Acetylation of lysine 382 and phosphorylation of serine 392 in p53 modulate the interaction between p53 and MDC1 in vitro

Abstract

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.

Figure 1. MDC1 and p53 interact following DNA damage through the tBRCT domain of MDC1.

(a) A scheme of the different domains of p53. (b) A scheme of the different domains of MDC1. Note that in A, and B the images are not to scale as p53 is about 5 times smaller than MDC1. (c) 293T control cells transfected with empty vector or cells overexpressing HA-tagged p53 were either untreated (-) or treated with NCS (+). Following 1 hr incubation, the proteins were extracted and subjected to co-IP using anti-HA antibodies. Control for the co-IP was done with anti-GST antibodies. Detection was done using antibodies directed against endogenous MDC1 and phopsho-Ser15 of p53 (ph-p53). (d) The tBRCT domain of MDC1 directly interacts with p53. GST pull-down assay was performed with GST-p53 and His-tBRCT or His-FHA. All recombinant proteins were expressed in bacteria. Proteins were separated on a SDS gel and stained with Coomassie blue. (e) Endogenous p53 and MDC1 interact. Protein extracts prepared from 293 cells that were induced with 5 Gray of ionizing radiation and left for recovery for 1 hr were used in an IP experiment using antibodies directed against MDC1-tBRCT (α-BRCT) or against MDC1-FHA (α-FHA). Bound proteins were detected in Western blot using anti-p53 antibodies. Inputs present 5% of the extract used in the experiment.

Figure 2. A C-terminus region (a.a. 318-393) of p53 directly binds MDC1-tBRCT.

(a) His-tBRCT retrieves p53 fragments consisting a.a. 318-393: Bacterially expressed His-tBRCT was incubated with different radio-labeled fragments of p53-HA expressed in reticulocytes (for details see schematic representations below). Following His pull-down reactions the labeled p53 fragments (in the input or those retrieved by His-tBRCT) were visualized by autoradiography. (b) p53 fragments containing a.a 318-393 bind tBRCT-MDC1: Fragments of p53 fused to GST (for details see schematic representations below) were expressed in bacteria and purified. Following incubation with radio-labeled His-tBRCT and GST pull-down reactions, His-tBRCT visualized by using autoradiography. Input is 5% of His-tBRCT added to the reaction. The same gels were used for autoradiography and Coomassie blue staining in B. (c) GST pull-down using a.a. 318-393 of p53 fused to GST (GST-p53Cter) for His-FHA or His-tBRCT, followed by Coomassie blue staining.

Figure 3. The CTD of p53 interacts with MDC1-tBRCT and this interaction is modulated by acetylation of K382 and phosphorylation of S392 in p53.

Peptide pull-down assay was performed with peptides corresponding to the CTD of p53 (a.a 361-393) that were either without PTMs (none), acetylated on K382 (AcK382), phosphorylated on S392 (pS392) or containing both PTMs, or with a peptide corresponding to the Tet domain of p53 in the presence of radio-labeled His-tBRCT. Bound proteins were visualized using autoradiography.

Figure 4. In p53 CTD, acetylation of K382, phosphorylation of S392 or both, contribute to the binding to MDC1-tBRCT.

(a) Cartoon representations of the p53 CTD peptides (Acetylated and phosphorylated peptide in blue or unmodified peptide in red), MDC1-tBRCT in cyan and phosphorous atom in gold. Left - initial conformations; right - representative snapshots of the molecular dynamics simulations. (b-e) Potential energy of the interactions between p53 CTD peptides and MDC1-tBRCT; The Lennard-Jones and the electrostatic contributions of each residue are shown in white and black, respectively. The peptides: (b) Ac-K382 and pS392. (c) Ac-K382. (d) pS392. (e) Unmodified. Error bars represent the standard deviation of the mean for the sum of the interactions.

Figure 5. Secondary structure of the CTD of p53.

CD spectra of p53 Tet peptide and p53 CTD peptides that are either unmodified, containing both AcK382 and pS392 or containing either AcK382 or pS392.

Figure 6. Suggested model and visualization of the p53-MDC1 interaction.

(a) MD derived interactions. tBRCT is shown in surface representation in light gray and the p53 CTD peptide is shown as a blue ribbon. Zoom-in panels of the Ac-K382 and pS392 are shown below; note that in the zoom-in panels the viewer angle is slightly rotated for visualization convenience. Red arrow points K1936 in MDC1. (b) Following genotoxic stress p53 (blue) undergoes K382 acetylation (red pentagon) and S392 phosphorylation (yellow pentagon). These residues mediate the interaction with MDC1 (gray) through its tBRCT domain (light gray).