Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis

Abstract

Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription.

Figure 1

The VP16 activation domain, but not other domains that activate transcription, confers protein instability. (A) Steady-state levels of GAL4-fusion activators. Human HeLa cells were transiently transfected with expression constructs encoding the indicated GAL4–fusion activators (lanes 2–9) or with pUC119 carrier DNA (lane 1). After transfection, cells were treated with either DMSO solvent (−PS1) or PS1 (+PS1). Cells then were harvested, and equal volumes of total cell lysates were resolved by SDS/PAGE. GAL4-fusion proteins were detected by immunoblotting. (B) Stability of GAL4-fusion activators. The indicated GAL4-fusion proteins were transiently expressed in HeLa cells and pulse–chase analysis was performed as described (1). Labeled GAL4–fusion proteins were recovered by denaturing immunoprecipitation, and visualized by SDS/PAGE followed by autoradiography.

Figure 2

Tandem reiteration of the VN8 module generates transcriptional activators of widely differing potencies. (A) Structure of synthetic activators used in this study. Each activator carried the GAL4 DBD fused to 1×, 2×, 3×, or 6× copies of the wild-type VN8 sequence, or three copies of mutant VN8 sequence (3×M). (B) Relative transcriptional potency of each activator. GAL4-fusion proteins were assayed for transcriptional activation in human HeLa cells as described in Materials and Methods. Relative transcriptional activity is shown on a logarithmic scale, setting the transcriptional activity of the GAL4 DBD alone (Δ) at 1.

Figure 3

The ability of VN8 modules to signal proteolysis correlates with their ability to activate transcription. (A) Steady-state levels of the GAL4-VN8 activators. Human HeLa cells were transiently transfected with expression constructs encoding the indicated GAL4-fusion proteins. After transfection, cells were either untreated (−LLnL) or treated with the proteasome inhibitor LLnL (+LLnL). Total proteins were prepared and HA-tagged GAL4-fusion proteins revealed by SDS/PAGE and immunoblotting analysis. (B) Stability of GAL4–VN8 activators. GAL4-VN8 proteins were transiently expressed in HeLa cells and their stabilities were determined by pulse–chase analysis as described in Materials and Methods.

Figure 4

The ubiquitylation status of GAL4-VN8 proteins is directly related to transcriptional potency. HeLa cells were transfected with expression constructs encoding the indicated GAL4-fusion proteins, either alone (odd-numbered lanes) or with an expression construct encoding polyhistidine-tagged human Ub (His-Ub; even-numbered lanes). After transfection, cells were treated with LLnL to allow the unstable GAL4-derivatives to accumulate. Cellular proteins were harvested as described in Materials and Methods, and HA-tagged GAL4-fusion proteins present in the total lysate (lanes 1–10) or the nickel-affinity-purified material (lanes 11–20) were revealed by SDS/PAGE and immunoblotting. The arrows indicate the position of His-Ub-GAL4–6×VN8 conjugates (lane 18).

Figure 5

The Cln2 and Cln3 degrons activate transcription in yeast. (A) Structure of the GAL4–Cln2 and –Cln3 activators. (B) Activation of His3p expression. The indicated GAL4-fusion proteins were expressed in yeast strain HF7c, and their ability to activate transcription was scored by yeast growth in the presence of 3AT. Yeast were grown either in media containing histidine (nonselective; Left) or media lacking histidine and containing 2 mM 3AT (selective media; Right). (C) Activation of β-galactosidase (β-gal) expression. Liquid cultures of HF7c cells carrying an empty expression vector (−) or vectors expressing the indicated GAL4–fusion proteins were assayed for β-gal expression as described in Materials and Methods. Relative β-gal activity is expressed relative to β-gal activity directed by the GAL4 DBD alone (Δ), which is set at 1.