C-terminal fragment (33-49)

Neuroimmunomodulation

We have studied the effect of pancreastatin and its C-terminal fragment (33-49) on mitogen-stimul... more We have studied the effect of pancreastatin and its C-terminal fragment (33-49) on mitogen-stimulated T lymphocyte proliferation. In a concentration range from 10(-12) to 10(-8) M they exhibit a dose-dependent stimulatory effect on concanavalin A-induced response with the maximal effect at 10(-8) M concentration. They were inactive in response to a B-cell mitogen, lipopolysaccharide, which points to an involvement of T but not B lymphocytes in their response. Pancreastatin can still produce a stimulatory effect when added 18 h after incubation of cultures with concanavalin A and apparently uses a diacylglycerol independent mechanism. When cells were preincubated for 4, 16 or 24 h with pancreastatin or its fragment and then stimulated with concanavalin A, a ten times lower concentration of peptides was needed (10(-9) M) to obtain the maximal response. This suggests that resting cells are more sensitive to pancreastatin and its fragment. Both peptides exhibit a very similar pharmacolo...

Research paper thumbnail of Neutrophil signal transduction in Met-enkephalin modulated superoxide anion release

Neuropeptides, 1996

The present study explored the involvement of signal transduction system(s) in Met-enkephalin (ME... more The present study explored the involvement of signal transduction system(s) in Met-enkephalin (MENK) modulated superoxide anion (O2-) release from human neutrophils. This opioid pentapeptide stimulated the O2- release in all samples if present at 10(-8) M concentration while in lower concentrations the stimulatory concentration was donor-dependent. The most abundant product of MENK degradation, Tyr-Gly-Gly (TGG), suppressed O2- release over a wide range of concentrations (10(-12)-10(-8) M). MENK induced O2- release was associated with a dose-dependent increase of diacylglycerol (DAG) concentration and protein-kinase C (PKC) translocation to the neutrophil membranes, with an increase of cytosolic Ca++, and could be abolished by H7, a PKC inhibitor. On the contrary, the suppressive effect of TGG was not associated with alteration of DAG concentration in neutrophil membranes. Superoxide anion release induced by low concentrations of MENK (10(12)-10(-10) M), could be blocked by NDGA, an inhibitor of the lipoxygenase pathway. We concluded that MENK-induced O2- release results mainly due to DAG/PKC pathway activation, although other secondary messengers might be involved.

Research paper thumbnail of A Type II Phosphoinositide 3-Kinase Is Stimulated via Activated Integrin in Platelets. A SOURCE OF PHOSPHATIDYLINOSITOL 3-PHOSPHATE

Journal of Biological Chemistry, 1998

We have observed that aggregation of human platelets, caused by activation of integrin alphaIIb b... more We have observed that aggregation of human platelets, caused by activation of integrin alphaIIb beta3 and its consequent binding of fibrinogen, stimulates a novel pathway for synthesis of phosphatidylinositol 3,4bisphosphate, thereby activating protein kinase B/Akt. Such synthesis depends upon both the generation of phosphatidylinositol 3-phosphate (PtdIns3P), which is sensitive to wortmannin (IC50 7 nM) and calpain inhibitors, and the phosphorylation of PtdIns3P by PtdIns3P 4-kinase. We now report that a recently characterized C2 domain-containing phosphoinositide 3-kinase isoform (HsC2-PI3K) is present in platelets and a leukemic cell line (CHRF-288) derived from megakaryoblasts, and is likely to be responsible for the stimulated synthesis of PtdIns3P observed in platelets. HsC2-PI3K, identifiable by Western blotting and immunoprecipitatable activity, is sensitive to wortmannin (IC50 6-10 nM), requires Mg2+, and shows strong preference for PtdIns over PtdIns4P or phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-PI3K is activated severalfold when platelets aggregate in an alphaIIb beta3-dependent manner or when platelet or CHRF-288 lysates are incubated with Ca2+. Activation is prevented by calpain inhibitors. CHRF-288, which cannot undergo activation of alphaIIb beta3 and thereby aggregate in response to platelet agonists, do not generate PtdIns3P or activate HsC2-PI3K under conditions that stimulate other phosphoinositide 3-kinases. HsC2-PI3K may thus be an important effector for integrin-dependent signaling.

Research paper thumbnail of Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2013

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Pl... more Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.

Research paper thumbnail of Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2003

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells ... more The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G1/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G1/S block, which correlates with G2/M phase of the cell cycle.

Research paper thumbnail of Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G1 phase of the cell cycle

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2007

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cel... more Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 minutes and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCβ 1 was detected with no change in the amount of PI-PLCβ 1b in nuclei isolated at 30 min and 11h after the addition of serum. PI-PLC inhibitor ET-18-OCH 3 and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCβ 1b activity occur in serum-stimulated cells during G 1 phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Research paper thumbnail of The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells

Research paper thumbnail of Unclear or nuclear: another role for the phosphatidylinositol cycle?

