Regulation of cell motility by mitogen-activated protein kinase.

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

[Lymphoma and renal failure]. / Linfoma e insufficienza renale.

The Authors examine the main causes of renal failure in lymphoma (infiltration of kidneys, glomerulonephritis, paraproteinemic nephropathy), the difficulties for a correct diagnosis with ultrasonography, urography and CT scan, the importance of renal biopsy. The histologic examination should be promptly performed in patients with sudden renal failure of unknown aetiology and clinical suspicion of malignancy, because renal function can improve dramatically with aggressive chemotherapeutic treatment of lymphoma.

Analysis of beta-catenin aggregation and localization using GFP fusion proteins: nuclear import of alpha-catenin by the beta-catenin/Tcf complex.

beta-Catenin plays essential roles in cell adhesion, by associating with cadherins, and as a signaling molecule, by interacting with the Tcf/LEF-1 family of transcription factors. In order to study the protein-protein interactions of beta-catenin in living cells, we fused it to green fluorescent protein (GFP). GFP-beta-catenin was incorporated into cell junctions but also accumulated in the nucleus, where it formed rod-like structures. The carboxyl-terminal armadillo repeats of GFP-beta-catenin were sufficient for nuclear localization, but formation of rods required the armadillo repeats and sequences in both the amino- and the carboxyl-terminal domains. Rod formation was prevented by coexpression of N-cadherin, APC, and Tcf-4, which bind to the armadillo repeats of beta-catenin, but not by coexpression of alpha-catenin, although alpha-catenin expression did prevent accumulation of beta-catenin in the nucleus. Interestingly, when alpha-catenin, beta-catenin, and Tcf-4 were coexpressed they colocalized in the nucleus, and this correlated with a decrease in beta-catenin/Tcf-dependent transcriptional activity. These results indicate that binding of beta-catenin to Tcf-4 overrides the function of alpha-catenin to sequester beta-catenin in the cytoplasm and suggest that alpha-catenin can regulate beta-catenin signaling in the nucleus.

alpha-catenin inhibits beta-catenin signaling by preventing formation of a beta-catenin*T-cell factor*DNA complex.

alpha-Catenin and beta-catenin link cadherins to the cytoskeleton at adherens junctions. beta-Catenin also associates with members of the T-cell factor (Tcf) family of transcription factors, and mutations in beta-catenin lead to activation of Tcf-dependent transcription and increased cell growth. Although the loss of alpha-catenin expression can also promote cell growth, the role of endogenous alpha-catenin in beta-catenin signaling is unclear. Here we show that loss of alpha-catenin expression in a colon cancer cell line correlates with increased Tcf-dependent transcription. The presence of alpha-catenin in colon cancer cell nuclei suggests that it inhibits transcription directly, and, in agreement with this, ectopic expression of alpha-catenin in the nucleus represses Tcf-dependent transcription. Furthermore, recombinant alpha-catenin disrupts the interaction between the beta-catenin.Tcf complex and DNA. We conclude that alpha-catenin inhibits beta-catenin signaling in the nucleus by interfering with the formation of a beta-catenin. Tcf.DNA complex.

Protein interactions with a gender-specific gene of Schistosoma mansoni: characterization by DNase I footprinting, band shift and UV cross-linking.

A schistosome gender specific gene (F-10), was used as a probe to characterize DNA binding proteins from adult male and female Schistoma mansoni. Using the band-shift and DNase I footprinting methods, it was found that proteins from male and female worms bound to the intact F-10 gene and to restriction fragments corresponding to different domains of the gene, generating relatively long protected sites. Clear differences between male and female proteins were only observed when nuclear proteins were tested. Thus, gender-specific binding was detected in fragments corresponding to the 5' and 3' ends. UV-induced cross-linking between schistosome proteins and a synthetic oligonucleotide bearing a steroid response element present in the 3' untranslated end of the F-10 gene, revealed a major DNA binding protein with a molecular mass of 30 kDa, in both male and female worms. These results suggested that the activation of transcription of the F-10 gene may depend essentially on nuclear proteins.

F-10 nuclear binding proteins of Schistosoma mansoni: structural and functional features.

By incubating total protein extracts of Schistosoma mansoni with 3H-17-beta-estradiol and 20-hydroxyecdysone, steroid binding proteins were detected in both male and female worms. The interaction of nuclear proteins with a restriction fragment of the gender and stage-specific gene F-10 was investigated using the 'band-shift' technique. Male and female nuclear proteins bound in a distinct way to the fragment of this gene containing putative regulatory consensus motifs. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the oestrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females a profile similar to that obtained with male worm protein extracts. This effect of Tamoxifen could not be correlated to inhibition of protein biosynthesis. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.

Glycogen synthase kinase-3 and Axin function in a beta-catenin-independent pathway that regulates neurite outgrowth in neuroblastoma cells.

We have sought to determine the roles of beta-catenin and the Wnt signaling pathway in neurite outgrowth using a model cell system, the Neuro-2a neuroblastoma cell line. Activation of the Wnt signaling pathway disrupts a multiprotein complex that includes beta-catenin, Axin, and glycogen synthase kinase-3 (GSK-3), which would otherwise promote the phosphorylation and degradation of beta-catenin. Stabilized beta-catenin accumulates in the cytosol and in the nucleus; in the nucleus it binds to TCF family transcription factors, forming a bipartite transcriptional activator of Wnt target genes. These events can be mimicked by lithium (Li(+)), which inhibits GSK-3 activity. Both Li(+) and the GSK-3 inhibitor SB415286 induced neurite outgrowth of Neuro-2a cells. Li(+)-induced neurite outgrowth did not require beta-catenin-/TCF-dependent transcription, and increasing levels of beta-catenin either by transfection or using Wnt-3A was not sufficient to induce neurite outgrowth. Interestingly, Axin, which is also a substrate for GSK-3, was destabilized by Li(+) and ectopic expression of Axin inhibited Li(+)-induced neurite outgrowth. Deletion analysis of Axin indicated that this inhibition required the GSK-3 binding site, but not the beta-catenin binding site. Our results suggest that a signaling pathway involving Axin and GSK-3, but not beta-catenin, regulates Li(+)-induced neurite outgrowth in Neuro-2a cells.

Molecular aspects of Schistosoma mansoni female maturation.

Incubation of total protein extracts of Schistosoma mansoni with 3H 17-beta-estradiol and 20-hydroxyecdysone, revealed steroid binding proteins in both, male and female worms. The interaction of nuclear proteins with restriction fragments of the gender and stage-specific gene F-10 was investigated using the "Band-Shift" technique. Distinct male and female nuclear proteins bound to the fragments of this gene. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the estrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females, a band profile similar to that obtained with male worm protein extracts. When Tamoxifen was injected into schistosome infected mice, the eggs produced by females presented an abnormal morphology, compatible with non-viable eggs. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.