Glucocorticoid-induced activation of caspase-8 protects the glucocorticoid-induced protein Gilz from proteasomal degradation and induces its binding to SUMO-1 in murine thymocytes.

In this study, we evaluated the possible cross-talk between glucocorticoid (GC)-induced leucine zipper (Gilz) and caspase-8 in dexamethasone (Dex)-treated thymocytes. We determined that expression of Dex-induced Gilz protein was reduced when caspase-8 activity was inhibited, and this effect was not partially due to altered Gilz mRNA expression. Inhibition of the proteasome abrogated this reduction in Gilz expression, suggesting that Dex-induced caspase-8 activation protects Gilz from degradation. We hypothesized that the caspase-8-dependent protection of Gilz could be due to caspase-8-driven sumoylation. As a putative small ubiquitin-like modifier (SUMO)-binding site was identified in the Gilz sequence, we assessed whether SUMO-1 interacted with Gilz. We identified a 30-kDa protein that was compatible with the size of a Gilz-SUMO-1 complex and was recognized by the anti-SUMO-1 and anti-Gilz antibodies. In addition, Gilz bound to SUMO ubiquitin-conjugating (E2)-conjugating enzyme Ube21 (Ubc9), the specific SUMO-1 E2-conjugating enzyme, in vitro and coimmunoprecipitated with Ubc9 in vivo. Furthermore, Gilz coimmunoprecipitated with SUMO-1 both in vitro and in vivo, and this interaction depended on caspase-8 activation. This requirement for caspase-8 was further evaluated in caspase-8-deficient thymocytes and lymphocytes in which Gilz expression was reduced. In summary, our results suggest that caspase-8 activation protects Gilz from proteasomal degradation and induces its binding to SUMO-1 in GC-treated thymocytes.

A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF.

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14(ARF). Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14(ARF) in a 'dose-dependent tug of war' fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14(ARF)) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14(ARF)) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.

Differential expression of CD44 isoforms during liver regeneration in rats.

BACKGROUND: CD44 is a transmembrane glycoprotein known to bind hyaluronic acid (HA). This molecule is a multifunctional cell surface glycoprotein involved in lymphocyte homing and activation, tumor growth and metastasis. We have investigated the qualitative modification of CD44 in the regenerating liver as a model for studying cellular proliferation in vivo. Molecules involved in cell adhesion and the extracellular matrix (ECM), which influence differentiation, growth, cell-cell interactions and cellular polarity, play an important role in the liver regeneration. We studied the modulation of CD44 gene expression and its post-transcriptional modifications, analyzing the expression of different isoforms containing exon v6 in the regenerating liver, in sham operated liver and in the hepatoma cells H-35. METHODS: The expression of CD44 and CD44v6 were analyzed in RNA extracted from regenerating liver at different times after partial hepatectomy (PH), and H-35 hepatoma cells by Northern blot, RT-PCR and Southern blot, and in protein extracts from regenerating liver by Western blot. H-35 hepatoma cells were assayed with the antibody cross-linked technique with CD44 antibodies. RESULTS: The standard CD44 form is expressed in regenerating liver and its levels were not modified following PH. However, our analysis revealed CD44 isoforms containing v6 in the first hours after PH as well as in the H-35 hepatoma cell line. H-35 cells treated with cross-linked anti-CD44 antibodies or HA show an increased rate of incorporation of [3H]thymidine (30 and 25%, respectively) with respect to the control. CONCLUSION: These findings suggest that CD44 may play a role in the proliferation of residual hepatocytes following PH.

Shc signaling in differentiating neural progenitor cells.

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).