Biochemical Society Transactions, 1993

Research paper thumbnail of Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells

Biochemical Journal, 1995

Differentiation of murine erythroleukaemia cells with the chemical agent DMSO leads to a cessatio... more 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 s...

Research paper thumbnail of Croatian Institute for Brain Research: a model of priority research project

Research paper thumbnail of The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells

Leukemia, 2006

The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed... more The pharmacological inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, several studies demonstrated the activation of PI3K in the nuclei of all-trans-retinoic acid (ATRA)-differentiated HL-60 cells, raising the possibility that PI3K/Akt-inhibitors may block antitumor properties of retinoids. The aim of the present study was to investigate the possible activation of nuclear Akt in ATRA-treated cells and to test the effects of Akt-inhibitors on ATRA-mediated differentiation. The Akt-activity was found to be increased in the nuclei and lysates of ATRA-differentiated HL-60 and NB4 cells. The down-modulation of the expression of Akt protein in HL-60 cells using siRNA reduces the CD11b expression in ATRA-treated cells. The treatment of both cell lines with the commercially available Akt inhibitors inhibited the growth of both control and ATRA-treated cells. Aktinhibitors had no inhibitory effects on ATRA-mediated growth arrest and the expression of CD11b in HL-60 cells, but increased the percentage of control cells expressing CD11b. In contrast, the presence of Akt inhibitors reduced the expression of CD11b in ATRA-treated NB4 cells.

Research paper thumbnail of Biphasic Activation of PKBα/Akt In Platelets

Journal of Biological Chemistry, 1998

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggreg... more Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin ␣ IIb ␤ 3 activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2). After aggregation, a larger amount of PtdIns(3,4)P 2 is generated. We report that this latter PtdIns(3,4)P 2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca 2؉-dependent protease calpain. Elevation of cytosolic Ca 2؉ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KII␣." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the ␤␥ subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to G␤␥ and is, in addition, activated. Both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 have been shown to stimulate PKB␣/Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKB␣/Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P 3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P 2 generated after aggregation. There is thus potential for both pre-and postaggregation-dependent signaling by PKB␣/Akt.

Research paper thumbnail of Acetylcholine and cholecystokinin receptors functionally couple by different G‐proteins to phospholipase C in pancreatic acinar cells

FEBS Letters, 1988

We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C fro... more We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin‐octapeptide (CCK‐OP) and GTPγS but not carbachol (CCh)‐induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP‐analogue, 8‐bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist‐induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP‐ribosylated a 40 kDa protein, which was inhibited by CCK‐OP but not by CCh. We conclude from these data that both CCK‐ and muscarinic acetylcholine receptors functionally couple to phospholipase ...

Research paper thumbnail of Short-Term and Long-Term-Effects of Phorbol 12-Myristate 13-Acetate and Different Inhibitors on the Ability of Bone Marrow Cells to Form Colonies In Vitro

cclm, 1995

The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated i... more The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated increase in the colony-forming potential of murine (CBA) bone marrow cells was studied using known modulators of the mitogenic signal. Pretreatment of cells for 60 minutes with staurosporine (1 μηιοΙ/1), an inhibitor of protein kinase C, completely prevented colony formation in the control group of cells and significantly reduced the number of colonies formed in the phorbol ester-treated group. Brief exposure (60 min) of cells to the phospholipase A 2 inhibitors, mepacrine (500 μιηοΐ/ΐ) and heparin (1 g/1), reduced the number of colonies formed in the control group and completely abolished the increase in the number of colonies formed after treatment of the cells with phorbol ester. When inhibitors of protein kinase C or phospholipase A 2 were present during the entire period of the colony forming assay (7 days), no colonies could be scored in either the control or phorbol ester-treated groups of bone marrow cells. Long-term treatment or temporary exposure (60 min) of cells to indomethacin (50 μπιοΙ/1), an inhibitor of cyclooxygenase, or nordihydroguaiaretic acid (50 μπιοΐ/ΐ), an inhibitor of lipoxygenase, had no effect on colony formation in both groups. Pretreatment of cells for 45 min with calcium ionophore A23187 (10 μιηοΐ/ΐ) failed to increase the number of colonies, compared with the control group. Moreover, simultaneous treatment of cells for 45 min with phorbol ester (500 nmol/1) and A23187 (10 μιηοΐ/ΐ) did not produce any further increase in the number of colonies, compared with the phorbol ester-treated group, suggesting that elevation of intracellular calcium is unimportant in the phorbol ester-mediated response. Dibutyryl cyclic adenosine monophosphate (50 μιηοΐ/ΐ) in the presence or absence of phorbol ester, failed to stimulate colony formation, indicating that cyclic AMP-dependent protein kinases are not involved in the signalling process. Temporary exposure (75 min) of bone marrow cells to okadaic acid (1 μπιοΐ/l), a potent inhibitor of serine/ threonine phosphatases, or to tyrphostine AG-115 (20 μηιοΙ/1), a tyrosine kinase inhibitor, did not effect colony growth in the control or phorbol ester-treated group. The results indicate that phospholipase A 2 activation is involved in the phorbol ester-mediated increase in colony formation, since, of the different agents applied, only staurosporine, an inhibitor of protein kinase C, and mepacrine and heparin, putative inhibitors of phospholipase A 2 , were capable of abolishing phorbol ester-mediated effects.

