Plakoglobin is a cytoplasmic protein and a homologue of ␤-catenin and Armadillo of Drosophila with similar adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the relatively well characterized role of ␤-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene expression, the signaling function of plakoglobin in regulation of cell growth is undefined. Here, we show that high levels of plakoglobin expression in plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics we observe a pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis-regulating proteins. The BCL-2 expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. Our data suggest that the growth regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death.

Pg is a homologue of β‐catenin and Armadillo, the product of the Drosophila segment polarity gene and has been shown to have both adhesive and signaling functions. It interacts with both classic and desmosomal cadherins. Pg interaction with the desmosomal cadherins is essential for desmosome assembly. Its precise role in the classic cadherin complexes is unclear, although Pg‐E–cadherin interaction appears to be necessary for the formation of desmosomes. In addition to cadherins in adhesion complexes, Pg interacts with a number of proteins involved in regulation of cell differentiation and proliferation such as Lef‐1/Tcf‐1 transcription factors and the tumor suppressor protein APC. In this study, we have introduced Pg cDNA into SCC9 cells, a Pg‐ and E‐cadherin‐deficient squamous cell carcinoma line, which also lacks desmosomes. These cells have both α‐catenin and β‐catenin, display unusual expression of N‐cadherin, and have the typical fibroblastic phenotype of transformed cells. Pg‐expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation coincided with the appearance of an epidermoid phenotype, with increased adhesiveness and a contact‐dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of N‐cadherin and a decrease in level and stability of β‐catenin, without any apparent effects on α‐catenin. These results show that, in the absence of E‐cadherin, Pg can efficiently use N‐cadherin to induce desmosome formation and epidermoid phenotype. They also suggest a role for Pg as one of the regulators of the intracellular β‐catenin levels and underscore the pivotal role of this protein in regulating cell adhesion and differentiation. Cell Motil. Cytoskeleton 40:87–100, 1998. © 1998 Wiley‐Liss, Inc.

Ago1, Dcr1 and Rdp1 are the core components of the RNA interference (RNAi) apparatus in the fission yeast Schizosaccharomyces pombe. They function in distinct gene-silencing pathways that direct homology-dependent degradation of mRNA and modification of chromatin. In addition, Ago1 and Dcr1 regulate enactment of Cdc2-dependent cell cycle checkpoints. The ability of the RNAi apparatus to perform multiple roles in these divergent pathways is sure to require dynamic localization of Ago1, Dcr1 and/or Rdp1. Although limited information is available, comprehensive studies regarding the relative localizations of Ago1, Dcr1 and Rdp1 are lacking. To this end, we employed live-cell imaging and immunoelectron microscopy to study the intracellular localizations of these proteins. In contrast to previous reports, our study results indicate that the bulk of Ago1 and Dcr1 form stable complexes and are associated with large, mobile, highly dynamic cytoplasmic elements. The majority of Rdp1 is localized to the nucleus, but a pool of Rdp1 is associated with the same cytoplasmic structures. The movements of these structures were dependent upon ATP and intact microtubules. Recruitment of the RNAi core proteins to these structures was not dependent upon siRNAs. Together, our data indicate that the enzymes required for the initiation and effector phases of RNA-dependent gene silencing are concentrated in a common intracellular location, an arrangement that would be expected to result in highly efficient post-transcriptional gene silencing.

Here, we provide evidence that the amino terminus of Ago1 binds to proteins that function in cell cycle regulation including 14-3-3 proteins. Interestingly, the amino terminus of human Ago2, the endonuclease that cleaves siRNA-targeted mRNAs, was also demonstrated to bind 14-3-3 proteins. Overexpression of the Ago1 amino terminus in yeast resulted in cell cycle delay at the G 2 /M boundary. Further investigation revealed that nuclear import of the mitosis-inducing phosphatase Cdc25 is inhibited by overexpression of the Ago1 amino terminus. Under these conditions, we found that the cyclin-dependent kinase Cdc2 is constitutively phosphorylated on tyrosine 15, thereby reducing the activity of this kinase, a situation that delays entry into mitosis. We hypothesize that 14-3-3 proteins are required for Argonaute protein functions in cell cycle and/or gene-silencing pathways.

The developmental success of interspecific Lepomis hybrids is used as an index of gene regulatory divergence between the green sunfish, L. cyanellus, and each of three other parental species, longear sunfish, L. megalotis, warmouth, L. gulosus, and bluegill, L. macrochirus. This gene regulatory divergence is compared to the degree of structural gene divergence among these four species (genetic distance [Nei, '78], D, ranged from 0.206 to 0.586). The developmental success of the hybrid embryos at the level of morphogenesis was higher than expected from the genetic distance between the parental species. The rates of morphogenesis of the hybrid embryos were the same as that for the green sunfish embryos. The percentage of embryos that hatched was relatively high in all crosses. However, two of the hybrid crosses resulted in enhanced percentages of hatched embryos. Slight increases in the extent of morphological abnormalities were observed in hybrids from crosses between more distantly related parental species. The schedules and levels of enzyme locus expression of the hybrids, assessed spectrophotometrically and electrophoretically for nine enzyme systems (encoded in a total of 14 loci), were different from each other and from those of the green sunfish embryos. Alterations in the time of first enzyme appearance and in the time of first increase in enzyme activity in the developing hybrid embryos were not correlated with genetic distance between parental species. However, the extents of alteration of enzyme activities over the entire period of hybrid embryogenesis were correlated with the genetic distance. We attribute the morphological and molecular anomalies observed in the hybrids to gene regulatory incompatibilities between species. Although the exact number of mutational differences and their relative developmental impacts are not known, some inferences can be drawn about the degree of divergence in gene regulation between species. It appears that an uncoupling of the rates of structural and regulatory gene evolution can occur between species of some taxa, an observation that has implications for the roles of gene regulatory differences in organismic evolution.