The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (beta ARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate beta ARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC-dependent modulation of beta ARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate beta ARK1 with an apparent Km for beta ARK1 of 6 nM. The ability of beta ARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of beta ARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in beta ARK activity ws demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 microM isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. beta ARK inhibitor heparin (160 micrograms/ml) prevented the augmenting effect of PMA on beta AR desensitization. These results show that beta ARK activity can be increased through phosphorylation by PKC, thus indicating that beta ARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously.
The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.
DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 40C as well as at 30'C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase I, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase U by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.Phorbol esters influence cellular functions at various levels (1, 2), presumably by activating protein kinase C (3, 4). Some cellular effects can be detected rapidly after the addition of phorbol esters. These effects include the regulation of ionic transport, release of bioactive substances, and receptor down-regulation (1,2,5). Other effects of phorbol esters target the genome, resulting in the regulation of DNA replication or in the modulation of gene expression. Examples of genes that appear to be induced by phorbol esters include ornithine decarboxylase in epidermal cells (6,7), interleukin 2 and IL-2 receptor in mouse T cells and Tlymphoma cells (8-10), c-fos in U937 monocytes and in HL-60 cells (11), actin and vimentin in K562 erythroleukemia cells (12), and calcitonin in thyroid medullary carcinoma cells (13). Genes that seem to be repressed by phorbol esters include globin in Friend erythroleukemia cells (14), glycophorin in K562 erythroleukemia cells (15), and c-myc in thyroid medullary carcinoma cells (13). Moreover, phorbol esters increase the frequency of initiation of DNA replication for bacteriophage X injected into Xenopus eggs (16).The effects of phorbol esters on gene transcription generally require more time to become manifest than the effects on ionic fluxes or receptors. Thus, the regulation of c-fos mRNA in U937 monocytes is maximal after 30 min (11) and that of glycophorin and actin genes becomes evident after about 1 hr (12, 15), whereas the effect on c-myc mRNA is observed 4 hr after exposure of the cells to the tumor promoter (13). The relative delay in these responses may reflect an indirect effect ofphorbol esters on gene transcription, requiring one or more intermediary steps or a cascade of reactions; alternatively, phorbol esters may alter gene transcription more directly by stimulating protein kinase C, which then may phosphorylate a polypeptide(s) involved in transcriptional regu...
The solution structure of an extended pleckstrin homology (PH) domain from the beta-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the beta-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal alpha-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended alpha-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gbetagamma subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains.
Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)
Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.
The thrombin-like serine protease and antithrombotle agent, Ancrod, was rapidly purified from the crude venom of Aki~'trodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analyais of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked 81ycosylatlon sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanasctreated peptides. Monoelonal antibodies raised against the denatured protein and HF.deglycosylated protein r~ognized Anerod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrlnogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.