G protein ␤␥ dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the ␤ subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified G␤␥ dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the G␤-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G i/o proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual G␤␥ dimers. In the retinal NDPK B-enriched fractions, a G␤-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated G␤␥. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified G␤␥. For identification of the phosphorylated histidine residue, bovine brain G␤␥ and G t ␤␥ were thiophosphorylated with guanosine 5 -O-(3-[ 35 S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin, separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine G␤ 1 that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with G␤␥ dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in G␤ 1 subunits.Heterotrimeric G proteins play a pivotal role in many signal transduction pathways in eukaryotic cells. They consist of a guanine nucleotide-binding ␣ subunit (40 -52 kDa), a ␤ subunit (33-43 kDa), and a ␥ subunit (6 -10 kDa). The latter two act as a functional unit and only dissociate upon denaturation. Both G␣ and G␤␥ are required for receptor-induced G protein activation and can trigger effector functions (for reviews see Refs. 1-3). Heterotrimeric G proteins are activated by a GDP/GTP exchange catalyzed by G protein-coupled receptors. Furthermore, we and other laboratories provided evidence that phosphotransfer reactions can participate in G protein activation in vitro by formation of GTP. There is amble evidence that nucleoside diphosphate kinase (NDPK) 1 contributes to G protein activation by replenishment of GTP from ATP and GDP (for reviews see Refs. 4 -6). Hypotheses suggesting a direct in situ phosphorylation of GDP bound to G␣ and monomeric G proteins (7-9) are most likely based on artifacts. Also complex formation of NDPK with G proteins and channeling of NDPKformed GTP into G␣ (10, 11) have not yet been proven beyond doubt.A phosphotransfer reaction that uses G␤ subunits as phosphorylated intermediates has been observed in various tissues (12)(13)(14)(15)(16). ...

We previously reported that, in the membranes of HL-60 cells during activation of G-proteins, a phosphate transfer reaction occurs which involves transient G-protein beta subunit (G beta) phosphorylation [Wieland, Nürnberg, Ulibarri, Kaldenberg-Stasch, Schultz and Jakobs (1993) J. Biol. Chem. 268, 18111-18118]. Here, the generality of this phenomenon is evaluated by studying membranes of various tissues obtained from different mammalian species. All membranes tested expressed at least G beta 1 and G beta 2 subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly G beta 1 or both subtypes at roughly equal concentrations. In contrast, significantly more G beta 2 immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak, G beta-specific immunoreactive signals. Conversely, these membranes showed the highest levels of G beta phosphorylation after incubation with [gamma-32P]GTP or 35S-labelled guanosine 5'-[gamma-thio]triphosphate. Interestingly, G beta-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. G beta phosphorylation was confirmed by immunoprecipitation with G beta-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated G beta-proteins differed in their apparent molecular size from unmodified G beta-proteins. Moreover, phosphorylated G beta-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish G beta phosphorylation. In contrast, reconstituted purified Gi/Go proteins showed no G beta phosphorylation. From these experiments we conclude that: (i) G beta phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated G beta-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) G beta phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.

The epitope recognized by monoclonal antibody MAb215 generated previously against Drosophila melanogaster RNA polymerase II was mapped to amino acid residues 806-820 of the largest, 215 kDa, subunit located in a region conserved within the largest subunits of pro-and eukaryotic RNA polymerases. The affinities of MAb215 and of a recombinant single-chain Fv fragment (scFv215) were determined for binding to the enzyme as well as the fusion protein and synthetic peptides used for epitope mapping. In addition, amino acid residues of the epitope important for binding to MAb215 were identified using peptides carrying single amino acid substitutions. The epitope is not involved in the polymerization reaction or the DNA unwinding process since no inhibitory effects of the monoclonal antibody were observed in nonspecific in vitro transcription using denatured calf thymus DNA or double stranded oligo dC-tailed T7 DNA as template. In contrast, MAb215 inhibits accurate in vitro transcription from the Krüppel gene promoter and from the adenovirus-2 major late promoter. Preincubation of template DNA with the nuclear extract had no effects on inhibition supporting the notion that the epitope does not participate directly in the formation of preinitiation complexes.The same inhibitory effects were observed using scFv215. The results provide further evidence that recombinant antibody fragments produced in Escherichia co//possess the same specificity and similar affinity as their parental antibodies and demonstrate that scFv fragments are useful tools for analysis of transcriptional processes.

The regulation of the cytoskeletal localization of guanine-nucleotide-binding protein a, subunits by formyl peptide receptors was studied in myeloid differentiated human leukemia (HL-60) cells. Stimulation of formyl peptide receptors with N-formyl-Met-Leu-Phe (met-Leu-Phe) transiently increased the amount of a, subunits in the Triton X-100-insoluble cytoskeleton. Similar to the biphasic regulation of the actin content, Met-Leu-Phe ( 2 10 nM) rapidly increased the cytoskeletal a, content (about threefold at 30 s), which was followed by a rapid reversal to control levels. The formyl peptide receptor increased the cytoskeletal content of both a, subtypes, a,2 and u,,, cells. In cells permeabilized withStaphylococcus aureus a-toxin, met-Leu-Phe increased binding of the stable GTP analogue, guanosine 5'-[y-thioltriphosphate (GTP [S]), to cytoskeletal proteins in a pertussis-toxin-sensitive manner, which was completely abolished by the F-actin-disrupting agent, cytochalasin B. Using the photoreactive GTP analogue, rn-acetylanilido-GTP, the formyl peptide receptor-regulated GTP binding sites at the cytoskeleton were identified as 40-kDa proteins, the molecular size of a, subunits. Cytoskeleton prepared from stimulated cells did not exhibit increased GTP [S] binding, which suggests that activated a, subunits are translocated to the cytoskeleton. Finally, in a-toxin-permeabilized HL-60 cells, met-Leu-Phe and GTP [S] cooperatively stimulated actin polymerization. In conclusion, evidence is provided that chemoattractant receptors cause translocation of activated a, subunits to the cytoskeleton coincidentally with F-actin formation. The data therefore argue for a potential role of translocated a, subunits in the process of receptorinduced actin polymerization.Keywords ; inhibitory guanine-nucleotide-binding protein ; formyl peptide receptors ; cytoskeleton ; actin polymerization; HL-60 cells.Phagocytic cells, such as neutrophils, play an important role in the inflammatory defense against invading microorganisms. These cells are able to adhere, migrate, and kill microorganisms by phagocytosis and secretion of microbicidal agents. Chemoattractants, e.g., bacterial-derived formyl peptides, initiate these cellular responses by binding to specific cell surface receptors coupled to pertussis-toxin-sensitive guanine-nucleotide-binding regulatory proteins (G proteins) [ I , 21. Activation of heterotrimeric G proteins results in dissociation into GTP-bound a subunits and free Py dimers, both of which can regulate effector activities [3-51. Human promyelocytic leukemia (HL-60) cells can be induced to differentiate into a mature myeloid form comparable to neutrophils by dibutyryl CAMP (Bt,cAMP) or dimethyl sulphoxide [7-91. Thus, they express chemoattractant receptors coupled to pertussis-toxin-sensitive G proteins and demonstrate many cellular responses similar to neutrophils.An initial process involved in phagocyte activation is a change in the cytoskeleton network, i.e. actin polymerization, which is rapidly induced by chemoatt...