Bromodomain motifs and "scaffolding"?

Bromodomain-containing multiprotein complexes share some of the properties of signal transduction scaffolds. Insights from MAP kinase signaling scaffolds, for example, may provide useful perspectives for future studies of bromodomain proteins. The regulatory processes of modification (phosphorylation, acetylation, ubiquitination), turnover, nuclear compartmentalization, feedback regulation and signaling pathway specificity are all likely to contribute to the mechanisms by which bromodomain-containing multiprotein complexes control transcription.

Duality in bromodomain-containing protein complexes.

Proteins that contain a motif called a bromodomain are implicated in both transcriptional activation and repression. The bromodomain of p/CAF, the only solution structure of a bromodomain that has been solved to date, reveals that the motif binds N-acetyl-lysine groups, presumably to anchor enzymatic functions to histones and by extension to chromatin. The enzymatic activities can either be encoded within the same polypeptide as the bromodomain motif, or associated with a multiprotein complex. Thus, a wide variety of chromatin-directed functions, including but not limited to phosphorylation, acetylation, methylation, transcriptional co-activation or recruitment, characterize the complexes that contain bromodomain motifs. Their versatility and ubiquity ensures diverse, rapid and flexible transcriptional responses.

RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F.

RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D11 cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.

Activation-induced nuclear translocation of RING3.

RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serum-starved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2F-regulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.

Stimulation of p85/RING3 kinase in multiple organs after systemic administration of mitogens into mice.

Using an autophosphorylation membrane assay, we examined activation of kinases in different organs after intraperitoneal injections of mitogens and cytokines into mice. In the multiple organs examined administration of either epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA) or interleukin-1beta (IL-1beta) activated a number of kinases. Most notably among those was a kinase of approximately 85 kDa (p85) that was activated by EGF, PMA and IL-1beta in the lung, kidney, brain, liver and heart. The size and properties of this enzyme are indistinguishable from the RING3 kinase that has a very high activity in leukocytes of patients with leukemia. In animals treated with PMA, antibodies against RING3 kinase immunoprecipitated PMA-responsive p85 activity from the lung and brain suggesting that p85 and RING3 kinases are the same enzymes. Activation of p85/RING3 kinase by growth factors in multiple organs might reflect involvement of this enzyme in the pathogenesis of leukemias and other proliferative diseases.

A novel, mitogen-activated nuclear kinase is related to a Drosophila developmental regulator.

Although the ultimate targets of many signal transduction pathways are nuclear transcription factors, the vast majority of known protein kinases are cytosolic. Here, we report on a novel human kinase that is present exclusively in the nucleus. Kinase activity is increased upon cellular proliferation and is markedly elevated in patients with acute and chronic lymphocytic leukemias. We have identified a human gene that encodes this nuclear kinase and find that it is closely related to Drosophila female sterile homeotic (fsh), a developmental regulator with no known biochemical activity. Collectively, these results suggest that this nuclear kinase is a component of a signal transduction pathway that plays a role in Drosophila development and human growth control.

Synthesis and Ca(2+)-release activity of D- and L-myo-inositol 2,4,5-trisphosphate and D- and L-chiro-inositol 1,3,4-trisphosphate.

Partial benzoylation of the 3,4-dibenzyl ethers of D- and L-chiro-inositol provided the 1,2,5-tri-O-benzoyl-3,4-di-O-benzyl-chiro-inositols. Inversion of the free axial hydroxyl group gave a mixture of chiral 1,3,4- and 1,2,4-tri-O-benzoyl-5,6-di-O-benzyl-myo-inositols [W. Tegge and C. E. Ballou, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 94-98]. Catalytic hydrogenolysis cleaved the benzyl ether groups of the 1,3,4-tri-O-benzoyl-5,6-di-O-benzyl-myo-inositols (D- and L-) to yield the 1,3,4-tri-O-benzoyl-myo-inositols, which were phosphorylated by a dibenzyl phosphoramidite method. Removal of all blocking groups gave the pure enantiomeric myo-inositol 2,4,5-trisphosphates. Syntheses of the chiro-inositol 1,3,4-trisphosphates, which are analogs of the myo-inositol 1,4,5-trisphosphates having an axial phosphate group at position 1, or analogs of the myo-inositol 2,4,5-triphosphates having an axial hydroxyl at position 1, were also devised starting with the 1,2,5-tri-O-benzoyl-3,4-di-O-benzyl-chiro-inositols. In a calcium-release assay with saponin-permeabilized rat basophilic leukemia cells, the D isomers of both of these analogs had EC50 values of 4 microM, compared with a value of 0.17 microM for D-myo-inositol 1,4,5-trisphosphate, whereas the L isomers had EC50 values of about 100 microM.

The Ca2+ release activities of D-myo-inositol 1,4,5-trisphosphate analogs are quantized.

Comparison is made between several synthetic stereo and positional isomers of D-myo-inositol 1,4,5-trisphosphate (D-myo-1,4,5-IP3) with respect to their ability to mobilize calcium from the internal stores of saponin-permeabilized rat basophilic leukemia cells. D- and L-myo-Inositol 1,4,5-trisphosphates, D- and L-myo-inositol 2,4,5-trisphosphates, D- and L-chiro-inositol 1,3,4-trisphosphates, D,L-trans-1,2-cyclohexane-diol bisphosphate, D,L-myo-inositol 4,5-bisphosphate, L-glycerol 1,2-bisphosphate, glycerol 1,3-bisphosphate and D,L-(1R,3R,4R)-1-phosphoryloxymethyl-trans-3,4-cyclohexanediol bisphosphate were tested. The analogs, each of which contains a vicinal trans-1,2-diol-bisphosphate motif, displayed potencies that were distributed over a 10(4)-fold range of concentration and fell into 4 distinct classes of activity.

Concanavalin A- and calcium-dependent phosphorylation of a protein of 80 kDa in mouse lymphocytes rendered permeable to exogenously added [gamma-32P]ATP.

The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [gamma-32P]ATP and other small molecules by treatment with 20 micrograms/ml alpha-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 microM calcium and 4 micrograms/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed.