Claudio Talora

Role of a white collar-1-white collar-2 complex in blue-light signal transduction

Mutations in either white collar‐1 (wc‐1) or white collar‐2 (wc‐2) lead to a loss of most blue‐light‐induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that wc‐1 and wc‐2 are transcription factors regulating the expression of light‐induced genes. The WC proteins form homo‐ and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that wc‐1 undergoes a change in mobility due to light‐induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized wc‐1. wc‐2 is unmodified and also does not change quantitatively in the time frame examined. Light‐dependent phosphorylation of wc‐1 also occurs in a wc‐2 mutant, suggesting that a functional wc‐2 is dispensable for this light‐specific event. These results suggest that light‐induced phosphorylation and degradation of wc‐1 could play a role in the transient expression of blue‐light‐regulated genes. Our findings suggest a mechanism by which wc‐1 and wc‐2 mediate light responses in Neurospora.

Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins.

The genes coding for white collar‐1 and white collar‐2 (wc‐1 and wc‐2 ) have been isolated previously, and their products characterized as Zn‐finger transcription factors involved in the control of blue light‐induced genes. Here, we show that the PAS dimerization domains present in both proteins enable the WC‐1 and WC‐2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the white collar (WC) proteins. A computer analysis of WC‐1 reveals a second domain, called LOV, also identified in NPH1, a putative blue light photoreceptor in plants and conserved in redox‐sensitive proteins and in the phytochromes. The WC‐1 LOV domain does not dimerize with canonical PAS domains, but it is able to self‐dimerize. The isolation of three blind wc‐1 strains, each with a single amino acid substitution only in the LOV domain, reveals that this region is essential for blue light responses in Neurospora. The demonstration that the WC‐1 proteins in these LOV mutants are still able to self‐dimerize suggests that this domain plays an additional role, essential in blue light signal transduction.

Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain

ABSTRACT The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2–gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).

Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia.

Notch signaling deregulation is linked to the onset of several tumors including T-cell acute lymphoblastic leukemia (T-ALL). Deregulated microRNA (miRNA) expression is also associated with several cancers, including leukemias. However, the transcriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorly understood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of several Notch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focused our attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kB consensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch and NF-kB are novel coregulatory signals of miR-223 expression, being able to activate cooperatively the transcriptional activity of miR-223 promoter. Notably, the Notch-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines. Moreover, we observed the inverse correlation of miR-223 and FBXW7 expression in a panel of T-ALL patient-derived xenografts. Finally, we show that miR-223 inhibition prevents T-ALL resistance to γ-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols.

Notch signaling and diseases: An evolutionary journey from a simple beginning to complex outcomes

Notch signaling pathway regulates a wide variety of cellular processes during development and it also plays a crucial role in human diseases. This important link is firmly established in cancer, since a rare T-ALL-associated genetic lesion has been initially reported to result in deletion of Notch1 ectodomain and constitutive activation of its intracellular region. Interestingly, the cellular response to Notch signaling can be extremely variable depending on the cell type and activation context. Notch signaling triggers signals implicated in promoting carcinogenesis and autoimmune diseases, whereas it can also sustain responses that are critical to suppress carcinogenesis and to negatively regulate immune response. However, Notch signaling induces all these effects via an apparently simple signal transduction pathway, diversified into a complex network along evolution from Drosophila to mammals. Indeed, an explanation of this paradox comes from a number of evidences accumulated during the last few years, which dissected the intrinsic canonical and non-canonical components of the Notch pathway as well as several modulatory extrinsic signaling events. The identification of these signals has shed light onto the mechanisms whereby Notch and other pathways collaborate to induce a particular cellular phenotype. In this article, we review the role of Notch signaling in cells as diverse as T lymphocytes and epithelial cells of the epidermis, with the main focus on understanding the mechanisms of Notch versatility.

Pre‐TCR‐triggered ERK signalling‐dependent downregulation of E2A activity in Notch3‐induced T‐cell lymphoma

Notch and basic helix–loop–helix E2A pathways specify cell fate and regulate neoplastic transformation in a variety of cell types. Whereas Notch enhances tumorigenesis, E2A suppresses it. However, whether and how Notch and E2A interact functionally in an integrative mechanism for regulating neoplastic transformation remains to be understood. It has been shown that Notch3‐induced T‐cell leukaemia is abrogated by the inactivation of pTα/pre‐T‐cell antigen receptor (pre‐TCR). We report here that Notch3‐induced transcriptional activation of pTα/pre‐TCR is responsible for the downregulation of E2A DNA binding and transcriptional activity. Further, the E2A messenger RNA and protein levels remain unaltered but the E2A inhibitor Id1 expression is augmented in thymocytes and T lymphoma cells derived from Notch3 transgenic mice. The increase in Id1 expression is achieved by pre‐TCR‐induced extracellular‐signalling‐regulated kinase 1/2. These observations support a model in which the upregulation of pre‐TCR signalling seems to be the prerequi‐site for Notch3‐induced inhibition of E2A, thus leading to the development of lymphoma in Notch3 transgenic mice.

Notch3 and the Notch3-upregulated RNA-binding protein HuD regulate Ikaros alternative splicing

Constitutive activation of the transmembrane receptor, Notch3, and loss of function of the hematopoietic transcription repressor, Ikaros (IK), play direct roles in T‐cell differentiation and leukemogenesis that are dependent on pre‐T‐cell receptor (pre‐TCR) signaling. We demonstrate the occurrence of crosstalk between Notch3 and IK that results in transcriptional regulation of the gene encoding the pTα chain of the pre‐TCR. We also show that, in the presence of the pre‐TCR, constitutive activation of Notch3 in thymocytes causes increased expression of dominantnegative non‐DNA‐binding IK isoforms, which are able to restrain the IK inhibition of Notch3s transcriptional activation of pTα. This effect appears to be mediated by Notch3s pre‐TCR‐dependent upregulation of the RNA‐binding protein, HuD. Notch3 signaling thus appears to play a critical role in the diminished IK activity described in several lymphoid leukemias. By exerting transcription‐activating and transcription‐repressing effects on the pTα promoter, Notch3 and IK cooperate in the fine‐tuning of pre‐TCR expression and function, which has important implications for the regulation of thymocyte differentiation and proliferation.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)Cθ is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-κB activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pTα/pre-TCR and constitutive activation of NF-κB characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKCθ is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-κB activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKCθ in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-κB activation. This paper therefore suggests that PKCθ mediates the activation of NF-κB by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Oxidative stress activation of miR-125b is part of the molecular switch for Hailey-Hailey disease manifestation.

Abstract:  Hailey–Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesion. Micro RNAs (miRNAs) are endogenous post‐transcriptional modulators of gene expression with critical functions in health and disease. Here, we evaluated whether the expression of specific miRNAs may play a role in the pathogenesis of HHD. Here, we report that miRNAs are expressed in a non‐random manner in Hailey–Hailey patients. miR‐125b appeared a promising candidate for playing a role in HHD manifestation. Both Notch1 and p63 are part of a regulatory signalling whose function is essential for the control of keratinocyte proliferation and differentiation and of note, the expression of both Notch1 and p63 is downregulated in HHD‐derived keratinocytes. We found that both Notch1 and p63 expression is strongly suppressed by miR‐125b expression. Additionally, we found that miR‐125b expression is increased by an oxidative stress‐dependent mechanism. Our data suggest that oxidative stress‐mediated induction of miR‐125b plays a specific role in the pathogenesis of HHD by regulating the expression of factors playing an important role in keratinocyte proliferation and differentiation.

Complex multipathways alterations and oxidative stress are associated with Hailey-Hailey disease

Background  Hailey–Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene that encodes the Ca2+/Mn2+‐adenosine triphosphatase ATP2C1 and HHD, we still have little understanding of how these mutations affect manifestations of the disease.