Pervanadate-triggered MAP kinase activation and cell proliferation are not sensitive to PD 98059. Evidence for stimulus-dependent differential PD 98059 inhibition mechanism.

A tight and stable complex with corresponding protein kinases and phosphatases establishes coupling between activators and inactivators. One such example is emerging from the studies of the Ras-dependent MAP kinase cascade signaling pathway. Pervanadate, a potent inhibitor of protein tyrosine phosphatase, stimulates MAP kinase and elicits cell proliferation in cultured mouse fibroblasts which is insensitive to PD 98059, the major inhibitor of upstream MEK, whereas serum- or TPA-triggered proliferation is sensitive to PD 98059. It is suggested that imbalanced coordination between protein kinase and protein phosphatase determines the cellular responses such as cell proliferation. The PD 98059-insensitive cell proliferation upon protein tyrosine phosphatase inhibition is attributed to a MEK bypass pathway.

cAMP-dependent protein kinase phosphorylates and activates nuclear Ca2+-ATPase.

A Ca2+-pump ATPase, similar to that in the endoplasmic reticulum, has been located on the outer membrane of rat liver nuclei. The effect of cAMP-dependent protein kinase (PKA) on nuclear Ca2+-ATPase (NCA) was studied by using purified rat liver nuclei. Treatment of isolated nuclei with the catalytic unit of PKA resulted in the phosphorylation of a 105-kDa band that was recognized by antibodies specific for sarcoplasmic reticulum Ca2+-ATPase type 2b. Partial purification and immunoblotting confirmed that the 105-kDa protein band phosphorylated by PKA is NCA. The stoichiometry of phosphorylation was 0.76 mol of phosphate incorporated/mol of partially purified enzyme. Measurement of ATP-dependent 45Ca2+ uptake into purified nuclei showed that PKA phosphorylation enhanced the Ca2+-pumping activity of NCA. We show that PKA phosphorylation of Ca2+-ATPase enhances the transport of 10-kDa fluorescent-labeled dextrans across the nuclear envelope. The findings reported in this paper are consistent with the notion that the crosstalk between the cAMP/PKA- and Ca2+-dependent signaling pathways identified at the cytoplasmic level extends to the nucleus. Furthermore, these data support a function for crosstalk in the regulation of calcium-dependent transport across the nuclear envelope.

Inhibition of p42/p44 mitogen-activated protein kinase by oxysterols in rat astrocyte primary cultures and C6 glioma cell lines.

We previously demonstrated that oxysterols inhibit the growth of experimental glioblastoma induced in the rat brain cortex. Mechanism of action of these compounds remains obscure. In this study, we investigated the effect of 7beta-hydroxycholesterol (7beta-OHCH) and 7ketocholesterol (7k-CH) on the growth and MAP kinase activity in three in vitro biological models: rat astrocyte primary cultures, primary cultures treated by dibutyryl-cAMP (reactive cells), and the C6 glioma cell line. The oxysterols are not lethal to primary astrocytes, even if MAP kinase activity is decreased, particularly when cells were treated with 7k-CH. Both oxysterols are toxic to reactive astrocytes, and as compared with untreated primary cultures, they amplified the MAP kinase activity decrease. However, the mechanism of action of oxysterols on reactive astrocytes seems not to be linked to the MAP kinase pathway. In highly proliferating C6 cell lines, only 7beta-OHCH has an antiproliferative effect and is cytotoxic. The inhibition of MAP kinase activity is a function of 7beta-OHCH concentration. PD098059, a MAP kinase pathway inhibitor, has only a time-limited antiproliferative effect on C6 cell growth. We conclude that in C6 cells, the MAP kinase activity decrease is correlated with the toxic effect of 7beta-OHCH and occurs at first stages of 7beta-OHCH action.

Pervanadate elicits proliferation and mediates activation of mitogen-activated protein (MAP) kinase in the nucleus.

There is growing evidence for the role of protein tyrosine phosphatases in controlling such fundamental cellular processes as growth and differentiation. Pervanadate is a potent inhibitor of protein tyrosine phosphatase which has been observed here to induce proliferation in C3H10T1/2 mouse fibroblasts. Pervanadate also translocated/activated p42/44 mitogen-activated protein (MAP) kinase to the cell nucleus. An almost similar pattern of nuclear p42/44 MAP kinase stimulation is seen with TPA. On the other hand, TPA treatment results in a rapid activation of cytosolic MAP kinase which declines with time. Thus pervanadate appears as a very useful tool for studying tyrosine phosphorylation.

