MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation

Tight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the \u394Np63\u3b1 protein. We found that MDM2 binds \u394Np63\u3b1 in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for \u394Np63\u3b1 nuclear export and subsequent degradation, whereas the MDM2 ring-finger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of \u394Np63\u3b1 by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the \u3b1 and \u3b2 tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous \u394Np63\u3b1 in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative \u394Np63\u3b1 protein

Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo

Promyelocytic leukemia protein is required for gain of function by mutant p53

10.1158/0008-5472.CAN-08-4010Cancer Research69114818-4826CNRE

MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation.

Tight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the DeltaNp63 alpha protein. We found that MDM2 binds DeltaNp63 alpha in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for Delt Np63 alpha nuclear export and subsequent degradation, whereas the MDM2 ring-finger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of Delta Np63 alpha by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the alpha and beta tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous DeltaNp63 alpha in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative DeltaNp63 alpha protein

E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis

Fulltext embargoed for: 12 months post date of publicationNeoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma

The role of PML in the control of apoptotic cell fate: a new key player at ER-mitochondria sites.

The development of malignant tumors results from deregulated proliferation or an inability of cells to undergo apoptotic cell death. Experimental works of the past decade have highlighted the importance of calcium (Ca(2+)) in the regulation of apoptosis. Several studies indicate that the Ca(2+) content of the endoplasmic reticulum (ER) determines the cell's sensitivity to apoptotic stress and perturbation of ER Ca(2+) homeostasis appears to be a key component in the development of several pathological situations. Sensitivity to apoptosis depends on the ability of cells to transfer Ca(2+) from the ER to the mitochondria. The physical platform for the interplay between the ER and mitochondria is a domain of the ER called the mitochondria-associated membranes (MAMs). The disruption of these contact sites has profound consequences for cellular function, such as imbalances of intracellular Ca(2+) signaling, cellular stress, and disrupted apoptosis progression. The promyelocytic leukemia (PML) protein has been previously recognized as a critical and essential regulator of multiple apoptotic response. Nevertheless, how PML would exert such broad and fundamental role in apoptosis remained for long time a mystery. In this review, we will discuss how recent results demonstrate that the elusive mechanism whereby the PML tumor suppressor exerts its essential role in apoptosis triggered by Ca(2+)-dependent stimuli can be attributed to its unexpected and fundamental role at MAMs in the control of the functional cross-talk between ER and mitochondri