Girolama La Mantia

The protein stability and transcriptional activity of p63α are regulated by SUMO-1 conjugation

Post-translational modification of proteins by the ubiquitin-like molecule SUMO-1 regulates their stability and activity with crucial implications for many cellular processes. Here we show that p63α, but not p63α and α, is sumoylated in vitro and in vivo at a single lysine residue, K637, in the post-SAM domain. SUMO-1 attachment targets ΔNp63α for proteasome mediated degradation while it does not influence p63α intracellular localization, as wild-type protein and a mutant carring the K637 mutated into arginine (K637R), have the same nuclear localization. Four natural p63 mutations, falling within the SAM and post-SAM domain of p63α, were found to be altered in their sumoylation capacity. The transcriptional activities of the natural mutants and of K637R were strongly increased compared to that of wild type p63, suggesting that sumoylation has a negative effect on p63 driven transcription. The findings that ΔNp63α protein levels are regulated by SUMO-1 and that this regulation is altered in natural p63 mutants, suggest that SUMO conjugation to p63 plays a critical role in regulating the biological activity of p63.

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 ΔNp63α protein. We found that MDM2 binds ΔNp63α in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for ΔNp63α 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 ΔNp63α by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the α and β tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous ΔNp63α 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 ΔNp63α protein.

The Human Tumor Suppressor ARF Interacts with Spinophilin/Neurabin II, a Type 1 Protein-phosphatase-binding Protein

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p14ARF (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.

Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias.

Ectodermal dysplasias (EDs) are a group of human pathological conditions characterized by anomalies in organs derived from epithelial-mesenchymal interactions during development. Dlx3 and p63 act as part of the transcriptional regulatory pathways relevant in ectoderm derivatives, and autosomal mutations in either of these genes are associated with human EDs. However, the functional relationship between both proteins is unknown. Here, we demonstrate that Dlx3 is a downstream target of p63. Moreover, we show that transcription of Dlx3 is abrogated by mutations in the sterile α-motif (SAM) domain of p63 that are associated with ankyloblepharon-ectodermal dysplasia-clefting (AEC) dysplasias, but not by mutations found in ectrodactylyectodermal dysplasia-cleft lip/palate (EEC), Limb-mammary syndrome (LMS) and split hand-foot malformation (SHFM) dysplasias. Our results unravel aspects of the transcriptional cascade of events that contribute to ectoderm development and pathogenesis associated with p63 mutations.

cDNA isolation, expression analysis, and chromosomal localization of two human zinc finger genes ☆

On the basis of sequence similarity in the repeated zinc finger domain, we have identified and characterized two human cDNA clones (ZNF7 and ZNF8), both encoding proteins containing potential finger-like nucleic acid binding motifs. Northern blot analysis indicates that both genes are expressed as multiple transcripts and they are ubiquitously present in many human cell lines of different embryological derivation. Moreover, their expression is modulated during in vitro induced terminal differentiation of human myeloid cell line HL-60. By in situ hybridization experiments, we have localized the ZNF7 gene to chromosome 8 (region q24) and the ZNF8 gene to the terminal band of the long arm of chromosome 20 (20q13).

Downregulation of ΔNp63α in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation

The tumor suppressor p14ARF inhibits cell growth in response to oncogenic stress in a p53¬dependent and independent manner. However, new physiologic roles for ARF activation have been proposed. We have previously demonstrated that ARF interacts with p63, influencing its transcriptional activity. p63 is a member of the p53 family involved in skin and limb development, as well as in the homeostasis of mature epidermis. Here, we show that, in human keratinocytes, as well as in tumor-derived cell lines, ARF targets ΔNp63α, the most abundantly expressed p63 isoform, to proteasomal degradation by stimulating its sumoylation. Interestingly, we have observed an increase of ARF expression in differentiating keratinocytes, that is concomitant to the already described upregulation of SUMO2/3. Remarkably, we found that ΔNp63α is preferentially sumoylated by SUMO2, instead of SUMO1, and p14ARF increases the efficiency of this process.