Research paper thumbnail of Inositides and the nucleus and inositides in the nucleus

Cell, 1993

Although there are many forms of evidence linking phosphoinositides to nuclear function, the subs... more Although there are many forms of evidence linking phosphoinositides to nuclear function, the substance of the links remains largely undefined. One link between inositide metabolism and the nucleus is suggested by the implication of inositol trisphosphate (IP3) in the process of nuclear envelope reassembly (Sullivan et al., 1993). That paper will be discussed below in its context, but this review will principally focus on another nuclear-inositide connect ion--a potential inositide cycle in the nucleus. It comes as something of a shock to see data that point to a phosphoinositide cycle entirely separate from the familiar one in the plasma membrane. Again contrary to expectation, the data suggest that the cycle is not in the nuclear membrane but appears to be within the nucleus. This aspect of inositide function has profound implications for the role of inositides in cell division and growth. For example, it makes us rethink the tumor-promoting actions of phorbol esters and the teratogenic effects of Li ÷ that have been associated with inositide homeostasis. In this article the evidence of a nuclear inositide cycle and what is known about its control are reviewed, and the role it may play in eukaryotic cell function is discussed. For a discussion of proposed nuclear functions for protein kinase C and what little is known about nuclear Ca 2÷, the reader is referred to a more comprehensive recent review (Irvine and Divecha, 1992). Evidence for a Nuclear Inositide Cycle When incubated with [y-32P]ATP, nuclei purified from rat liver (Smith and Wells, 1984) or Friend cells (Cocco et al., 1987) incorporate radioactivity into PtdlP, PtdlP2, and PtdOH. These findings suggest that all the enzymes necessary for making polyphosphoinositides are present in nuclei. The pattern of incorporation of radioactivity into nuclear lipids differs between differentiated and undifferentiated Friend cells (Cocco et al., 1987); the lack of any difference in the patterns in whole-cell homogenates argues against simple contamination of the nuclei with other cellular membranes. However, the best case for a distinct inositide cycle in the nucleus may come from data on Swiss 3T3 cells (Divecha et al., 1991). Stimulation of quiescent Swiss 3T3 cells with insulin-like growth factor 1 (IGF-1) has no detectable effect on the levels of PtdlP2 or diacylglycerol in the plasma membrane, but leads to a decrease in PtdlP2 and an increase in diacylglycerol in the

Research paper thumbnail of Hepatocyte growth factor activates phosphoinositide 3-kinase C2β in renal brush-border plasma membranes

Biochemical Journal, 2002

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid met... more Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH3 and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2β activity, which is sensitive to wortmannin (10nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices wi...

Research paper thumbnail of Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2β to the nucleus

Biochemical Journal, 2009

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2α and PI3K-C2β act acutely downstr... more Although the class II phosphoinositide 3-kinase enzymes PI3K-C2α and PI3K-C2β act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2β translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2β resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2β co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2β levels and lipid kinase activity following epidermal growth factor s...

Research paper thumbnail of Activation of phosphoinositide 3-kinase C2β in the nuclear matrix during compensatory liver growth

Advances in Enzyme Regulation, 2006

In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable... more In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable PI3K-C2b activity is observed, which is sensitive to wortmannin (10 Mm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On western blots PI3K-C2b revealed a single immunoreactive band of 180 kD, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed in the nuclear matrix, suggesting that observed activation of enzyme is achieved by proteolysis. As it is know that PI3K-C2a is associated with nuclear speckles [Didichenko SA, Thelen M. Phosphatidylinositol 3-kinase C2a contains a nuclear localization sequence and associates with nuclear speckles. J Biol Chem 2001;276:48135-42.], the data presented in this report show that in the nuclear matrix PI3K-C2b is activated during the compensatory liver growth, which clearly demonstrates that different class II PI3K enzymes have different subnuclear localization and therefore might have different intranuclear functions.

