TAp63alpha indirectly regulates a cutaneous HPV promoter through complex formation with Jun family members

The p63alpha isoforms of the p53 family have been demonstrated to play a crucial role in the development and differentiation of the skin. We show that expression of the TAp63alpha isoform leads to an upregulation of the cutaneous papillomavirus HPV 20 promoter, which is increased at least three-fold when c-Jun is co-expressed, in contrast to a minimal increase in activity in the presence of c-Jun alone. Co-expression of TAp63alpha with JunB or JunD, respectively, and in combination, leads to a reduction in the viral promoter activation measured by the expression of TAp63alpha alone. JunB and JunD also inhibits the additive effect exerted on the TAp63alpha activation by c-Jun. Co-immunoprecipitation assays demonstrate a complex formation of c-Jun, JunB and JunD with TAp63alpha through the SAM domain mediating protein-protein interactions, which is characteristic for p63alpha. Co-expression of p53 mutant R248W not only downregulates the differential modulation of the viral promoter by TAp63alpha alone and in the presence of the Jun family members, but leads to a reduction in the protein levels of the overexpressed c-Jun, JunB, JunD, as well as TAp63alpha. This model system provides insight into yet unknown pathways through which TAp63alpha and Jun may cooperate in the pathogenesis of HPV associated cutaneous lesions.     

Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized mostly by tumors of the parathyroids, pancreas and anterior pituitary. The gene responsible, MEN1, encodes Menin, a 610 aminoacid nuclear protein with no sequence homology to other proteins. Although a mouse knock-out model is available, the function of Menin is still elusive. Proteins of known function are shown to interact with Menin: JunD, nuclear factor-KappaB, Smad3, Pem, Nm23H1, glial fibrillary acidic protein, Vimentin, and probably P53. Their partnership with Menin may correspond to a regulation of their activity, but their relevance to the various traits of MEN1 pathogenicity is not established. This raises fundamental issues on the regulation pathways implicated in this complex endocrine disease.     

Jun, the oncoprotein

Cellular Jun (c-Jun) and viral Jun (v-Jun) can induce oncogenic transformation. For this activity, c-Jun requires an upstream signal, delivered by the Jun N-terminal kinase (JNK). v-Jun does not interact with JNK; it is autonomous and constitutively active. v-Jun and c-Jun address overlapping but not identical sets of genes. Whether all genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined. The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology. Genetic screens and functional tests remain a necessity for the identification of genes that control the oncogenic phenotype.     

The chicken junD gene and its product

We have isolated and characterized the chicken junD gene. It does not contain an intron; its upstream regulatory sequences lack the AP-1-binding site seen in c-jun but include two CRE elements. Downstream untranslated sequences do not show the destabilizing signal ATTTA. The amino acid sequence of the chicken JunD protein is closely related to that of mouse JunD in the dimerization and DNA contact surfaces of the carboxy-terminal region; additional homologies to mouse JunD are seen in acidic and amphipathic amino-terminal domains. Chicken JunD contains stretches of oligoglycines, alanines and prolines, possibly acting as hinges that connect functionally distinct domains of the protein. Chicken junD is broadly expressed at low basal levels in differentiated tissues and at somewhat higher levels in cultured fibroblasts. The cDNA clone of junD was transcribed and translated in vitro. The resulting JunD protein migrates in between 40 and 50 kDa in an SDS gel and can be precipitated with an antibody prepared against a polypeptide consisting of the carboxy-terminal 100 amino acids of mouse c-Jun.     

Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun.

Chicken c-Jun proteins synthesized in vitro in reticulocyte extract consist of several electrophoretic isoforms resulting from phosphorylation which can be specifically reversed by purified protein phosphatase 2A (PP2A). Using the phosphatase inhibitors okadaic acid and microcystin-LR, we conclude that the isoforms seen in vitro represent a balance between the action of an unidentified kinase(s) which phosphorylates c-Jun and dephosphorylation by an endogenous PP2A-like phosphatase. c-Jun proteins are also subject to phosphorylation in vivo in chick embryo fibroblasts (CEF), which can be reversed by PP2A. In contrast, the viral Jun oncoprotein encoded by ASV17 is not subject to PP2A-sensitive phosphorylation in vitro and is hypophosphorylated in comparison with c-Jun in ASV17-transformed CEF. Hybrids between c-Jun and v-Jun demonstrate that differential phosphorylation in vitro is a consequence of deletion of 27 amino acids in the N-terminal third of v-Jun. The deletion is important for oncogenic activation and lies in a domain, termed delta, which regulates c-Jun transactivation function. PP2A-sensitive phosphorylation in vitro correlates with the differential responsiveness of c-Jun and v-Jun to a recently identified cell type-specific inhibitor of transactivation function.     

