Phosphorylation of an actin · tropomyosin · troponin complex from human skeletal muscle

A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ-³²P]ATP, Mg²⁺, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10⁻⁷ M Ca²⁺ and protein kinase 0.1 mole of [³²P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [³²P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca²⁺, 5 · 10⁻⁵ M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [³²P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [³²P]phosphate. Increasing the Ca²⁺ concentration did not significantly decrease the [³²P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [³²P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca²⁺.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3beta

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in the brains of individuals with Alzheimer’s disease (AD). We recently found that the glycosylation might precede hyperphosphorylation of tau in AD. In this study, we investigated the effect of glycosylation on phosphorylation of tau catalyzed by cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase-3β (GSK-3β). The phosphorylation of the longest isoform of recombinant human brain tau, tau₄₄₁, at various sites was detected by Western blots and by radioimmuno-dot-blot assay with phosphorylation-dependent and site-specific tau antibodies. We found that cdk5 phosphorylated tau₄₄₁ at Thr-181, Ser-199, Ser-202, Thr-205, Thr-212, Ser-214, Thr-217, Thr-231, Ser-235, Ser-396, and Ser-404, but not at Ser-262, Ser-400, Thr-403, Ser-409, Ser-413, or Ser-422. GSK-3β phosphorylated all the cdk5-catalyzed sites above except Ser-235. Deglycosylation by glycosidases depressed the subsequent phosphorylation of AD-tau (i) with cdk5 at Thr-181, Ser-199, Ser-202, Thr-205, and Ser-404, but not at Thr-212; and (ii) with GSK-3β at Thr-181, Ser-202, Thr-205, Ser-217, and Ser-404, but not at Ser-199, Thr-212, Thr-231, or Ser-396. These data suggest that aberrant glycosylation of tau in AD might be involved in neurofibrillary degeneration by promoting abnormal hyperphosphorylation by cdk5 and GSK-3β.     

Tumour-promoting phorbol esters inhibit agonist-induced phosphatidate formation and Ca flux in human platelets

Tumour-promoting phorbol esters (phorbol-12-myristate-13-acetate, PMA; phorbol-12,13-dibutyrate, PDBu) but not 4β-phorbol, activate protein kinase C. Using human platelets pre-labelled with quin2 or ³²PO₄ we examined the effects of these compounds on human platelet cytosolic free Ca²⁺ ([Ca²⁺]_j) and on [³²]phosphatidic acid ([³²P]PtdOH). PMA and PDBu, but not 4β-phorbol inhibited thrombin-, PAF- and vasopressin-induced elevation of [Ca²⁺], and [²⁺P]PtdOH formation. It is suggested that protein kinase C may act to terminate the transduction processes that link receptor occupancy to cellular activation.     

The involvement of ecto-ATPase activity in the phosphorylation of intracellular proteins by the addition of extracellular [P]ATP in PC12 cells

We have investigated the possibility that ecto-phosphorylation by extracellular ATP may play a role in the development of PC12 cells. To test this model and to identify putative target membrane proteins, intact PC12 cells were radiolabeled by the addition of 20 μM [γ-³²P]ATP. An analysis of the labeled proteins revealed that a 57 kDa protein was the most abundant phosphorylated protein even within time periods as short as 3 min and continued to be labeled over and above the level of other proteins. This protein was identified as tyrosine hydroxylase by immunoprecipitation with antiserum to tyrosine hydroxylase. When intact cells were incubated with either [γ-³²P]ATP or ³²P_i of comparable specific radioactivity, the overall protein labeling pattern and the degree of phosphorylation of tyrosine hydroxylase were similar. There were no discrete proteins that were labeled by [γ-³²P]ATP and not by ³²P_i that would provide evidence for ecto-kinase activity in PC12 cells. Also, the addition of nonradioactive P_i reduced the incorporation of radioactivity into the protein from extracellular [γ-³²P]ATP. These results suggested that the phosphorylation of tyrosine hydroxylase by extracellular [γ-³²P]ATP required the initial hydrolysis of ATP and the subsequent incorporation of the ³²P_i into the intracellular ATP pool. To support this interpretation, we have demonstrated directly the presence of ecto-ATPase activity in intact PC12 cells by measuring the hydrolysis of extracellular [γ-³²P]ATP. Nearly 50% of the total ATP added (20 μM) was hydrolyzed within 10 min under conditions identical to those used to demonstrate intracellular protein phosphorylation. PC12 cells express both a Ca²⁺-dependent ecto-ATPase activity and a Mg²⁺-dependent ecto-ATPase activity. In addition, extracellular ATP is degraded enzymatically not only to ADP, but sequentially to adenosine. Our results also point out the difficulties inherent in attempts to identify ecto-kinase activity in cells that also contain ecto-ATPase activities.     

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [³²P]phosphorus-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [γ-³²P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.     

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.     

