Journal of Molecular Biology
SMAR1 Forms a Ternary Complex with p53-MDM2 and Negatively Regulates p53-mediated Transcription
Introduction
The major biological mission of checkpoints is to allow time to repair the damage so that checkpoint-arrested cells can eventually resume cell-cycle progression and continue their physiological program. Cell-cycle arrest, following exposure to chemotherapeutics or ionizing radiation is considered to be a major pathway by which p53 suppresses tumor formation. In the absence of stress, the relatively few active p53 molecules appear to be rather ineffective as transcriptional activators, although they do contribute to the maintenance of basal levels of several p53 target genes.1 A number of modulators for p53 functions have been reported, including kinases,2., 3., 4. components of proteasomal degradation machinery,5., [6] viral proteins,7., 8. and transcriptional inhibitors. One of the critical regulators of p53 protein is MDM2, identified as a RING domain containing ubiquitin ligase.9 Recently, MDM2 has been shown to associate with histone ubiquitylation and function as a basal transcriptional repressor in addition to its association with histone deacetylase1 (HDAC1).10 A dual mechanism for MDM2-mediated regulation of p53 has been reported, which can target p53 from nucleus to cytosol after monoubiquitination followed by polyubiquitination-initiated degradation,11 or mask the activation domain of p53 through protein–protein interactions that can recruit MDM2 to promoters, where it interferes with basal transcription machinery,[12], 13. The repression of p53-mediated transcription by MDM2 has been shown to occur via deacetylation of p53 in all the three lysine residues (Lys320, Lys373, and Lys382) critical for transcriptional regulation by p53.14 Apart from maintaining a low level of p53 in unstressed cells, MDM2 is involved in switching off the p53 stress response after the damage is repaired. Moreover, nuclear degradation of p53 and MDM2 occurs in cells during down-regulation of the p53 response after many types of DNA damage.[15], 16. Thus, activation of the p53 circuit upon stress and subsequent deactivation may occur via multiple regulatory circuits.
In the current study, we investigated the possible role of SMAR1, a matrix attachment region binding protein (MARBP), in suppressing p53 response in the post stress recovery phase. This stems from the fact that SMAR1 is a known interacting partner of the HDAC1–mSin3A complex, involved in deacetylation of histones and nonhistone proteins like p53.[17], 18. Our recent studies demonstrate that a minimal domain of SMAR1 interacts with p53 and brings about its stabilization.19., [20] Moreover, SMAR1 is a stress-responsive protein and exists in a positive feedback loop with p53, stabilizing it after stress.18
Here, we report that SMAR1 interacts both with p53 (phosphorylated at Ser15) and MDM2 independently. This interaction leads to a ternary complex formation during a narrow window period of post stress recovery. We find that the formation of ternary complex is a crucial step in down-regulation of the transcriptional activity of p53. The deacetylation of p53 by SMAR1 and MDM2 in the ternary complex dampens the transcriptional response of p53 required to facilitate re-entry into the cell cycle following DNA damage. On the basis of these results, we summarize that the interaction of SMAR1 with MDM2 and p53 adds yet another layer of complexity crucial in understanding the control of p53 response post DNA damage repair.
Section snippets
SMAR1 interacts with the N-terminus of p53
SMAR1 directly binds, stabilizes and activates p53, causing cell-cycle arrest.19., 21. To explore the domain specificity of the p53 and SMAR1 interaction, p53 null H1299 cells were transfected with full-length and various deletion constructs of p53. Upon immunoprecipitation (IP) analysis, we find that the full-length p53 binds to SMAR1 (Fig. 1a, lane 1), while the truncated protein lacking the initial 27 residues was unable to bind to SMAR1, as shown in Fig. 1a, lane 3. The C-terminal deletion
Discussion
This study addresses the role of SMAR1 in regulating p53 response after DNA damage involving a ternary complex formation with MDM2. Since p53 response to stress is regulated by a number of modifications, like phosphorylation, acetylation, methylation, ubiquitination, sumoylation, etc.,25., 26., 27., 28. the first step was to determine the specific residues involved in SMAR1-p53 interaction. While the N terminal 27 residues of p53 appear to be critical for SMAR1 binding, chemical shift
Cell culture transfections and reagents
MCF7, 293, HCT 116 p53+/+ or p53-/- and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. Transfections were done using 1 μg of the indicated plasmids per 35 mm dish (unless indicated otherwise) using Lipofectamine 2000 (Invitrogen). In the case of siRNA treatment, 100 nM SMAR1 siRNA or scrambled siRNA were transfected 12 h before treatments. TSA (200 M; Sigma) was added to the culture medium
Acknowledgements
We thank Dr G. C. Mishra, the Director of NCCS for his generous support of the experiments. pGCT MDM2, pCDNA MDM2 truncations were a kind gift from Dr C. J. Sherr. GST MDM2 constructs and the truncations were a kind gift from Dr Laitto, Helsinki University. Deletion constructs of p53 were a kind gift from Dr R.T Hay, University of St. Andrews. The p53 inducible plasmid construct p21 luc and p53 Wt construct were kind gifts from Dr B. Vogelstein, John Hopkins Oncology Center, USA. This work is
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