Control of ASPP2/53BP2L Protein Levels by Proteasomal Degradation Modulates p53 Apoptotic Function

Zhu, Zhiyi; Ramos, Jason; Kampa, Kerstin M.; Adimoolam, Shanthi; Sirisawad, Mint; Yu, Zhiyong; Chen, Dexi; Naumovski, Louie; Lopez, Charles D.

doi:10.1074/jbc.m503736200

Cited by 27 publications

(39 citation statements)

References 35 publications

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“…ASPP2 knockdown, on the other hand, did not significantly increase the survival of unstressed cells (Zhu et al, 2005), which was confirmed in this study. On UV treatment of ASPP2 knockdown cells, Ddx42p showed no obvious protective effect.…”

Section: Ddx42p Counteracts Aspp2supporting

confidence: 87%

“…In otherwise unstressed cells, ASPP2 overexpression causes little reduction in cell viability, which corresponds to former results (Zhu et al, 2005). After varying doses of UV stress, the fraction of viable cells is decreased on ASPP2 overexpression, and simultaneous overexpression of mutant Ddx42p(1-737) did not rescue cells.…”

Section: Ddx42p Counteracts Aspp2supporting

confidence: 81%

“…The N-terminal Ddx42p binding site (aa 415-489) resides within a region of ASPP2 that is subject to ubiquitination (aa 401-847; Zhu et al, 2005). However, ubiquitination does not interfere with complex formation as shown by similar ubiquitination states of total ASPP2 and the Ddx42p-complexed ASPP2 fraction, respectively (Figure 3c).…”

Section: Ddx42p Physically Interacts With Aspp2mentioning

confidence: 96%

“…The ank-SH3 region binds to ASPP2 interaction partners, which include the p53 family (Bergamaschi et al, 2004) and other proteins related to apoptosis/proliferation such as Bcl-2 (Naumovski and Cleary, 1996), nuclear factor-kB subunit 3 (Yang et al, 1999) and amyloid b-precursor protein-binding protein 1 (APP-BP1; Chen et al, 2003). Cellular ASPP2 status is tightly controlled at transcriptional (Lopez et al, 2000;Stanelle et al, 2002) and protein levels (Zhu et al, 2005), and the proline-rich region of ASPP2 competes with other binding partners . In some carcinomas, mostly those expressing wild-type p53, the ASPP2 gene is silenced by hypermethylation (Liu et al, 2005), which could be linked to tumour progression.…”

Section: Introductionmentioning

confidence: 99%

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The DEAD box protein Ddx42p modulates the function of ASPP2, a stimulator of apoptosis

2009

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Ddx42p is a recently characterized mammalian DEAD box protein with unknown cellular function. We found that in human cells Ddx42p physically interacts with ASPP2, a major apoptosis inducer known to enhance p53 transactivation of proapoptotic genes. The proteins interact via a domain within the carboxy-terminal part of Ddx42p and a mid-amino-terminal sequence as well as the ankyrin-SH3 region of ASPP2. Overexpression of Ddx42p interferes with apoptosis induction by ASPP2, whereas Ddx42p knockdown reduces the survival rate of cultured human cells. In addition, ASPP2 is found in cytoplasm and nucleus at low Ddx42p level, and predominantly in cytoplasm at high concentration of Ddx42p, respectively. Our results show that Ddx42p is capable of modulating ASPP2 function.

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Section: Ddx42p Counteracts Aspp2supporting

confidence: 87%

Section: Ddx42p Counteracts Aspp2supporting

confidence: 81%

Section: Ddx42p Physically Interacts With Aspp2mentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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The DEAD box protein Ddx42p modulates the function of ASPP2, a stimulator of apoptosis

2009

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“…Treatment of cells with bortezomib, a specific inhibitor of the proteasome which is clinically active in different tumour types, increased ASPP2 protein and protein half-life but not RNA levels (Zhu et al, 2005). Treatment with anthracycline, a chemotherapy agent that can induce DNA damage and inhibit the proteasome, also increased ASPP2 protein but not RNA levels.…”

Section: Regulation Of Aspp2 Expression At the Protein Levelmentioning

confidence: 99%

ASPP: a new family of oncogenes and tumour suppressor genes

2007

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The apoptosis stimulating proteins of p53 (ASPP) family consists of three members, ASPP1, ASPP2 and iASPP. They bind to proteins that are key players in controlling apoptosis (p53, Bcl-2 and RelA/p65) and cell growth (APCL, PP1). So far, the best-known function of the ASPP family members is their ability to regulate the apoptotic function of p53 and its family members, p63 and p73. Biochemical and genetic evidence has shown that ASPP1 and ASPP2 activate, whereas iASPP inhibits, the apoptotic but not the cellcycle arrest function of p53. The p53 tumour suppressor gene, one of the most frequently mutated genes in human cancer, is capable of suppressing tumour growth through its ability to induce apoptosis or cell-cycle arrest. Thus, the ASPP family of proteins helps to determine how cells choose to die and may therefore be a novel target for cancer therapy.

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Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts

Halter

Tona²,

Bhadriraju³

et al. 2007

Cytometry Pt A

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To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters. Key termsgreen fluorescent protein; protein degradation rate constant; proteolysis; live cell microscopy; quantitative microscopy; micropatterning; NIH 3T3 cells; destabilized GFP INTRACELLULARLY synthesized fluorescent protein markers such as green fluorescent protein (GFP) are popular probes for assessing the activation of genes. Although these probes are most often observed in cells under static conditions using flow cytometry or fluorescence microscopy, GFP intensity can also be measured in adhered living cells and provide information about dynamic intracellular signaling and protein synthesis events (1-5). For example, GFP intensity used as a reporter for gene activity can provide information about processes that give rise to variations in expression level distributions exhibited by cell populations (6-9). We have observed that populations of monoclonal fibroblasts engineered to express GFP when the tenascin-C promoter is activated show a reproducible distribution of different expression levels for GFP in highly controlled environments (10). This type of reporter information can be used in the development of systems biology models that include intrinsic and extrinsic noise in cellular processes and facilitate understanding how these variations in gene activity occur.How accurately GFP fluorescence indicates rapidly changing promoter activity will be at least in part dependent on rates of GFP degradat...

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Control of ASPP2/53BP2L Protein Levels by Proteasomal Degradation Modulates p53 Apoptotic Function

Cited by 27 publications

References 35 publications

The DEAD box protein Ddx42p modulates the function of ASPP2, a stimulator of apoptosis

The DEAD box protein Ddx42p modulates the function of ASPP2, a stimulator of apoptosis

ASPP: a new family of oncogenes and tumour suppressor genes

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts

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