Basal Dynamics of p53 Reveal Transcriptionally Attenuated Pulses in Cycling Cells

Loewer, Alexander; Batchelor, Eric; Gaglia, Giorgio; Lahav, Galit

doi:10.1016/j.cell.2010.05.031

Cited by 222 publications

(349 citation statements)

References 54 publications

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“…All potentially can generate bursts of activity, as in the p53 system (4,39). In analogy to our observations in Xenopus experiments, where the bursts changed in number but not shape in response to pathway inhibition, the p53 bursts are present in normally cycling cells and become more frequent with DNA damage (4).…”

Section: Discussionmentioning

confidence: 56%

“…Proper temporal control of pathway activity is crucial for normal development; however, the dynamic aspects of signaling are difficult to infer from population data and have lagged behind dissection of pathway components (2,3). The few cases that have been examined have revealed rich dynamics that could not have been predicted from knowledge of the molecular interactions or from bulk measurements of protein modifications or mRNA levels (2)(3)(4).…”

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confidence: 99%

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Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4

Warmflash

Zhang²,

Sorre³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated RSmads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.A small number of signaling pathways are used repeatedly throughout metazoan development, and their effects depend upon timing and context (1). Extensive biochemical characterization of these developmental signaling pathways has elucidated the sequence of events leading from ligand binding at the cell surface to regulation of transcription. Proper temporal control of pathway activity is crucial for normal development; however, the dynamic aspects of signaling are difficult to infer from population data and have lagged behind dissection of pathway components (2, 3). The few cases that have been examined have revealed rich dynamics that could not have been predicted from knowledge of the molecular interactions or from bulk measurements of protein modifications or mRNA levels (2-4).The TGF-β pathway is essential for developmental processes including mesoderm specification and dorsal-ventral axis formation and is dysregulated in a variety of cancers. It also is an important model for pathway crosstalk and dynamics, because it has two branches that share several components including receptors and transcription factors (5, 6). Binding of ligands specific to each branch to receptor complexes leads to the phosphorylation of branch-specific transcription factors: TGF-β/activin/nodal ligands induce the phosphorylation of Smad2/3, whereas bone morphogenic proteins (BMPs) activate Smad1/5/8. Phosphorylation of the receptor-activated Smads (R-Smads) from either branch of the pathway results in complex formation with Smad4, nuclear accumulation, and transcriptional activation.The prevailing model is that R-Smads carry pathway information with Smad4 mirroring their activity (7,8). R-Smad phosphorylation is necessary for the nuclear accumulation and transcriptional activity of both the R-Smads and Smad4. Termination of signaling often is presumed to be caused by either degradation or dephosphorylation of activated R-Smads (9, 10), and it further is assumed that the continuous pres...

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Section: Discussionmentioning

confidence: 56%

mentioning

confidence: 99%

Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4

Warmflash

Zhang²,

Sorre³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…There is accumulating evidence indicating that acetylation of p53 plays a major role in activating p53 function during stress responses (2,13,14). Following our early findings of C terminus p53 acetylation, we and others recently showed that p53 is also acetylated by Tip60 (also known as KAT5)/MOF (human ortholog of males absent on the first) at residue Lys120 (K120) within the DNA-binding domain (15)(16)(17).…”

