Effect of Phosphorylation on the Structure and Fold of Transactivation Domain of p53

Abstract

(1). A major response is to halt cell cycle progression with corresponding activation of genes needed for DNA repair. A second kind of response is apoptosis (2). Genotoxic stresses activate many cellular kinases that phosphorylate various serine and threonine residues in p53, as well as enzymes that cause lysine acetylations (3). Presently, at least 12 serine/threonines and 2 lysines are known to be modified in p53 and there may be more modification sites. This signaling system and the code of post-translational modification are still incompletely understood (1). An understanding of the structural basis of such signal integration and transduction remains a distant dream.p53 is a multidomain protein. The N-terminal domain 1-73 is responsible for transactivation function and binding of such proteins as Mdm2/Hdm2, which down-regulate the levels of p53 (4). The N-terminal domain of p53 is emerging as one of the most important regions in p53 function. One of the major pathways of transactivation is through interaction with p300/ CBP 1 and recruitment of this protein to the required promoter region. p300/CBP has well known histone acetyltransferase activity, which results in histone acetylation and remodeling of the chromosome in that region leading to enhanced transcription (5). Clearly, interactions of the N-terminal domain of p53 with Mdm2 and p300/CBP and possibly with other regulatory proteins are of crucial importance in understanding the various roles played by p53 as the guardian of the genome.Although crystal or NMR structures of other domains have been reported (6, 7), very little is known about the structure of this important domain. Only very recently, an NMR study of this domain has shown this to be disordered, although the authors concluded that secondary structural elements are present (8). It now appears that there are at least eight phosphorylation sites within the N-terminal subdomain: serines 6, 9, 15, 20, 33, 37, and 46 and threonine 18 (1). Functions of several of these phosphorylations are not clear. However, it appears that the Ser-15 phosphorylation plays a crucial role in the transactivation process (9 -11). Phosphorylation of Ser-15 occurs very soon after initial stressing of the cells (12). Among * This work was supported in part by the Nissan Science Foundation and a grant-in-aid for scientific research (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ 1 The abbreviations used are: CBP, cAMP-responsive element-binding protein-binding protein; Pep53, peptide corresponding to residues 17-28 of human p53; Bpep53, same peptide as Pep53 except five ␣-aminoisobutyric acid residues incorporated in designated positions; Aib, ␣-aminoisobutyric acid; Ser15P, p53-(1-39) domain phosphorylated at serine 15; Thr18P, p53-(1-39) domain phosphorylated at threonine 18; Ser20P, p53-(1-39) domain phosphorylated at serine ...

p53 C-Terminal Phosphorylation by CHK1 and CHK2 Participates in the Regulation of DNA-Damage-induced C-Terminal Acetylation

“…Most of the acetylation events occurred within the C-terminal 100 amino acids and are contributed to by p300/CBP and PCAF Liu et al, 1999;Ito et al, 2001). The functional interaction between acetylation and phosphorylation has been demonstrated previously; phosphorylation at the p53 N terminus potentiated acetylation at the C terminus (Lambert et al, 1998;Sakaguchi et al, 1998), possibly through an enhanced interaction between p53 and the acetylases as a result of phosphorylation (Lambert et al, 1998;Kar et al, 2002). In our study, we have demonstrated that p53 C-terminal phosphorylation by CHK1 and CHK2 may also modulate C-terminal acetylation.…”

“…Because N-terminal phosphorylation at Ser15 and Ser20 has been shown to augment p53 acetylation, possibly through an increased interaction with p300/CBP (Lambert et al, 1998;Sakaguchi et al, 1998;Kar et al, 2002), we therefore examined these mutants for their N-terminal phosphorylation status. Consistently, the 377/378A mutant displayed significantly higher Ser20 phosphorylation ( Figure 6B, lane 5) and showed a better interaction with p300 ( Figure 6C, lane 3) compared with the other mutants.…”

“…The observation that both acetylation and phosphorylation are induced by DNAdamaging agents has led to the speculation that these modification events may be interdependent. Indeed, phosphorylation at the p53 N terminus has been shown to enhance its interaction with acetylase p300/CBP and to potentiate p53 acetylation (Lambert et al, 1998;Sakaguchi et al, 1998;Kar et al, 2002).…”

“…The observation that both acetylation and phosphorylation are induced by DNAdamaging agents has led to the speculation that these modification events may be interdependent. Indeed, phosphorylation at the p53 N terminus has been shown to enhance its interaction with acetylase p300/CBP and to potentiate p53 acetylation (Lambert et al, 1998;Sakaguchi et al, 1998;Kar et al, 2002). The mammalian cell cycle checkpoint kinases CHK1 and CHK2 are structurally distinct, but functionally overlapping, Ser/Thr kinases that are activated by a variety of genotoxic assaults (Rhind and Russell, 2000;Bartek and Lukas, 2003).…”

Leucine‐rich hydrophobic clusters promote folding of the N‐terminus of the intrinsically disordered transactivation domain of p53

“…Its transactivation domain, located at the N-terminus of the protein, is responsible for binding proteins such as MDM2, which down-regulates the levels of p53 [16]; hence, the transactivation domain of p53 is emerging as one of the most important regions in p53 function. Nuclear magnetic resonance (NMR) [17,18], combined NMR and small-angle X-ray scattering [19], paramagnetic relaxation enhancement [20] and computer simulations [21] have shed some light on the structural features of the transactivation domain of p53. More recently, Lowry et al proposed an ensemble of three-dimensional structures of the 71-residue transactivation domain of p53 based on distance constraints derived from paramagnetic relaxation enhancement experiments [20].…”