Intracellular acidification mediates the proliferative response of PC12 cells induced by potassium ferricyanide and involves MAP kinase activation.

Potassium ferricyanide is known to elicit cell growth and mitogenesis in various cells by stimulating a transplasma membrane electron-transport system. When serum-starved PC12 cells were treated with potassium ferricyanide, stimulation of mitogenesis was evidenced by enhanced DNA synthesis, as well as by increased cell numbers. Intracellular pH (pH(i)) of PC12 cells was measured at 37 degrees C by microfluorimetric analysis of 2',7'-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). The resting pH(i) of unstimulated cells was 7.52 (external pH 7.40). Addition of potassium ferricyanide (100 microM) decreased pH(i) by about 0.25 pH units. Lowering pH(i) to a similar extent, either by decreasing external pH (pH(o)) or by adding a weak acid, also elicited a mitogenic response, indicating that intracellular acidification by itself has growth factor-mimicking, mitogenic effects. Nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) triggered proliferation without changes in pH(i). The mitogenic treatments eliciting intracellular acidification did not activate protein kinase C (PKC) but stimulated the p42/p44 mitogen-activated protein (MAP) kinase. Our results indicate that 2 distinct mitogenic pathways are active in PC12 cells: the first is independent of pH(i) and involves activation of the PKC pathway and the second requires a permissive pH(i) value around 7.25 and involves activation of the p42/p44 MAP kinase pathway.

Calcium transported to isolated rat liver nuclei by nicotinamide adenine dinucleotide is insensitive to thapsigargin.

Calcium uptake by isolated nuclei was mediated by nicotinamide adenine dinucleotide. Oxidized nicotinamide nucleotide analogues were more effective mediators of nuclear calcium uptake. Thapsigargin inhibited ATP-mediated nuclear calcium transport without affecting NAD-mediated nuclear calcium uptake. Whilst DBHQ did not influence ATP-induced calcium transport, it did stimulate NAD-mediated nuclear calcium entry. Calcium channel blockers did not influence the action of NAD. This study provides a further mechanism for nuclear calcium transport regulated by changes in the cytosolic NAD(+)/NADH ratio.

High affinity inositol 1,3,4,5-tetrakisphosphate receptor from rat liver nuclei: purification, characterization, and amino-terminal sequence.

Inositol 1,3,4,5-tetrakisphosphate (InsP4) mediates nuclear calcium signalling [Köppler P., Matter, N., Malviya A.N. (1993) J. Biol. Chem. 268, 26248-26252], and a distinct high affinity InsP4 binding site is identified with rat liver nuclei [Köppler, P., Mersel, M., & Malviya, A.N. (1994) Biochemistry 33, 14707-14713] as compared with other rat liver membrane fractions. A novel InsP4 receptor protein derived from rat liver nuclei has been purified to apparent homogeneity employing preparative isoelectric focusing, electrophoretic mobility, nondenaturating polyacrylamide gel electrophoresis, and electroelution. Isoelectric focusing indicated an isoelectric pH around 4.3 +/- 0.2 which was further confirmed by bidimensional electrophoresis. The high affinity nuclear InsP4 receptor was identified as a 74 kDa protein both on the SDS-PAGE and on the bidimensional electrophoresis. Partial microsequence analysis showed that the N-terminal end of nuclear InsP4 receptor consists of amino acids: PNHKNEIAGNFS. The 74 kDa nuclear InsP4 receptor protein is a distinct protein from the other InsP4 receptors purified from other sources and documented in the literature.

Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signaling by inositol 1,4,5-trisphosphate. Discrete distribution of inositol phosphate receptors to inner and outer nuclear membranes.

Transient rise in nuclear calcium concentration is implicated in the regulation of events controlling gene expression. Mechanism by which calcium is transported to the nucleus is vehemently debated. Inositol 1,4,5-trisphosphate (InsP3) and inositol-1,3,4,5-tetrakisphosphate (InsP4) receptors have been located to the nucleus and their role in nuclear calcium signaling has been proposed. Outer nuclear membrane was separated from the inner membrane. The two membrane preparations were, as best as possible, devoid of cross contamination as attested by marker enzyme activity, Western blotting with antilamin antibody, and electron microscopy. InsP4 receptor and Ca(2+)-ATPase were located to the outer nuclear membrane. InsP3 receptor was located to the inner nuclear membrane. ATP or InsP4 induced nuclear calcium uptake. External free calcium concentration, in the medium bathing the nuclei, determined the choice for ATP or InsP4-mediated calcium transport. We present a mechanistic model for nuclear calcium transport. According to this model, calcium can reach the nucleus envelope either by the action of ATP or InsP4. However, the calcium release from the nucleus envelope to the nucleoplasm is mediated by InsP3 through the activation of InsP3 receptor, which is located to the inner nuclear membrane. The action of InsP3 in this process was instantaneous and transient and was sensitive to heparin.

Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells.

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.

Subcellular localization of specific inositol 1,3,4,5-tetrakis([3H]phosphate) binding sites in rat liver membrane fractions: a comparative evaluation of pH sensitivity and binding characteristics.

Inositol 1,3,4,5-tetrakis([3H]phosphate) ([3H]IP4) binding sites were investigated in plasma membranes, nuclei and microsomes derived from the rat liver. The pH optimum for maximum [3H]IP4 binding was not the same for plasma membranes, pH 7.5, nuclei, pH 6.5, and microsomes, pH 8.0. Evidence is presented demonstrating that inositol 1,3,4,5-tetrakis(phosphate) (IP4) was the most effective inositol phosphate in displacing the binding of the [3H]IP4 in all the membrane fractions studied. Furthermore, the rank order of inhibition in various membrane fractions was identical; i.e., IP5, Ins(3,4,5,6), and IP3. This suggests that similar types of putative IP4 receptor proteins are dealt with in the plasma membranes, nuclei, and microsomes. Scatchard analysis of saturation isotherms revealed a single binding site in the plasma membranes and in the microsomes, whereas two binding sites marked by distinct KD and Bmax values were found in the nuclei. The density of putative IP4 binding sites in the plasma membranes corresponded to that of the high-affinity ones in the nuclei. Microsomes contained fewer binding sites as compared with plasma membranes or nuclei. On the basis of the pH sensitivity of [3H]IP4 binding and the KD and Bmax values in various membrane compartments, it is proposed that inositol 1,3,4,5-tetrakis(phosphate) receptor proteins are similar but not identical in membrane fractions in rat liver. Plasma membrane [3H]IP4 binding was displaced with IP4 and IP6, revealing IC50 values of 8 +/- 2 and 150 +/- 20 nM, respectively, indicating that rat liver plasma membrane IP4 receptor is not clathrin assembly protein AP-2.(ABSTRACT TRUNCATED AT 250 WORDS)

The nuclear inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate receptors.

IP3R is located to the inner nuclear membrane. Nuclear IP3R is recognized as a 220 kD immunoreactive protein by antisera raised against purified rat brain IP3R. Antisera against C-terminal 95-108 peptide fragment derived from rat brain IP3R does not reveal immunoreactivity in the nucleus. Nuclear IP3R is sensitive to heparin and is phosphorylated by nuclear PKC, enhancing the efficiency of IP3 in nuclear calcium release. There are two IP4 binding sites located to the nuclear envelope. The nuclear IP4R is sensitive to pH and pH 6.5 is found optimum for the ligand binding. The high affinity IP4R is associated with the outer nuclear membrane and mediates nuclear calcium uptake by IP4. Low affinity IP4R is identified with the inner nuclear membrane and is not involved in IP4 mediated calcium entry into the nucleus. The nature of IP4R associated with the outer nuclear membrane as compared with the one identified with the inner nuclear membrane remains to be elucidated.

Effects of cadmium on nuclear protein kinase C.

Cadmium is a carcinogen whose genotoxicity is only weak. Besides its tumor-initiating capacity, cadmium may be tumor-promoting, since it interferes with several steps of cellular signal transduction. We have investigated effects of cadmium(II) on protein kinase C (PKC), which is a key enzyme in the control of cellular growth and differentiation. Tumor-promoting phorbol esters cause an activation and translocation of PKC from the cytosol to the plasma membrane and to the nucleus of mammalian cells. In mouse 3T3/10 T 1/2 fibroblasts, cadmium(II) potentiated the effect of phorbol ester on nuclear binding and activation of PKC. Furthermore, in a reconstituted system consisting of rat liver nuclei and rat brain PKC, cadmium stimulated the binding of the enzyme to a 105-kDa protein. We propose a model in which cadmium(II) substitutes for zinc(II) in the regulatory domain of PKC, thus rendering the putative protein-protein binding site exposed. Further work is required to elucidate the potential role of the nuclear PKC binding protein(s) in the control of cell proliferation.

Regulation of signalling pathways to the nucleus by dopaminergic receptors.

Dopamine regulates postsynaptic gene expression in the central nervous system. The pattern of gene expression is different from chronic vs acute stimulation of dopaminergic receptors. Signalling to the nucleus through dopamine receptors involves different second messenger systems, and each receptor subtype regulates multiple effectors. Long term adaptive changes in neuronal function following administration of dopaminergic drugs such as antipsychotic agent or drugs of abuse is one such example of molecular plasticity triggered by dopaminergic receptors. Role of dopaminergic receptors in the control of transcriptional events and immediate early gene regulation are reviewed.

Neuroleptics differentially induce jun family genes in the rat striatum.

The effect of neuroleptics in single administration on the expression of genes of the jun family was studied in the rat striatum. jun B, but not jun D was dose-dependently and transiently induced. This effect was blocked by pretreatment with a specific dopamine D2 agonist. The expression of c-jun was also stimulated, although this did not reach statistical significance. Thus dopamine D2 receptors differentially regulate the expression of jun family members in the striatum.

Evidence for stereospecific inositol 1,3,4,5-[3H]tetrakisphosphate binding sites on rat liver nuclei. Delineating inositol 1,3,4,5-tetrakisphosphate interaction in nuclear calcium signaling process.

3H-Labeled inositol 1,3,4,5-tetrakisphosphate (IP4) binding sites are observed on nuclei isolated from rat liver and devoid of any microsomal, mitochondrial, or plasma membrane constituents. A pH of about 6.5 is found optimum for maximum [3H]IP4 specific binding that is sensitive to changes in pH. The [3H]IP4 binding on the nuclei can be distinguished into a high affinity site and a low affinity site. The two binding sites are characterized by distinct KD and Bmax (1.6 nM versus 57.0 nM KD; 0.25 pmol/mg protein and 3.7 pmol/mg protein Bmax). IP4 is capable of 45Ca2+ uptake even in the absence of ATP. The calcium uptake by nuclei is highly sensitive to IP4 since it is achieved even at 1 nM IP4 concentration. Furthermore, data are documented demonstrating that a rapid and transient 45Ca2+ release by inositol 1,4,5-trisphosphate (IP3) from the intact nuclei can be reversed by IP4. The presence of IP3 potentiates the action of IP4 in nuclear calcium reuptake as attested by the rate of calcium uptake by IP4 in the absence of IP3 (0.16 nmol/s/mg of protein) and in the presence of IP3 (4.0 nmol/s/mg of protein). A novel mechanism of nuclear calcium signaling is proposed where IP4 brings calcium into the nuclei mediated by its specific putative binding sites.

Impaired gene transcription and nuclear protein kinase C activation in the brain and liver of aged rats.

The expression of the hsp70 and c-fos genes and the activation of nuclear protein kinase C (PKC) were studied in young and aged whole rats under heat-shock conditions. The induction of hsp70 and c-fos genes by heat shock were decreased several fold in the brain as well as in the liver of senescent animals. Nuclear run-off transcription assay indicated that this age-related impairment could be attributed to a block at the level of transcription. Nuclear PKC activation by heat shock was not apparent in old animals. Nuclear PKC involvement in the repression of transcription during senescence is postulated.

Nuclear protein kinase C and signal transduction.

Protein kinase C (pKC) is a family of enzymes, consisting of ten isoenzymes. Some of the members of the pKC family are not dependent on calcium for their activity and also do not bind diacylglycerol. Protein kinase C is either translocated to the nucleus or present endogenously. Both calcium-dependent as well as calcium-independent isoenzymes are located in the nucleus. Protein kinase C has specific functions in the events activated within the nucleus during signal transduction. Three lines of approach have been taken to discern the nuclear function of pKC: pathways of activation of cytosolic pKC regulating nuclear events; translocation of pKC to the nucleus from the cytosol, and activation of native pKC in isolated nuclei. Protein kinase C contains a nuclear targeting bipartite motif and has a role in the nuclear calcium signaling process. Targeting and binding of pKC to the sites of replicational and posttranscriptional activity may be one of the mechanisms of the pKC signaling process. Protein kinase C-mediated activation of nuclear phosphatases and dephosphorylation of target nuclear proteins are the areas where much less attention has been paid. Exploring these avenues may lead to new insights into the molecular mechanism of nuclear signal transduction.