Dynamics of Posttranslational Modifications of p53

The latest experimental evidence indicates that acetylation of p53 at K164 (lysine 164) and K120 may induce directly cell apoptosis under severe DNA damage. However, previous cell apoptosis models only studied the effects of active and/or inactive p53, that is, phosphorylation/dephosphorylation of p53. In the present paper, based partly on Geva-Zatorsky et al. (2006) and Batchelor et al. (2008), we propose a new cell apoptosis network, in which p53 has three statuses, that is, unphosphorylated p53, phosphorylated p53, and acetylated p53. The time delay differential equations (DDEs) are formulated based on our network to investigate the dynamical insights of p53-induced cell apoptosis. In agreement with experiments (Loewer et al. (2010)), our simulations indicate that acetylated p53 accumulates gradually and then induces the proapoptotic protein Bax under enough DNA damage. Moreover, phosphorylated p53 oscillates and initiates cell repair during DNA damage

Dynamics of p53: A Master Decider of Cell Fate

Cellular stress-induced temporal alterations?i.e., dynamics?are typically exemplified by the dynamics of p53 that serve as a master to determine cell fate. p53 dynamics were initially identified as the variations of p53 protein levels. However, a growing number of studies have shown that p53 dynamics are also manifested in variations in the activity, spatial location, and posttranslational modifications of p53 proteins, as well as the interplay among all p53 dynamical features. These are essential in determining a specific outcome of cell fate. In this review, we discuss the importance of the multifaceted features of p53 dynamics and their roles in the cell fate decision process, as well as their potential applications in p53-based cancer therapy. The review provides new insights into p53 signaling pathways and their potentials in the development of new strategies in p53-based cancer therapy

Modeling the Basal Dynamics of P53 System

The tumor suppressor p53 has become one of most investigated genes. Once activated by stress, p53 leads to cellular responses such as cell cycle arrest and apoptosis.Most previous models have ignored the basal dynamics of p53 under nonstressed conditions. To explore the basal dynamics of p53, we constructed a stochastic delay model by incorporating two negative feedback loops. We found that protein distribution of p53 under nonstressed condition is highly skewed with a fraction of cells showing high p53 levels comparable to those observed under stressed conditions. Under nonstressed conditions, asynchronous and spontaneous p53 pulses are triggered by basal DNA double strand breaks produced during normal cell cycle progression. The first peaking times show a predominant G1 distribution while the second ones are more widely distributed. The spontaneous pulses are triggered by an excitable mechanism. Once initiated, the amplitude and duration of pulses remain unchanged. Furthermore, the spontaneous pulses are filtered by ataxia telangiectasia mutated protein mediated posttranslational modifications and do not result in substantial p21 transcription. If challenged by externally severe DNA damage, cells generate synchronous p53 pulses and induce significantly high levels of p21. The high expression of p21 can also be partially induced by lowering the deacetylation rate.Our results demonstrated that the dynamics of p53 under nonstressed conditions is initiated by an excitable mechanism and cells become fully responsive only when cells are confronted with severe damage. These findings advance our understanding of the mechanism of p53 pulses and unlock many opportunities to p53-based therapy

HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis

Doctor of Philosophy

dissertationProper cell fate decisions require precise and coordinated changes in gene expression. Alterations in gene expression proceed through the functions of transcription factors, their associated coregulators and histone modifying enzymes. However, how these complex and diverse groups of proteins and enzymes coordinate their respective functions at target genes remains largely unknown. To gain additional insights into the coordinated activities of transcription factors and histone modifying enzymes, we studied how the transcription factor growth factor independence 1 (GFI1) carries out transcriptional repression through interaction with coregulator histone modifying enzymes. GFI1 is a transcriptional repressor and master regulator of normal and malignant hematopoiesis. GFI1 is comprised of a transcriptionally repressive N-terminal Snail/Slug/GFI1 (SNAG) domain, a C-terminal concatemer of DNA binding zinc fingers and a linker region which separates them. The relatively simple protein domain structure of GFI1 makes it an ideal transcription factor for studying mechanisms of transcriptional repression. We describe here two novel mechanisms of transcriptional repression by GFI1, both of which occur through posttranslational modification. First, we identify and characterize a SUMOylation event carried out by the SUMO2/3, UBC9, and PIAS3 SUMOylation machinery. SUMOylation occurs at K239 within a type I SUMO consensus element in the linker region of GFI1. We find that SUMO iv defective GFI1 derivatives fail to complement Gfi1 depletion phenotypes in zebrafish developmental erythropoiesis and in granulocyte differentiation in cultured human cells. SUMO defective GFI1 derivatives also display impaired LSD1/CoREST binding and fail to repress the GFI1 target gene MYC during granulocyte differentiation and enforced MYC expression blocks GFI1 mediated granulocyte differentiation. Second, we show SMYD2 mediated methylation at K8 within the GFI1 SNAG domain is a critical determinant of GFI1 transcriptional repression and contributes to GFI1 hematopoietic differentiation and leukemia cell survival functions. The methylation defective GFI1 SNAG domain lacks repressor function due to a failure of LSD1 recruitment and accumulation of promoter H3K4 dimethyl marks. Our findings here suggest GFI1 SUMOylation and methylation are part of a series of regulatory inputs that regulate GFI1 function. From these data we propose SNAG domain methylation and linker region SUMOylation coordinate LSD1/CoREST recruitment and enable CoREST dependent activation of LSD1 H3K4 demethylase activity for repression of GFI1 target genes. Our findings add GFI1 to the growing roster of transcription factors regulated by posttranslational modification and provides a rare mechanistic understanding into how these modifications regulate transcription factor functions through the recruitment histone modifying enzyme effectors

