Second messengers and phosphoproteins最新文献

We have examined the ability of recombinant human epidermal growth factor (EGF) and bradykinin (BK) to stimulate formation of inositol polyphosphates and sn-1,2-diacylglycerol (DAG), and mobilize intracellular Ca2+ ([Ca2+]i) in adult human keratinocytes (KC). Inositol polyphosphates were resolved by high performance liquid chromatography coupled with flow detector spectroscopy. Free intracellular calcium was quantitated using digital ratio imaging fluorescence microscopy of fura-2 loaded KC. The mass amount of DAG was quantitated using the DAG kinase reaction. When comparing maximal doses of BK (0.1 microM) and EGF (200 ng/ml), BK stimulated larger increases in all second messengers measured. The majority of cells responded rapidly to BK with global increases in [Ca2+]i. Cells responding to EGF were fewer in number and slower to respond with the Ca2+ signal being less pronounced. Treatment of cells with pertussis toxin (PTX) for 24 h significantly attenuated the BK-stimulated inositol polyphosphate formation and [Ca2+]i while the EGF response remained unaffected in both parameters. BK (10(-9) to 10(-6) M) did not stimulate DNA synthesis in KC as measured by [3H]-thymidine incorporation when cultures were treated for 5 days. These results demonstrate that the coupling and biochemical signals produced by stimulation of BK and EGF receptors in human KC are different and suggests that stimulation of second messenger formation from inositol lipid hydrolysis may not be an absolute requirement for the initiation of cell proliferation.

A method has been developed for measuring specific protein phosphatase activity in biological samples using synthetic, phospho-Kemptide and phospho-GS-peptide. This method uses ion-exchange chromatography to determine phosphatase activity by quantifying the release of [32P]phosphate directly. The method was used to measure phosphatase activity of rat kidney, adrenals, heart, and liver cytosol and the activity of purified alkaline phosphatases, protein phosphatase 1, and protein phosphatase 2A. Ion-exchange chromatography was also used for the preparation of the radiolabeled phosphopeptide substrates. This method results in high recovery and specific activity of the labeled peptides. These techniques should be useful in isolating and characterizing specific protein phosphatases found in cells.

We have identified multiple members of the protein tyrosine phosphatase family in three subcellular compartments from rat liver; membrane, cytoskeleton and cytosol. Characterization based on substrate specificity, size, and reactivity with an anti-peptide antiserum against human placental PTP1B indicate the presence of at least three PTPases in Triton X-100 extracts of particulate membranes. Of these, one of 600 kDa possesses characteristics of a transmembrane, receptor-like enzyme. A fourth particulate PTPase (70 kDa) represents a distinct cytoskeletal PTPase. Cytosol contains one main PTPase species which was detected as a 41 kDa protein in Western immunoblots. These data indicate the existence of multiple hepatic PTPases whose differences in structure and subcellular localization may reflect functional heterogeneity.

A genetic selection procedure has been developed which makes the growth of E. coli dependent on expression of a cGMP phosphodiesterase cDNA. E. coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP. If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source. The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly. Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E. coli guaA auxotroph in the presence of cGMP. Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.

Tyrosine kinase activity was studied in the crude cytosolic and particulate fraction of normal mucosa and squamous cell carcinomas of the upper aero-digestive tract. In the presence of exogenously added phosphorylation substrate (Glu,Tyr4:1), the cytosolic tyrosine kinase activity was 6-fold higher in tumors compared to normal mucosa (p = 0.001), and in the particulate fraction the increase was 8-fold in tumors compared to normal mucosa. Different proposed tyrosine kinase inhibitors, including genistein, quercetin and the alpha-cyanocinnamide ST 638, were tested for their ability to inhibit phosphorylation of the synthetic tyrosine phosphorylation substrate. Phosphorylation of Glu,Tyr4:1 in tumors (cytosolic fraction) was reduced to 77.8 +/- 8.7% of the control value by 10 microM ST 638 (p less than 0.05), and to 50.7 +/- 10.4% by 100 microM quercetin (p less than 0.01). In normal mucosa (cytosolic fraction) the corresponding values were 41.7 +/- 16.6% in the presence of 10 microM ST 638 (p less than 0.05) and 32.1 +/- 5.8% in the presence of 100 microM quercetin (p less than 0.05). These inhibitors had no effect on the tyrosine kinase activity in the particulate fractions. Phosphorylation of endogenous proteins in the crude cytosolic fraction was evaluated by SDS-polyacrylamide gel electrophoresis after alkali treatment of the gels. Autoradiography of the gels treated in this manner revealed bands corresponding to phosphorylated proteins with apparent molecular weight of 18, 23, 37-38, 42-44 (double band), 53-55 (double band), 61 and 92-94 (double band) kD. Quercetin (100 microM) markedly reduced the phosphorylation of these proteins, while no effect of ST 638 could be seen. Heparin (20 micrograms/ml) stimulated the phosphorylation of three proteins with apparent molecular weight of 39 and about 72 kD, respectively, and inhibited the phosphorylation of 2 proteins with molecular weight of 92 and 53 kD in tumors. These features were observed in both tumors and normal tissue, with the exception that heparin only stimulated the 72 kD band in normal mucosa and that the phosphorylation was markedly higher in tumors. In summary, our results show an increased tyrosine phosphorylation in squamous cell carcinomas of the upper aero-digestive tract compared to normal oral mucosa. These differences and their origin might be of vital importance in the regulation of events leading to malignant transformation.

Cyclic GMP mediates vascular smooth muscle relaxation to a variety of drugs and naturally-occurring substances. The reduction of intracellular Ca2+ levels is believed to underlie this action, but the mechanism of this effect is unknown. In order to test the hypothesis that inhibition of guanine nucleotide-binding protein function is involved in the actions of cGMP, the effects of cGMP-dependent protein kinase on the phosphorylation of both pertussis toxin-sensitive (Gi/Go) and insensitive (Gz) G-proteins were examined in vitro. None of these proteins were effective substrates for either cGMP- or cAMP-dependent protein kinases, despite the fact that assay conditions were designed to detect poorly phosphorylated substrate proteins. In line with these observations, atriopeptin II did not inhibit angiotensin II-treated inositol phosphate formation in cultured vascular smooth muscle cells. These results suggest that phosphorylation by cGMP-dependent protein kinase of these G-proteins is not the major mechanism by which cGMP reduces intracellular Ca2+ levels in vascular smooth muscle.

Incubation of the murine macrophage tumour cell line PU5-1.8 in K+ (140 mM)-HEPES buffer induced depolarization of the membrane and the translocation of protein kinase C (PKC) to the subnuclear region. Membrane depolarization also induced an increase of intracellular free Ca2+ levels ([Ca2+]i) which was due to the Ca2+ influx. The amount of K(+)-mediated Ca2+ uptake was dependent on the Ca2+ concentration gradient as measured by indo-1 fluorescence and 45Ca2+ fluxes. The depolarization-mediated Ca2+ influx was suppressed by voltage sensitive Ca2+ channel blockers such as nifedipine and verapamil. Furthermore, in Na(+)-HEPES buffer, incubation of cells with a dihydropyridine agonist [3H]PN200-110 produced a dose-dependent saturable binding. On the other hand, short-term incubation of cells with phorbol 12-myristate 13-acetate (PMA) abolished the early phase of 45Ca2+ influx and the rise of indo-1 fluorescence. Depleting cells of PKC or incubating them with PKC inhibitors, H7 and sphingosine, enhanced the uptake of 45Ca2+ and the rise of indo-1 fluorescence. These observations suggest that membrane depolarization caused an activation of PKC and induced Ca2+ influx through the activation of dihydropyridine-sensitive, voltage-operated Ca2+ channels. Data also show that PKC may act as a negative modulator in controlling the Ca2+ response by closing the voltage-operated Ca2+ channel and/or by enhancing the Ca(2+)-ATPase activity during membrane depolarization in PU5-1.8 cells.

After photolabelling of purified bovine brain particulate cGMP stimulated phosphodiesterase (PDE) with [32P]cGMP, incubation with V8 Endoproteinase Glu-C produced several fragments; most of the [32P] was associated with smaller fragments (approximately 12-14 kDa), and some occasionally with larger fragments (approximately 55-57 kDa). Partial amino acid sequences were determined for the smaller photolabelled fragments and other peptides. On Western immunoblots, affinity-purified antibodies against a synthetic peptide with a sequence matching part of that of the approximately 12-14 kDa photolabelled material reacted with intact PDE and the approximately 12-14 kDa and approximately 55-57 kDa fragments. Several partial cDNA clones encoding the cGMP-stimulated PDE were isolated from a Lambda Zap II bovine brain cDNA library. Deduced amino acid sequence from one cDNA clone, lambda cGS 3-1, as well as the partial sequence of the approximately 12-14 kDa and other fragments, exhibited considerable identity with amino acid residues 311-921 of the cardiac cGMP-stimulated PDE (Trong et al., 1990), including the putative cGMP-binding domain (Charbonneau et al., 1990). These results further define this cGMP-binding domain and suggest that different cGMP-stimulated PDEs will exhibit considerable homology, at least in their cGMP-binding region(s) and catalytic domains.

Thiol titrations of bovine brain calcineurin, phosphatase with Ellman's reagent revealed the presence of 5 exposed sulfhydryl groups on the native protein, and 10 sulfhydryl groups on the denatured protein. Attempts were made to identify the location of the free thiols within the catalytic and regulatory domains of the enzyme. Our data indicates that free sulfhydryl groups are absent from the vicinity of the Mg2+ and calmodulin binding sites as well as from the active site of the enzyme. However, the fact that the number of free thiols decreased in the presence of Ca2+ and Mn2+, to 4 and 2 respectively, possibly indicates that either free thiols are at or near these domains or become inaccessible as a consequence of conformational changes induced by the metal ions. The Ca2+ and Ca2+/Mg2+ stimulated activities of calcineurin were monitored during modification with Ellman's reagent, iodoacetate and iodoacetamide. Upon modification of 1-2 of the free thiols the activity of the enzyme increased 1.3 to 10.5-fold depending on the thiol reagent and the stimulatory metal ions employed. Modification of the remainder of the free thiols resulted in a decrease in activity. These results suggest that 1-2 thiols are essential for the full expression of calcineurin activity.

Subcellular liver fractions from rats receiving a subcutaneous injection of turpentine, which causes a local inflammation, show an increased synthesis of Prostaglandin E2 and Prostaglandin F2 alpha which reaches a peak 90 minutes and 3 hours after treatment, respectively. Stimulation of phospholipase A2 activity of liver cell preparations seems to be responsible for the supply of arachidonic acid necessary to feed PG synthesis: this stimulation is accompanied by unchanged levels of diacylglycerol lipase, diacylglycerol kinase and protein kinase C activities and by an unchanged content of diacylglycerol in the liver tissue. This picture does not favour the hypothesis of an involvement of phospholipase C in the early stages after turpentine treatment. Determinations of GTP-ase activity in plasma membrane-rich liver preparations give ambiguous results, which do not allow any conclusion on the possible role of G-proteins in phospholipase A2 activation.