Research paper thumbnail of Nuclear diacylglycerol, the cell cycle, the enzymes and a red herring (or how we came to love phosphatidylcholine)

Biochemical Society Transactions, 1997

Research paper thumbnail of Stimulation of T-cell proliferation by pancreastatin and its C-terminal fragment (33-49)

Neuroimmunomodulation

We have studied the effect of pancreastatin and its C-terminal fragment (33-49) on mitogen-stimul... more We have studied the effect of pancreastatin and its C-terminal fragment (33-49) on mitogen-stimulated T lymphocyte proliferation. In a concentration range from 10(-12) to 10(-8) M they exhibit a dose-dependent stimulatory effect on concanavalin A-induced response with the maximal effect at 10(-8) M concentration. They were inactive in response to a B-cell mitogen, lipopolysaccharide, which points to an involvement of T but not B lymphocytes in their response. Pancreastatin can still produce a stimulatory effect when added 18 h after incubation of cultures with concanavalin A and apparently uses a diacylglycerol independent mechanism. When cells were preincubated for 4, 16 or 24 h with pancreastatin or its fragment and then stimulated with concanavalin A, a ten times lower concentration of peptides was needed (10(-9) M) to obtain the maximal response. This suggests that resting cells are more sensitive to pancreastatin and its fragment. Both peptides exhibit a very similar pharmacolo...

Research paper thumbnail of Neutrophil signal transduction in Met-enkephalin modulated superoxide anion release

Neuropeptides, 1996

The present study explored the involvement of signal transduction system(s) in Met-enkephalin (ME... more The present study explored the involvement of signal transduction system(s) in Met-enkephalin (MENK) modulated superoxide anion (O2-) release from human neutrophils. This opioid pentapeptide stimulated the O2- release in all samples if present at 10(-8) M concentration while in lower concentrations the stimulatory concentration was donor-dependent. The most abundant product of MENK degradation, Tyr-Gly-Gly (TGG), suppressed O2- release over a wide range of concentrations (10(-12)-10(-8) M). MENK induced O2- release was associated with a dose-dependent increase of diacylglycerol (DAG) concentration and protein-kinase C (PKC) translocation to the neutrophil membranes, with an increase of cytosolic Ca++, and could be abolished by H7, a PKC inhibitor. On the contrary, the suppressive effect of TGG was not associated with alteration of DAG concentration in neutrophil membranes. Superoxide anion release induced by low concentrations of MENK (10(12)-10(-10) M), could be blocked by NDGA, an inhibitor of the lipoxygenase pathway. We concluded that MENK-induced O2- release results mainly due to DAG/PKC pathway activation, although other secondary messengers might be involved.

Research paper thumbnail of A Type II Phosphoinositide 3-Kinase Is Stimulated via Activated Integrin in Platelets. A SOURCE OF PHOSPHATIDYLINOSITOL 3-PHOSPHATE

Journal of Biological Chemistry, 1998

We have observed that aggregation of human platelets, caused by activation of integrin alphaIIb b... more We have observed that aggregation of human platelets, caused by activation of integrin alphaIIb beta3 and its consequent binding of fibrinogen, stimulates a novel pathway for synthesis of phosphatidylinositol 3,4bisphosphate, thereby activating protein kinase B/Akt. Such synthesis depends upon both the generation of phosphatidylinositol 3-phosphate (PtdIns3P), which is sensitive to wortmannin (IC50 7 nM) and calpain inhibitors, and the phosphorylation of PtdIns3P by PtdIns3P 4-kinase. We now report that a recently characterized C2 domain-containing phosphoinositide 3-kinase isoform (HsC2-PI3K) is present in platelets and a leukemic cell line (CHRF-288) derived from megakaryoblasts, and is likely to be responsible for the stimulated synthesis of PtdIns3P observed in platelets. HsC2-PI3K, identifiable by Western blotting and immunoprecipitatable activity, is sensitive to wortmannin (IC50 6-10 nM), requires Mg2+, and shows strong preference for PtdIns over PtdIns4P or phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-PI3K is activated severalfold when platelets aggregate in an alphaIIb beta3-dependent manner or when platelet or CHRF-288 lysates are incubated with Ca2+. Activation is prevented by calpain inhibitors. CHRF-288, which cannot undergo activation of alphaIIb beta3 and thereby aggregate in response to platelet agonists, do not generate PtdIns3P or activate HsC2-PI3K under conditions that stimulate other phosphoinositide 3-kinases. HsC2-PI3K may thus be an important effector for integrin-dependent signaling.

Research paper thumbnail of Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2013

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Pl... more Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.

Research paper thumbnail of Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2003

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells ... more The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G1/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G1/S block, which correlates with G2/M phase of the cell cycle.

Research paper thumbnail of Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G1 phase of the cell cycle

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2007

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cel... more Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 minutes and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCβ 1 was detected with no change in the amount of PI-PLCβ 1b in nuclei isolated at 30 min and 11h after the addition of serum. PI-PLC inhibitor ET-18-OCH 3 and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCβ 1b activity occur in serum-stimulated cells during G 1 phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Research paper thumbnail of The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells