Volume 3, Issue 1, Pages 77-86 (January 1999) Allosteric Regulation of Even-skipped Repression Activity by Phosphorylation  Chi Li, James L Manley  Molecular Cell  Volume 3, Issue 1, Pages 77-86 (January 1999) DOI: 10.1016/S1097-2765(00)80176-8

Figure 1 Phosphorylation of Eve Inhibits Its Repression Activity (a) Silver staining of a protein gel containing 200 ng of the Eve (wt) proteins used in transcription experiments shown in (b). The numbers at the left indicate the molecular weights of protein markers. A schematic diagram of Eve protein is shown. Full-length Eve is divided into six regions according to Han and Manley 1993. wt, wild type. (b) In vitro transcription assays were carried out with the minimal set of general transcription factors (dTBP, dTFIIB, dTFIIF30) and RNA polymerase II. Two G-less cassette templates were used in the reactions. The template with three Eve binding sites upstream of Drosophila Adh promoter, pNP3-Adh40/10-G380, was the test template, and the one without Eve binding sites, pAdh40/10-G280, was the control template. The indicated Eve proteins (0, 40, or 80 nM) were added to reaction mixtures, respectively. (c) Analysis of purified Eve (wt) proteins (200 ng) by SDS-PAGE. The sizes of molecular weight marker are indicated. (d) Transcription from the Adh promoter was reconstituted with the minimal set of factors as described in (b). pNP3-Adh40/10-G380 and pAdh40/10-G280 served as templates, and 0, 40, or 80 nM of the Eve proteins shown in (c) was included in reaction mixtures. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)

Figure 2 Phosphorylation Does Not Affect Eve DNA-Binding Activity, but Inhibits Its Interaction with TBP (a) DNA-binding activity of Eve proteins examined by gel shift assays. The DNA probe used was an end-labeled 80 bp DNA fragment containing three Eve binding sites. Reaction mixtures contained 0, 4, 16, or 64 nM of the Eve proteins shown in Figure 1A. Conditions for the gel shift assays are described in Experimental Procedures. (b) Binding of purified Eve proteins to GST-dTBP. The indicated Eve proteins (200 ng) were incubated with either 2 μg GST or 2 μg GST-dTBP bound to glutathione-agarose beads. After extensive washing, protein complexes were eluted and detected by Western blotting with anti-Eve antibodies. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)

Figure 3 Neither the Homeodomain Nor the Repression Domain of Eve Is Phosphorylated (a) Silver staining of a protein gel containing 200 ng of the indicated Eve-BCD protein, which is diagrammed at the bottom of the panel. The molecular weights of protein markers are indicated to the left of the gel. (b) In vitro transcription assays were carried out in the minimal system as described in Figure 1B. Reaction mixtures contained 0, 40, or 80 nM of the indicated Eve derivative. Eve-BCD proteins shown in (a) were added into each reaction mixtures. (c) Protein gel containing 200 ng of the indicated Eve-BC2D2 proteins, which were tested for repression activity in (d). Molecular weights of protein markers are indicated on the left. A schematic diagram of the protein is also shown. (d) The Eve-BC2D2 proteins were analyzed for repression activity in the minimal transcriptional system. Reaction mixtures contained 0, 40, or 80 nM of the indicated proteins. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)

Figure 4 Phosphorylation of the Eve N Terminus, Not the C Terminus, Prevents Repression (a) Analysis of Eve-BCDEF proteins by SDS-PAGE. Proteins (200 ng) were visualized by silver staining. Molecular weights of protein marker are indicated on the left. A schematic diagram of the protein is shown. (b) Eve-BCDEF proteins shown in (a) were tested for their transcriptional repression activity at concentrations of 30 nM (lanes 2, 6, and 10), 60 nM (lanes 3, 7, and 11), or 90 nM (lanes 4, 8, and 12). (c) Silver staining of a protein gel containing 200 ng of the indicated Eve-ABC2D2 proteins. The sizes of molecular weight marker are indicated, and schematic diagram of Eve-ABC2D2 is also shown. (d) In vitro transcription assays in the minimal system. Reaction mixtures contained 0, 20, 40, or 80 nM of the indicated Eve-ABC2D2 proteins. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)

Figure 5 Lithium Specifically Inhibits Eve Phosphorylation In Vitro The indicated recombinant proteins were incubated with HeLa nuclear extract in the presence of γ-32P-ATP in the presence of 0, 5, 10, 20, 40, or 80 mM LiCl. After incubation, proteins were repurified by either Ni2+ agarose beads (Eve-wt and Eve-ABC2D2) or glutathione agarose beads (GST-ASF) and analyzed by SDS-PAGE. The sizes of molecular weight marker are indicated. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)

Figure 6 Lithium Blocks Inhibition of Eve Repression Activity (a) Silver staining of a protein gel containing 200 ng of the indicated Eve (wt) proteins. The sizes of molecular weight markers are indicated. (b) In vitro transcription assays were carried out to test Eve proteins shown in (a). Eve (0, 40, or 80 nM) was added to reaction mixtures as indicated. (c) The protein gel shows Eve-ABC2D2 proteins (200 ng), visualized by silver staining. The positions of molecular weight markers are indicated to the left of the gel. (d) Repression activity of the indicated Eve-ABC2D2 was tested by in vitro transcription assays. Reaction mixtures contained 0, 60, or 120 nM of the indicated Eve-ABC2D2 proteins. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)

Figure 7 GSK-3 Plus CKII Are Sufficient for Eve Phosphorylation and Inhibition of Repression Activity (a) Analysis of purified Eve-ABC2D2 proteins that had been incubated with the indicated kinases. Proteins were visualized by silver staining. Molecular weights of protein markers are indicated on the left. The primary sequence of the Shaggy/GSK-3 consensus sites in the Eve N terminus is diagrammed at the bottom of the panel. The potential sites for Shaggy/GSK-3 and CK II are indicated by arrows. (b) The repression activity of Eve-ABC2D2 proteins shown in (a) were examined in the minimal transcription system. Reaction mixtures contained 0, 40, or 80 nM of the Eve-ABC2D2 protein. Molecular Cell 1999 3, 77-86DOI: (10.1016/S1097-2765(00)80176-8)