Barbour S. Warren

ATP-Dependent Mobilization of the Glucocorticoid Receptor during Chromatin Remodeling

ABSTRACT Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.

Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

ABSTRACT Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000).

Involvement of functional protein kinase C in the mitogenic response to the H-ras oncogene product.

Microinjection of purified protein kinase C (PKC) into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol-12,13-dibutyrate restores the mitogenic response of the cells to phorbol-12,13-dibutyrate (G. Pasti, J.C. Lacal, B.S. Warren, S.A. Aaronson, and P.M. Blumberg, Nature [London] 324:375-377, 1986). Our present studies demonstrate that the mitogenic activity of the H-ras oncogene in H-ras p21-microinjected quiescent cells is markedly reduced under conditions in which PKC is downregulated by chronic phorbol ester treatment. The ability to reconstitute the mitogenic response upon microinjection of both H-ras p21 and PKC implies involvement of functional PKC in the mitogenic activity of the H-ras oncogene product.

Loss of mouse fibroblast cell response to phorbol esters restored by microinjected protein kinase C.

The phorbol esters in addition to being among the most potent mouse skin tumour promoters profoundly affect many different biological systems1,2. It is postulated that they act through activation of protein kinase C (ref. 3–5), but substantial heterogeneity in their pharmacological and binding behaviour in some systems6 has caused concern about whether this is their only target. Evidence linking protein kinase C activation with biological responses to the phorbol esters includes similarity in structure–activity relations for binding and response7; in vitro phosphorylation of specific proteins by protein kinase C at the same sites at which phorbol ester treatment induces phosphorylation in intact cells3; and correlation in certain cell types between down regulation of protein kinase C on chronic phorbol ester treatment and loss of cellular responsiveness to the phorbol ester8–10. Here we report that micro-injection of purified protein kinase C into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol 12,13-diburyrate (PDBu) restores the mitogenic response of the cells to PDBu, directly establishing the involvement of protein kinase C in this response.

Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C

Phorbol ester treatment of intact neutrophils both stimulates protein kinase C (PK-C) and causes the rapid proteolytic conversion to a cytosolic, co-factor independent fragment, protein kinase M (PK-M). In intact neutrophils, phorbol ester treatment activates the NADPH-oxidase, the enzyme responsible for the oxidative burst. Addition of purified PK-M to resting neutrophil light density membranes activated the NADPH-oxidase in the presence of PS, ATP and Mg2+. A 3.5-fold greater stimulation of oxidase (ca. 25 nmoles O2-/min/mg membrane protein) was obtained with comparable PK-M concentrations to that observed with the reconstituted PK-C system, and approximately 1/3 that obtained with arachidonic acid (AA) or SDS. In contrast to the reconstituted system using PK-C, PMA and Ca++ were neither required nor affected activity. The effect of PS was unexpected, since PK-M does not require phospholipids for enzymatic activity, and likely represents the action of PS on the oxidase itself or on another component in the plasma membrane fraction. Our studies demonstrate for the first time that purified PK-M permits reconstitution of a physiologic phorbol ester response.

Protein Kinase C as the Site of Action of the Phorbol Ester Tumor Promoters

The phorbol esters are among the most potent tumor promoters in the two-stage model for mouse skin carcinogenesis(1–3) They induce numerous biochemical and cellular responses in mouse skin, of which increased cellular proliferation and hyperplasia are particularly prominent. In in vitro systems likewise, the phorbol esters induce a wide spectrum of responses, including various alterations in cellular proliferation, differentiation, and intercellular communication(4–6) The phorbol esters thus represent valuable tools to explore cellular function. The elucidation of their mechanism of action at the molecular level is relevant not only to the study of experimental chemical carcinogenesis but also to broader areas of research in cellular growth and differentiation.