Experimental Galactose Toxicity: Effects on Synaptosomal Phosphatidylinositol Metabolism

Abstract Experimental galactose toxicity was induced by weaning rats onto an isocaloric 40% galactose diet. Synaptosomes were prepared from cerebra of rats at 2-9 weeks post-weaning and incubated with [³³P]P_i and myo -[2-³H]inositol in the presence or absence of 0.2 mM-acetylcholine. The acetylcholine-stimulated [³³P]P_i labeling of phosphatidylinositol and the changes in amounts of phosphatidylinositol were similar in the normal and galactose-toxic rats; however, acetylcholine-stimulated myo -[2-³H]inositol labeling of phosphatidylinositol was markedly decreased in the galactose-toxic rats. The impairment of acetylcholine-stimulated myo -[2-³H]inositol incorporation into phosphatidylinositol observed after 2 weeks on the diet did not vary after more prolonged exposure to galactose.     

Evidence for the simulaneous translocation of muscarinic acetylcholine receptor and G protein by carbachol

Interactions between guanine nucleotide regulatory proteins (G proteins) and muscarinic acetylcholine receptors (mAChRs) were studied in vivo following carbachol treatment. Rat brain homogenates were separated by high speed ultracentrigation into heavy and light membrane and 300,000 g supernate franctions. The G proteins were partially purified by Sephadex-G200 and heptylamine-Sepharose and the mAChRs by (3,2′-aminobenzhydryloxy)-tropane-(ABT)-affinity chromatographies. Radioligand binding assays showed that acute carbachol induced a biphasic translocation of the mAChRs and G proteins into the light membrane fraction with an initial release at 5–10 min and a second phase at 60 min. Portions of the released mAChRs and the G proteins, were found in the 300,000 g supernates and light membranes and were eluted in the same peak fractions from a Sephadex G-200 column. This dually labelled peak dissociated in the presence of digitonin, suggesting close association between the mAChR and G protein. ABT-affinity chromatography yielded dually labelled mAChR-G protein fractions which eluated as a single radioactive peak on a second ABT column. the partially purified G proteins from these fractions were photoaffinity labelled with 8-azidoguanosine-5′-triphosphate, [γ-³²P]. SDS-PAGE autoradiography revealed the presence of G_0ξ and G_i which may be released simultaneously with the mAChRs from the plasma membrane. In addition, a 110,000 molecular weight polypeptide was dually labelled by [³H]-PrBCM and [γ-³²P]-8-azido-GTP suggesting the presence of a “mAChR-G protein complex.” These findings provide direct evidence for the release of mAChRs and G proteins and a mAChR-G protein complex by agonist occupation of the mAChRs.     

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Synthesis, spectral studies and in vitro assessment for antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones

We report here the synthesis, characterization and in vitro antiamoebic activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones (TSC), 1–5, and their bidentate complexes [Ru(η⁴-C₈H₁₂)(TSC)Cl₂] 1a–5a. The biological studies of these compounds were investigated against HK-9 strain of Entamoeba histolytica and the concentration causing 50% cell growth inhibition (IC₅₀) was calculated in the micromolar range. The ligands exhibited antiamoebic activity in the range (2.05–5.29 μM). Screening results indicated that the potencies of the compounds increased by the incorporation of ruthenium(II) in the thiosemicarbazones. The complexes 1a–5a showed antiamoebic activity with an IC₅₀ of 0.61–1.43 μM and were better inhibitors of growth of E. histolytica, based on IC₅₀ values. The most promising among them is Ru(II) complex 2a having 1,2,3,4-tetrahydroquinoline as N⁴ substitution.     

-Subunits of Ns are released from the plasma membrane following cholera toxin activation

Cholera toxin (CT) and islet-activating protein (IAP, a Bordetella pertussis toxin) were employed to test the hypothesis that GTP-binding regulatory proteins are released from plasma membranes to a greater extent when ‘activated’ than when ‘inactivated’. CT, which activates N_s (the stimulatory GTP-binding regulatory protein of the adenylate cyclase system), catalyzed the incorporation of radioactivity from [³²P]NAD into 45 and 47.5 kDa peptides associated with rat liver plasma membranes. Following ADP-ribosylation and centrifugation at 100000 × g for 1 h, approx. 30–35% of these CT-labelled peptides were no longer associated with the plasma membranes, but were recovered from the supernatant fraction. IAP, which inactivates N_i (the inhibitory GTP-binding regulatory protein of the adenylate cyclase system) catalyzed the incorporation of radioactivity from [³²P]NAD into a 41 kDa peptide associated with the membranes. However, in contrast to the CT-labelled peptides, typically less than 5% of the lAP-labelled peptide was found in the 100000 × g supernatant fraction, but rather was almost exclusively associated with the membrane pellet. The data indicate that the -subunits of N_s are released from the plasma membrane following activation, and support the hypothesis that the βγ-subunits act to anchor the -subunits to the plasma membrane. Cholera toxin Islet-activating protein GTP-binding protein     

