Timothy S. Finco

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.

Ras proteins function in stimulating cell proliferation and differentiation through the activation of Raf-dependent and Raf-independent signal transduction pathways and the subsequent activation of specific transcription factors. The transcription factor NF-kappaB has been widely studied as a regulator of genes involved in immune and inflammatory responses. A variety of stimuli activate NF-kappaB through the induced phosphorylation and degradation of the inhibitor IkappaB followed by nuclear translocation of NF-kappaB. We show here that oncogenic forms of Ha-Ras activate NF-kappaB, not through induced nuclear translocation, but rather through the activation of the transcriptional function of the NF-kappaB RelA/p65 subunit. Importantly, RelA/p65 -/- cells are inefficient in the activation of kappaB-dependent gene expression in response to oncogenic Ras expression. Furthermore, IkappaBalpha expression blocks focus formation in NIH3T3 cells induced by oncogenic Ras. These results demonstrate that NF-kappaB is a critical downstream mediator of Ha-Ras signaling and oncogenic potential.

The ubiquitous transcription factor NF-kappa B is regulated by its cytoplasmic inhibitor I kappa B. A variety of cellular stimuli cause the dissociation of NF-kappa B from I kappa B, allowing NF-kappa B to translocate to the nucleus and regulate gene expression. Although the activation of NF-kappa B in vivo is associated with the phosphorylation and degradation of I kappa B alpha, it has remained unclear how each of these events contributes to this process. Recently, studies utilizing protease inhibitors have suggested that the proteolysis of I kappa B alpha is a necessary event in the activation of NF-kappa B. We demonstrate in this study that these and an additional protease inhibitor also completely repress inducible phosphorylation of I kappa B alpha. This surprising result suggests a more complex role of proteases in NF-kappa B activation. In addition, data presented here indicate that many of these inhibitors also directly modify NF-kappa B and inhibit its DNA binding activity. Due to the pleiotropic effects of these protease inhibitors, it is difficult to conclude from their use how I kappa B alpha phosphorylation and degradation contribute to NF-kappa B activation. In the present study, a more direct approach demonstrates that phosphorylation of I kappa B alpha alone is not sufficient for NF-kappa B activation.