Activation of major histocompatibility complex class I mRNA containing an Alu-like repeat in polyoma virus-transformed rat cells

Class I genes of the major histocompatibility complex (MHC) appear to be activated in mouse cells transformed by the DNA tumour virus simian virus 40 (SV40)1. Conversely, suppression of MHC class I genes has been reported in adenovirus-12-transformed baby kidney rat cells2. We have now investigated the expression of genes encoded by the rat MHC locus in rat fibroblast cells transformed by polyoma virus (Py). Using a mouse genomic H–2 clone as a probe in Northern transfer hybridization analysis, we have observed a high level of expression of rat MHC class I messenger RNA in all the transformed rat cell lines analysed. The class I 1.6-kilobase (kb) mRNA activated in Py-transformed rat cells appears to contain an Alu-like type II repeat element, as the same 1.6-kb mRNA is detected using either the H–2 class I sequence or a repetitive Alu-like type II element as a probe. High levels of heterogeneous poly(A)+ transcripts of 0.5–0.8 kb are also observed in Py-transformed rat cells using probes containing an Alu-like type II repetitive element.

The promiscuity of ARF interactions with the proteasome.

The tumor suppressor ARF is one of the most important oncogenic stress sensors in mammalian cells. Its effect is exerted through the interaction with different cellular partners, often resulting in their functional inactivation. This review focuses on the role played by the proteasome in ARF regulation of protein turnover and the function of most of its interacting partners. Specific proteasome components appear to be involved in the regulation of ARF turnover, bringing to light a complex network of interactions between ARF and the proteasome.

Inhibition of p63 Transcriptional Activity by p14ARF: Functional and Physical Link between Human ARF Tumor Suppressor and a Member of the p53 Family

ABSTRACT The ARF/MDM2/p53 pathway is a principal defense mechanism to protect the organism from uncontrolled effects of deregulated oncogenes. Oncogenes activate ARF, which interacts with and inhibits the ubiquitin ligase MDM2, resulting in p53 stabilization and activation. Once stabilized and activated, p53 can either induce or repress a wide array of different gene targets, which in turn can regulate cell cycle, DNA repair, and a number of apoptosis-related genes. Here we show that, unlike p53, p63, a member of the p53 family, directly interacts with p14ARF. Through this interaction ARF inhibits p63-mediated transactivation and transrepression. In p63-transfected cells, ARF, which normally localizes into nucleoli, accumulates in the nucleoplasm. Based on these observations, we suggest that stimuli inducing p14ARF expression can, at the same time, activate p53 and impair p63 transcriptional activity, altering the pattern of p53 target gene expression. Here we show, for the first time, a physical and functional link between the p14ARF tumor suppressor protein and p63, a member of the p53 family.

Status and expression of the p16INK4 gene in human thyroid tumors and thyroid‐tumor cell lines

The p16INK4 tumor‐suppressor gene (also known as CDKN2, CDK41 and MTSI) encodes a negative regulator of the cell cycle. This gene, located in 9p21, is mutated or homozygously deleted in a high percentage of tumor cell lines and specific types of primary tumors. We have examined the status of the p16INK4 gene in 31 thyroid tumors and 7 thyroid cell lines. No DNA abnormalities were found in primary tumors. Conversely, p16INK4 gene structural alterations, deletions and point mutations were found in 4 thyroid cell lines. The expression of the 2 different p16INK4 mRNAs, the p16α and p16β transcripts, was determined by RNA‐PCR experiments. All the primary thyroid tumors expressed the β transcript, while the p16α was barely detectable. The thyroid cell lines always expressed the p16β transcript, while the α transcript was absent or, whenever present, coded for a mutated form of the p16INK4 gene product. Taken together, our results suggest that loss of p16INK4 function is not directly involved in the process of thyroid‐tumor development, but it probably gives cells in tissue culture a selective growth advantage.