Synthesis and binding properties of oligodeoxynucleotides containing phenylphosphon(othio)ate linkages

A method for the synthesis of chimeric oligodeoxynucleotides comprised of phosphodiester and phenylphosphonate [3′-O-P(=O)(C₆H₅)-O-5′] or phenylphosphono-thioate [3′-O-P(=S)(C₆H₅)-O-5′] linkages has been developed. Synthesis was performed using suitably protected nucleoside phenylphosphonamidites as building blocks following an adjusted solid-phase phosphoramidite synthesis protocol. The new oligodeoxy-nucleotide analogues were characterized by electrospray ionization- and matrix-assisted laser desorption mass spectrometry, as well as by ³¹P NMR spectroscopy. Additionally, their binding properties to complementary oligodeoxynucleotides has been studied.     

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu⁵]PF4, [Ala²]PF4, [Gly²]PF4, [Ala²,Leu⁵]PF4, and [Gly²,Leu⁵]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu⁵]PF4 [Al²]PF4 = [Ala²,Leu⁵]PF4 [Gly²]PF4 = [Gly²,Leu⁵]PF4. Leu⁵ for Ile⁵ substitutions in PF4 did not alter the activity of this peptide; however, Gly²/Ala² for Pro² substitutions reduced, bud did not abolish, peptide activity. Peptide stability studies revealed that [Gly²]PF4(2–7) and -(3–7) and [Ala²]PF4(2–7), -(3–7), and -(4–7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro² in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases.     

Subcellular localization and some properties of the enzymes hydrolysing inositol polyphosphates in rat liver

The hydrolysis of inositol [³²P]trisphosphate (IP₃) and inositol [³²P]bisphosphate (IP₂) has been examined in subcellular fractions of rat liver. IP₃ was degraded by an enzyme located in the plasma membrane which did not degrade IP₂. Cytosolic fractions were found to degrade both IP₃ and IP₃. The IP₃ phosphatase activity of liver plasma membranes displayed a neutral pH optimum, was Mg²⁺ dependent and was not inhibited by high concentrations of Li⁺. Half-maximal activity of the enzymes hydrolysing IP₃ and IP₂ was obtained with substrate concentrations in the range 1–2μM. The significance of these observations to the proposed Ca²⁺ -mobilizing role of IP₃ is discussed.     

Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N-Methyl-d-Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain

Abstract: To clarify the regulatory mechanism of the N -methyl- d -aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l -glutamate-(or NMDA)-induced increase of (+)-5-[³H]methyl-10, 11-dihydro-5 H -dibenzo[a, d]cyclohepten-5, 10-imine maleate ([³H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l -[³H]glutamate, cis -4-[³H](phospho-nomethyl)piperidine-2-carboxylate ([³H]CGS-19755; competitive NMDA receptor antagonist), [³H]glycine, α-[³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [³H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l -glutamate-induced increase of [³H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l -glutamate-induced [³H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca²⁺ influx through the channel.     

Multi-site phosphorylation in ox-kidney branched-chain 2-oxoacid dehydrogenase complex

Tryptic [³²P]phosphopeptides were prepared from [³²P]phosphorylated ox-kidney branched-chain complex and analysed by high-voltage paper electrophoresis at pH 1.9. In the maximally phosphorylated complex 3 tryptic [³²P]phosphopeptides were identified (TA, TB, TC). R_F-values relative to N⁶-dinitrophenyllysine were (mean ± SEM for 25 obs.): TA, 1.53 ± 0.03; TB, 1.07 ± 0.02; TC, 0.65 ± 0.01. Relative rates of phosphorylation were TA> TB> TC. Phosphorylation of TA reached a maximum when about 66% of the complex was inactivated. Phosphorylation of TB and TC was associated mainly with 66–95% inactivation of the complex.     

Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format

p56(lck) is a lymphocyte-specific tyrosine kinase that plays an important role in both T-cell maturation and activation. We have developed a homogeneous assay in which p56(lck) catalyzes the transfer of the gamma-phosphate group from [gamma-(33)P]ATP to a biotinylated peptide substrate. The labeled peptide is then captured on a streptavidin-coated scintillation proximity assay (SPA) bead or imaging proximity bead. The SPA is counted in a microplate scintillation counter and the imaging proximity assay is counted in a charge-coupled device-based imaging system called LEADseekertrade mark, recently launched as a homogeneous imaging system by Amersham Pharmacia Biotech. We show, via time-dependence assays and inhibitor studies, that this assay can be performed in 1536-well microplate format using imaging proximity as the method of detection. The results compare favorably with the same assay performed in 384-well microplate format using both SPA and imaging proximity as the detection methods. From this study, we conclude that a kinase assay can be performed in 384- and 1536-well format using imaging as the detection method, with significant time savings over standard scintillation counting. In addition, we show cost saving advantages of 1536- over 384-well format in terms of reagent usage, higher throughput, and waste disposal.