Redox regulation of protein phosphorylatio

In previous studies it has been shown that restoration of contact inhibition by fibroblast interferon in human HT1080 fibrosarcoma cells is coincidental with the enhancement of protein disulfide isomerase (PDI) expression and activity. PDI, an oxidoreductase, functions in a redox-responsive manner. The cooperative interaction between PDI and the cellular redox state in modulation of disulfide bond formation implicates the redox state in growth and contact inhibition controls. In the present study it was demonstrated that the DNA binding activity of the transcription factor Jun as well as its phosphorylation by casein kinase II (CKII) were redox-dependent. It was also shown that autophosphorylation of CKII, known to influence its activity, is regulated by redox state. In vitro kinase assays revealed that CKII-catalyzed phosphorylation increased the affinity of Jun for the DNA. Upon phosphorylation by CKII, the DNA binding activity of Jun remained redox-dependent although the modulatory effect of the redox environment appeared to be modified by CKII. Conformational analyses indicated that CKII maintained an intact structure only within the redox potential range that was permissive to its autophosphorylation and its catalysis of Jun phosphorylation. Outside of the permissive redox range the structural integrity of the enzyme was lost. Collectively, the data suggested that the redox state influenced Jun's activity by profoundly affecting the autophosphorylation as well as the structure of the CKII complex. To determine whether changes in the redox environment sufficient to cause the observed changes in CKII and Jun actually occurs with the nuclei, the redox potentials of fibroblast nuclei at different stages of the growth cycle were determined. Results indicated that as the density of a proliferating population of cells increased, the intranuclear environment gradually advanced from a high reducing towards a less reducing state. Evidence for alterations in the nuclear redox state demonstrates the biological relevance of redox modulation of the transcription factor-DNA binding and other molecular events and suggests that a redox mechanism may be involved in the regulation of growth.