Identification of N-terminally truncated stable nuclear isoforms of CDC25B that are specifically involved in G2/M checkpoint recovery

CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage. ΔN-CDC25B isoforms are expressed from a distinct promoter not involved in expression of canonical full-length isoforms. Remarkably, in contrast to the high lability and spatial dynamism of the full-length isoforms, ΔN-CDC25B isoforms are highly stable and exclusively nuclear, strongly suggesting the existence of two pools of CDC25B phosphatases in the cell that have functionally distinct properties. Using isoform-specific siRNA, we found that depleting full-length isoforms, but not ΔN-CDC25B isoforms, delays entry into mitosis. Thus, in an unperturbed cell cycle, the full-length isoforms are exclusively responsible for activating cyclin B-CDK1. Strikingly, in the late response to DNA damage, we found a CHK1-dependent shift in accumulation of CDC25B isoforms toward the ΔN-CDC25B species. Under this physiological stress condition, the ΔN-CDC25B isoform was found to play a crucial, nonredundant function in restarting the cell cycle after DNA damage-induced G2 phase arrest. Our findings reveal the existence of a previously unrecognized CDC25B isoform that operates specifically in the nucleus to reinitiate G2/M transition after DNA damage.     

Expression of tyrosine kinase Etk/Bmx and its relationship with AP-1- and NF-kappaB-associated proteins in hepatocellular carcinoma

Etk/Bmx is a cytoplasmic tyrosine kinase, which was first identified in human bone marrow cells. It has been found to play an important role in the regulation of differentiation and tumorigenicity in some cancers. The aim of this study was to investigate the significance of Etk/Bmx expression in hepatocellular carcinoma (HCC) and the relationship between Etk/Bmx and activated protein-1 (AP-1)- and nuclear factor-kappaB (NF-kappaB)-associated proteins. We used immunohistochemisty to examine 40 cases of human HCC along with corresponding nontumor tissues to assess Etk/Bmx, Jun family (c-Jun, JunB, JunD), Fos family (c-Fos, FosB, Fra-1) and NF-kappaB p65 expression in these samples. Etk/Bmx expression was present in 12 of 40 (30%) HCC specimens, 4 of which among the 25 well-differentiated tumors and 8 among the 15 poorly differentiated tumors, respectively. In contrast, 6 of 40 (15%) cases expressed Etk/Bmx in adjacent nontumor tissues. Expression level and cellular localization of Etk/Bmx were different in cancer cells and nontumor cells. Etk/Bmx expression was correlated with histological differentiation, but not with clinicopathological features including tumor size, HBV infection, cirrhosis, and metastasis. There was a close relationship between Etk/Bmx and c-Fos expression in HCC. Etk/Bmx immunopositivity was independent of c-Jun, JunD, FosB, Fra-1 and NF-kappaB p65. Our results indicated that Etk/Bmx may have different biological roles in tumor and nontumor cells, and may be involved in regulating hepatocyte differentiation by c-Fos activation in HCC.     

The isoform-specific stretch of hSos1 defines a new Grb2-binding domain

hSos1 isoform II, defined by the presence of a 15 amino acid stretch in its carboxy-terminal region, exhibits higher Grb2 affinity than hSos1 isoform I. In this study, we investigated the cause for this difference and observed that, in addition to the four currently accepted Grb2-binding motifs, a number of additional, putative SH3-minimal binding sites (SH3-MBS) could be identified. The isoform II-specific 15 amino acid stretch contained one of them. Indeed, we demonstrated by site-directed mutagenesis that these SH3-MBS were responsible for the Grb2 interaction, and we found that C-terminal fragments of the two hSos1 isoforms (lacking the four cannonical Grb2-binding motifs, but containing the SH3-minimal binding sites) were able to bind Grb2, with the isoform II fragment showing higher Grb2 affinity than the corresponding isoform I fragment. Furthermore, we provide evidence that C-terminal truncated mutants of either hSos1 isoform, containing only the SH3-minimal binding sites, were able to originate in vivo stable complexes with Grb2. Although, Grb2-binding remains higher in both full-length isoforms, compared to the C-terminal truncated mutants, these mutants were also able to activate Ras, supporting a potential role of this C-terminal region as negative modulator of Sos1 activity. These findings document the existence of a new, functional, SH3-minimal binding site located in the specific stretch of hSos1 isoform II which may be responsible for the increased Grb2 affinity of this isoform in comparison to isoform I, and for the physiological properties differences between both isoforms. Moreover, these SH3-minimal binding sites may be sufficient to attain stable and functional hSosl-Grb2 complexes.     

Characterization of the short isoform of Helios overexpressed in patients with T-cell malignancies

In an earlier report, we demonstrated overexpression of a short isoform of Helios, Hel-5, which lacks three of four N-terminal zinc fingers, in patients with adult T-cell leukemia/lymphoma. Here, we characterized Hel-5 using immunoprecipitation, and gel shift and luciferase promoter assays, and found that Hel-5 lacks the repressor function observed with a full-length isoform of Helios. Moreover, Hel-5 associates with the full-length isoforms of the Ikaros gene family, Ikaros, Aiolos and Helios, and inhibits their DNA binding activity when present in excess, leading to dominant-negative effects on the full-length isoforms of the Ikaros gene family. Our results suggest a critical role for Helios in the mechanism of leukemogenesis.