The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site.

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.

A novel A-isoform-like inositol 1,4,5-trisphosphate 3-kinase from chicken erythrocytes exhibits alternative splicing and conservation of intron positions between vertebrates and invertebrates.

Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides. Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein. The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively). These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes. Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence. This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone. In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme. This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates. Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

Epigenetic activation of Gi-2 protein, the product of a putative protooncogene, mediates tumor promotion in vitro.

Promotion of 'initiated' JB6 epidermal cells to the tumor phenotype can be effected by 12-O-tetradecanoylphorbol-13-acetate treatment, by stimulation of epidermal growth factor (EGF) receptor activity with EGF or transforming growth factor alpha and by exposure to the isoquinoline derivative H7. When these cells were incubated with pertussis toxin (PTX), induction of anchorage-independent growth by all four promoting substances was suppressed. The inhibition is specific since cell proliferation is not affected, suggesting that activation of a Gi protein is essential for promotion of the epidermal cells. This interpretation is strongly supported by the observation that the wasp poison mastoparan, which is known to mimic receptor-mediated activation of certain Gi proteins, also promoted anchorage independence. Immunological data and partial amino acid sequence analysis of ADP-ribosyl alpha i isolated from PTX-treated JB6 cells indicate that a Gi-2 protein is a mediator to tumor promotion in this system. The inhibitory action of 4-bromophenacyl bromide may point to a coupling of the Gi protein to phospholipase A2. From our data we infer that promoters induce the tumor phenotype in 'initiated' JB6 epidermal cells by activating epigenetically the same Gi protein that in a number of adrenal and ovarian tumors appears to be persistently activated by mutational events.

3-Aminobenzamide inhibits cytotoxicity and adhesion of phorbol-ester-stimulated granulocytes to fibroblast monolayer cultures.

Damage of 3T3 fibroblasts as induced by short-term co-cultivation with O2(-)-producing granulocytes, stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was compared with that induced by treatment with enzymically generated O2- and with the alkylating agent dimethyl sulfate. The action of stimulated granulocytes was different in several aspects: (a) DNA fragmented by the products of TPA-stimulated granulocytes showed a biphasic alkaline elution pattern while fragmentation induced by alkylation or by enzymically produced O2- was monophasic. (b) Poly(ADP-ribosyl)ation of nuclear proteins after treatment with TPA-stimulated granulocytes exhibited a lag phase and was, in most experiments, less pronounced than after equitoxic dimethyl sulfate treatment. (c) 3-Aminobenzamide, the most widely used inhibitor of ADP-ribosylation, partially protected target cells from the cytotoxic effects of TPA-stimulated granulocytes, while it enhanced alkylation-induced and O2(-)-induced cytotoxicity. Protection by 3-aminobenzamide in the granulocyte system was apparently not mediated by an inhibition of nuclear poly(ADP-ribosyl)ation. Other inhibitors, like benzamide and nicotinamide, augmented cytotoxicity of TPA-stimulated granulocytes. The unique effect of 3-aminobenzamide in this system appeared to relate to TPA-induced adhesion of the neutrophils to surfaces. In the presence of 1 mM 3-aminobenzamide, but not of benzamide, the adhesion of stimulated granulocytes to 3T3 monolayer cultures was markedly reduced or even abolished. This effect was also seen in granulocyte preparations depleted of monocytes. Since 3-aminobenzamide at the doses applied does not inhibit TPA-induced superoxide production in isolated granulocytes, its specific anticytotoxic effect appears to result from a 'dilution' of granulocyte-derived damaging agents into the medium. Our data suggest that prevention of granulocyte adhesion is likely to reduce tissue damage and carcinogenesis in areas of chronic inflammation.

ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions.

Various types of ADP-ribosyl protein conjugates were synthesized and their chemical stability was compared with that of cysteine-linked ADP-ribosyl groups as formed by incubation of transducin or Gi/Go proteins with NAD and pertussis toxin. Treatment with 0.1 mM HgCl2 specifically cleaved the cysteine-linked conjugates. This may provide a tool for the quantitation of modified Gi/Go proteins as well as of other acceptors modified by ADP-ribose at cysteine residues in the presence of other ADP-ribosyl proteins.

2'-Phosphoadenylylation of eukaryotic proteins: a type of covalent modification.

An enzymatic system in rat liver microsomal preparations has been detected that catalyzes the transfer of the 2'-phospho-AMP moiety from NADP to endogenous polypeptides; the major acceptor is a polypeptide of about 40 kDa (p40). Modification of the acceptor by 2'-phospho-AMP residues was deduced from the simultaneous transfer of 2'-[33P]phosphate and [3H]adenine residues from double-labeled NADP, while no incorporation of radioactivity into p40 was seen with NADP species labeled in the NMN moiety. The true substrate of this phosphoadenylylation reaction was 2'-phospho-ADP-ribose rather than NADP, because labeled phospho-ADP-ribose was as efficient as or more efficient than NADP in forming modified p40. Also, NADP was rapidly converted to phospho-ADP-ribose during incubation with microsomes. Furthermore, isonicotinic acid hydrazide, an inhibitor of NADP glycohydrolase, prevented phosphoadenylylation from NADP, but not from phospho-ADP-ribose, and glycohydrolase-resistant NADPH could not substitute for NADP. Transferase activity was found in liver and brain microsomes and, to a smaller extent, in the cytosol fractions. In Ehrlich ascites tumor cells, most of the activity resided in the cytosol, from which it could be partially purified. The apparent Km for phospho-ADP-ribose was about 2 X 10(-4) M, and the pH optimum was around 7. Divalent cations like Mg2+ and Mn2+ inhibited the reaction. In all compartmental preparations, activity was eliminated by heating or short treatment with alkali or acid. In submitochondrial particles from rat liver, a system with different characteristics led to the phosphoadenylylation of several endogenous polypeptides.

Nonenzymic ADP-ribosylation of specific mitochondrial polypeptides.

The apparent NAD:protein ADP-ribosyl transferase activity of mitochondria and submitochondrial particles from beef heart and rat liver is simulated by a reaction sequence that consists of an enzymic hydrolysis of NAD to ADP-ribose (ADP-Rib) by NAD glycohydrolase(s) and a nonenzymic ADP-ribosylation of acceptor proteins by the free ADP-Rib formed. The nonenzymic ADP-ribosylation of mitochondrial proteins showed two pH optima and exhibited the same remarkable selectivity as the reaction with NAD. The predominant acceptor in beef heart mitochondria was a 30-kDa protein, whereas in mitochondrial extracts of rat liver a 50-55 kDa polypeptide served as an acceptor. No authentic ADP-Rib transferase activity could be detected even when free ADP-Rib was trapped by NH2OH. Once formed, the mitochondrial ADP-Rib conjugates were resistant to hydroxylamine. NH2OH-resistant mono(ADP-Rib)-protein conjugates as found in most cells may also be products of nonenzymic ADP-ribosylation. In mouse tissues, their amounts relate to protein and NAD contents, and they increase specifically and reversibly in the hypothyroid status. Furthermore, intact rat liver mitochondria contain a mono(ADP-Rib)-polypeptide (50-55 kDa) that appeared to be identical with the polypeptide reacting with ADP-Rib in vitro.