Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition - PubMed (original) (raw)
. 2000 Feb 1;14(3):366-76.
Affiliations
- PMID: 10673508
- PMCID: PMC316366
Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition
D O Cowley et al. Genes Dev. 2000.
Abstract
Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events.
Figures
Figure 1
Model of Ets-1 autoinhibition. (A) Schematic of Ets-1 showing DNA-binding domain (ETS domain, white), regions required for autoinhibition (inhibitory, black) and calcium-dependent phosphorylation (hatched). Expanded: secondary structural elements of the inhibitory module and ETS domain. α-Helices are indicated by barrels; β strands by arrows. (B) Structural model. α-Helices H1, HI-1, HI-2, and H4 form the inhibitory module. DNA binding is coupled to a conformational change that includes loss of structure in helix HI-1 and exposure of proteolytic cleavage sites (asterisks, Petersen et al. 1995; Jonsen et al. 1996). (C) Amino acid sequence spanning sites of calcium-dependent phosphorylation (hatched in A). In vivo calcium-dependent phosphorylation sites are indicated (Rabault and Ghysdael 1994).
Figure 2
Calcium-dependent phosphorylation and inhibition of Ets-1 DNA binding. (A) Phosphorylation of Ets-1 by nuclear extract and CaMKII. (Top) PhosphorImage of radiolabeled kinase reaction products separated by SDS-PAGE. (Bottom) Apparent phosphorylation stoichiometry of Ets-1 treated as in lanes 2 and 4 of top panel. Mean (±
s.d.
) of three experiments. (B) Calcium dependence of Ets-1 phosphorylation by nuclear extract. Ets-1 was treated with nuclear extract and [γ-32P]ATP, with or without calcium and analyzed as in A. (C) Nuclear extract treatment inhibits Ets-1 DNA binding 50-fold. Flag–Ets-1 was incubated with nuclear extract without (mock-treated) or with (phosphorylated) ATP and repurified on α-Flag agarose. (Top) Repurified protein was tested for DNA binding by EMSA. A degradation product with DNA-binding activity is indicated (*). (Bottom) Binding curves were generated by measuring the free DNA signal. _Kd_s (inset) were determined by curve-fitting to [_DP_]/[_Dt_] = 1/(1 + (Kd/[_P_])). (D) CaMKII treatment inhibits Ets-1 DNA binding 50-fold. (Top) EMSA of Ets-1 treated with CaMKII without (mock-treated) or with (phosphorylated) unlabeled ATP. Reaction products were used in DNA-binding assays without repurification. (Bottom) Binding curves were generated from mean (±
s.d.
) of three EMSA performed as in top panel and analyzed as in C. (E) Ets-1 DNA-binding inhibition is reversed by phosphatase treatment. Phosphorylated, repurified Flag–Ets-1 was mock-treated or incubated with calf intestinal phosphatase (CIP). EMSA analysis was used to derive DNA-binding curves and equilibrium dissociation binding constants as in C.
Figure 3
Mutational disruption of the Ets-1 inhibitory module. (A) Sequence and secondary structure of inhibitory helices HI-1 and H4. Y307P and L429A mutations are marked with asterisks and designated on helical wheel diagrams. (B) DNA-binding affinities of Ets-1, Y307P, and L429A. Binding curves and Kd values (inset) were derived from EMSA of bacterially expressed, purified proteins. (C) Trypsin proteolysis of Ets-1, Y307P, and L429A. (Top) Coomassie blue-stained SDS-PAGE of protease digestion products; (bottom) cleavage positions of T3, T4, and T5 fragments are indicated relative to inhibitory helix HI-1 (hatched) and the ETS domain (ETS, white). Note that T5 represents a doublet with products cleaved at position 302 or 310.
Figure 4
A disrupted inhibitory module impairs phosphorylation-dependent repression of DNA binding. (A) Phosphorylation of wild-type Ets-1 and inhibitory-module mutants by nuclear extract or CaMKII. Bars indicate mean phosphate incorporation (±
s.d.
) from three experiments. (B) Equilibrium DNA-binding studies of L429A. L429A was treated with nuclear extract without (mock-treated) or with (phosphorylated) ATP. Reaction mixtures were diluted into DNA-binding buffer and analyzed by EMSA. (C) Kd of mock-treated and phosphorylated L429A and Y307P. L429A and Flag–Y307P were treated with nuclear extract and analyzed as in B; L429A was treated with CaMKII and analyzed as in Fig. 2D. Kd values were obtained from mean data points of three experiments. (D) Phosphorylation-dependent inhibition of wild-type Ets-1, L429A, and Y307P. Mean (±
s.d.
) inhibition values were determined from Kd values of two [extract-treated wild-type (WT)] or three (all others) individual EMSA experiments. Extract-treated wild-type represents inhibition of Flag–Ets-1 repurified from nuclear extract as in Fig. 2C.
Figure 5
CaMKII phosphorylation stabilizes the Ets-1 inhibitory module. (A) Schematic of full-length Ets-1 and carboxy-terminal proteolytic fragments from endoproteinase Lys-C digestion. Potential Lys-C cleavage sites are indicated by vertical lines (see Fig. 3A for sequence). Previously characterized trypsin cleavage sites are labeled as in Fig. 3C. Calcium-dependent phosphorylation sites are indicated by S. α-Helices of the ETS domain and inhibitory module are indicated by rectangles. (B) Western analysis of Lys-C partial proteolysis. Mock-treated (top) or phosphorylated (bottom) Ets-1 was digested with indicated amounts of endoproteinase Lys-C and analyzed by SDS-PAGE and Western blotting with antiserum specific to the Ets-1 carboxyl terminus. (C) Quantitation of Western blots. The ratio of ΔN302/ΔN246 was obtained by densitometry of Western blots from B. Data show mean (±
s.d.
) of two experiments.
Figure 6
Model of phosphorylation-dependent inhibition of Ets-1 DNA binding. Electrostatic interactions between phosphoserines (P) and basic residues of the inhibitory module stabilize the inhibitory conformation, shifting the equilibrium toward the folded state. DNA binding is repressed by the higher energetic cost of the conformational change that accompanies DNA binding.
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