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Little consideration has been given to the possibility that there could be variations in protein expression that alter susceptibility to tumorigenesis without causing other obvious phenotypic effects. Therefore, we sought to determine if haploinsufficiency for the anti-apoptotic protein Bcl-x(L) would affect tumorigenesis. We chose to study Bcl-x(L) because although bcl-x+/- mice were thought to be phenotypically normal, we and others found that haploinsufficiency for Bcl-x(L) lowers fibroblast resistance to apoptosis in tissue culture. Since resistance to certain forms of apoptosis is required for tumor formation, this suggested that decreased Bcl-x(L) expression would afford protection against tumorigenesis. Indeed, we demonstrate here that bcl-x+/- mice are strikingly resistant to carcinogen-induced tumorigenesis. However, we found that they pay a price for their resistance in that they are more susceptible to clinically relevant forms of tissue injury--they suffer increased hepatic injury in a model of binge alcohol abuse and in response to TNF-alpha treatment. These findings are important because they suggest that even minor variations in Bcl-x(L) expression could affect susceptibility to cancer and other diseases. Additionally, they indicate that the potential for increased susceptibility to tissue injury must be considered in the design of chemopreventative and antineoplastic strategies that involve inhibition of Bcl-x(L) activity.

Deamidation of two asparagines activates a conditional PEST sequence to target Bcl-xL for degradation., The cellular concentration of Bcl-xL is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-xL undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-xL is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-xL for degradation. Additionally, we show that degradation of deamidated Bcl-xL is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-xL, underscoring its importance in Bcl-xL regulation. Our findings strongly suggest that deamidation-regulated Bcl-xL degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli., Author Summary Cellular levels of the pro-survival protein Bcl-xL are an important determinant of cellular susceptibility to many death stimuli, including most cancer therapies. We previously showed that human Bcl-xL undergoes deamidation – the conversion of two neutral asparaginyl side-chains into negatively charged aspartyl side-chains – a process that occurs spontaneously but is accelerated by the treatment of tumor cells with DNA-damaging agents. Here, we show that deamidation activates a hitherto undetected signal sequence within Bcl-xL that targets it for degradation by a pathway involving the proteolytic enzyme calpain. This increased degradation of Bcl-xL, and the consequent enhanced cellular susceptibility to programmed cell death, may contribute to the ability of DNA-damaging agents to kill tumors. We also demonstrate that deamidation of Bcl-xL has likely been conserved from the simplest metazoans to humans, underscoring the importance of deamidation in the regulation of Bcl-xL.

The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.