H Althoefer | PubMed Health

PubMed1997

Constitutively active Galphaq and Galpha13 trigger apoptosis through different pathways.

We investigated the effect of expression of constitutively active Galpha mutants on cell survival. Transfection of constitutively active Galphaq and Galpha13 in two different cell lines caused condensation of genomic DNA and nuclear fragmentation. Endonuclease cleavage of genomic DNA was followed by labeling the DNA fragments and subsequent flow cytometric analysis. The observed cellular phenotype was identical to the phenotype displayed by cells undergoing apoptosis. To distinguish between the apoptosis-inducing ability of the two Galpha-subunits, the signaling pathways involved in this cellular function were investigated. Whereas Galphaq induced apoptosis via a protein kinaseC-dependent pathway, Galpha13 caused programmed cell death through a pathway involving the activation of the small G-protein Rho. Both of the pathways leading to apoptosis were blocked by overexpression of bcl-2. In contrast to other apoptosis-inducing systems, expression of constitutively active Galphaq and Galpha13 triggered apoptosis in high serum as well as in defined medium.

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PubMed1995

Mcm1 is required to coordinate G2-specific transcription in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, MCM1 encodes an essential DNA-binding protein that regulates transcription of many genes in cooperation with different associated factors. With the help of a conditional expression system, we show that Mcm1 depletion has a distinct effect on cell cycle progression by preventing cells from undergoing mitosis. Genes that normally exhibit a G2-to-M-phase-specific expression pattern, such as CLB1, CLB2, CDC5, SWI5, and ACE2, remain uninduced in the absence of functional Mcm1. In vivo footprinting experiments show that Mcm1, in conjunction with an Mcm1-recruited factor, binds to the promoter regions of SWI5 and CLB2 at sites shown to be involved in cell cycle regulation. However, promoter occupation at these sites is cell cycle independent, and therefore the regulatory system seems to operate on constitutively bound Mcm1 complexes. A gene fusion that provides Mcm1 with a strong transcriptional activation domain causes transcription of SWI5, CLB1, CLB2, and CDC5 at inappropriate times of the cell cycle. Thus, Mcm1 and a cooperating, cell cycle-regulated activation partner are directly involved in the coordinated expression of multiple G2-regulated genes. The arrest phenotype of Mcm1-depleted cells is consistent with low levels of Clb1 and Clb2 kinase. However, constitutive CLB2 expression does not suppress the mitotic defect, and therefore other essential activities required for the G2-to-M transition must also depend on Mcm1 function.

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