多分子及び一分子測定によるp53の標的探索ダイナミクスの解明

Tohoku University髙橋聡課

Simulating Molecular Mechanisms Of the Mdm2-Mediated Regulatory Interactions: a Conformational Selection Model Of the Mdm2 Lid Dynamics

Diversity and complexity of MDM2 mechanisms govern its principal function as the cellular antagonist of the p53 tumor suppressor. Structural and biophysical studies have demonstrated that MDM2 binding could be regulated by the dynamics of a pseudo-substrate lid motif. However, these experiments and subsequent computational studies have produced conflicting mechanistic models of MDM2 function and dynamics. We propose a unifying conformational selection model that can reconcile experimental findings and reveal a fundamental role of the lid as a dynamic regulator of MDM2-mediated binding. In this work, structure, dynamics and energetics of apo-MDM2 are studied as a function of posttranslational modifications and length of the lid. We found that the dynamic equilibrium between closed and semi-closed lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size. Our results revealed that these factors may regulate p53-MDM2 binding by fine-tuning the thermodynamic equilibrium between preexisting conformational states of apo-MDM2. In agreement with NMR studies, the effect of phosphorylation on MDM2 interactions was more pronounced with the truncated lid variant that favored the thermodynamically dominant closed form. The phosphomimetic mutation S17D may alter the lid dynamics by shifting the thermodynamic equilibrium towards the ensemble of semi-closed conformations. The dominant semi-closed lid form and weakened dependence on the phosphorylation seen in simulations with the complete lid can provide a rationale for binding of small p53-based mimetics and inhibitors without a direct competition with the lid dynamics. The results suggested that a conformational selection model of preexisting MDM2 states may provide a robust theoretical framework for understanding MDM2 dynamics. Probing biological functions and mechanisms of MDM2 regulation would require further integration of computational and experimental studies and may help to guide drug design of novel anti-cancer therapeutics

Posttranslational modifications of the retinoblastoma tumor suppressor protein as determinants of function

The retinoblastoma tumor suppressor protein (pRB) plays an integral role in G1-S checkpoint control and consequently is a frequent target for inactivation in cancer. The RB protein can function as an adaptor, nucleating components such as E2Fs and chromatin regulating enzymes into the same complex. For this reason, pRB\u27s regulation by posttranslational modifications is thought to be critical. pRB is phosphorylated by a number of different kinases such as cyclin dependent kinases (Cdks), p38 MAP kinase, Chk1/2, Abl, and Aurora b. Although phosphorylation of pRB by Cdks has been extensively studied, activities regulated through phosphorylation by other kinases are just starting to be understood. As well as being phosphorylated, pRB is acetylated, methylated, ubiquitylated, and SUMOylated. Acetylation, methylation, and SUMOylation play roles in pRB mediated gene silencing. Ubiquitinylation of pRB promotes its degradation and may be used to regulate apoptosis. Recent proteomic data have revealed that pRB is posttranslationally modified to a much greater extent than previously thought. This new information suggests that many unknown pathways affect pRB regulation. This review focuses on posttranslational modifications of pRB and how they influence its function. The final part of the review summarizes new phosphorylation sites from accumulated proteomic data and discusses the possibilities that might arise from this data. © The Author(s) 2013