mentioning

confidence: 99%

Differential effects on p53-mediated cell cycle arrest vs. apoptosis by p90

Dai

Tang

Jung

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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p53 functions as a central node for organizing whether the cell responds to stress with apoptosis or cell cycle arrest; however, the molecular events that lead to apoptotic responses are not completely understood. Here, we identified p90 (also called Coiled-Coil Domain Containing 8) as a unique regulator for p53. p90 has no obvious effects on either the levels of p53 or p53-mediated cell cycle arrest but is specifically required for p53-mediated apoptosis upon DNA damage. Notably, p90 is crucial for Tip60-dependent p53 acetylation at Lys120, therefore facilitating activation of the proapoptotic targets. These studies indicate that p90 is a critical cofactor for p53-mediated apoptosis through promoting Tip60-mediated p53 acetylation.T he p53 tumor suppressor acts as the major sensor for a regulatory circuit that monitors signaling pathways from diverse sources, including DNA damage, oncogenic events, and other abnormal cellular processes (1, 2). p53 monitors and responds to a multitude of stress signals by coordinating cell growth arrest or apoptosis (3-5). Central to p53 regulation of these cellular processes is its activity as a transcription factor, although transcription-independent functions of p53 are also critical under some biological settings. The activation of p53 transcription activity requires multiple steps, including sequence-specific DNA binding, antirepression and acquirement of combinations of posttranslational modifications, and recruitment of corepressors/coactivators in a promoter-specific manner (6). To suppress tumor growth, p53 induces either cell growth arrest or apoptosis depending on the cellular context. The molecular mechanisms that govern the choice between growth arrest and apoptosis are extremely important but not well understood (4, 7). As a key player in the stress response, p53 demands an exquisitely complicated network of control and fine-tuning mechanisms to ensure correct, differentiated responses to the various stress signals encountered by cells (2,5,(8)(9)(10).p53 was the first nonhistone protein known to be regulated by acetylation and deacetylation (11,12). There is accumulating evidence indicating that acetylation of p53 plays a major role in activating p53 function during stress responses (2, 13, 14). Following our early findings of C terminus p53 acetylation, we and others recently showed that p53 is also acetylated by Tip60 (also known as KAT5)/MOF (human ortholog of males absent on the first) at residue Lys120 (K120) within the DNA-binding domain (15-17). K120 acetylation is crucial for p53-mediated apoptosis but has no obvious effect on p21 expression, an essential target of p53-mediated growth arrest. Notably, although Tip60 is required for K120 acetylation of p53 in vivo, the levels of K120 acetylation are dynamically regulated in vivo and the interaction between p53 and Tip60 is not very stable, indicating that additional regulators may play a role in controlling K120 acetylation and subsequent p53-mediated apoptotic response (18)(19)(20). Through biochemical pur...

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“…Images were acquired every 15 min for 12-24 h using the Nikon Elements software. Image analysis was performed with ImageJ (NIH) and Matlab (MathWorks) as described 33 .…”

Section: Live Cell Microscopymentioning

confidence: 99%

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

et al. 2014

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Levels of residual structure in disordered interaction domains determine in vitro bindingaffinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage.These results establish that residual structure is an important determinant of signaling fidelity in cells.Intrinsically disordered protein domains often mediate protein-protein interactions that result in disorder-to-order transitions via coupled folding and binding reactions 1 . In addition, many disordered interaction domains exhibit defined levels of transient secondary structure resembling their complex-bound states when free in solution 2 . These levels of residual structure affect binding energies, also by reducing the loss of conformational entropy associated with disorderto-order transitions 3 . Accordingly, higher levels of residual structure in disordered interaction domains increase in vitro binding affinities 4,5 . Here we ask to what extent residual structure contributes to protein binding affinities in cells and whether engineered changes to residual structure affect protein function at the cellular level. To answer these questions, we designed p53 mutants with higher residual helicity within their disordered, N-terminal transactivation domains (TADs) and investigated their effects on cellular Mdm2 binding and p53's ability to induce 2 target gene expression and cell cycle arrest. p53 is activated by many forms of cellular stress, including DNA double-strand breaks (DSBs) and functions as a major tumor suppressor and cell cycle regulator 6 . In the absence of DNA damage, cellular p53 levels are kept low by targeted proteasomal degradation mediated by the E3 ubiquitin ligase Mdm2, which interacts with p53TAD and subsequently ubiquitinates p53's C-terminal regulatory domain 7 . Upon DNA damage, post-translational modifications of p53TAD and Mdm2, together with Mdm2 degradation, disrupt the p53-Mdm2 complex. This leads to p53 accumulation and the expression of p53 target genes that regulate DNA repair, cell cycle arrest, senescence or apoptosis 8,9 . One of these target genes is Mdm2, whose expression establishes a negative feedback loop that shapes cellular p53 dynamics and thereby controls cell fate decisions 10 .In its free form, p53TAD exists in equilibrium between disordered and partially helical conformations 11-13 , whereas residues 19-25 form a stable amphipathic α-helix in the Mdm2 complex 14 (Fig. 1a). To increase the binding affinity between p53 and Mdm2 without altering the binding interface, we designed p53TAD mutants with higher levels of residual helicity by mutating conserved proline residues flanking the Mdm2 binding site (i.e., Pro12, Pro13 or Pro27) to alanines (Supplementary Results, Supplementary Fig. 1a). Using NMR spectroscopy, we determined that wild-type (WT) p53TAD helicity (28%) increased to 64% when we replaced Pro27 with ...

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Basal Dynamics of p53 Reveal Transcriptionally Attenuated Pulses in Cycling Cells

Cited by 222 publications

References 54 publications

Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4

Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4

Differential effects on p53-mediated cell cycle arrest vs. apoptosis by p90

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

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