These data were interpreted to suggest that phosphorylation is necessary for the formation of a transcriptional complex and for activation of the CYP1A1 gene. In agreement with this view, pretreatment of mouse hepatocyte cultures and hepatoma cells with staurosporine inhibited TCDD-activated transactivation of CYP1A1 in a dose-dependent manner (Chun et al., 1994). Tyrphostin AG213, a specific tyrosine kinase inhibitor, had no effects on TCDD-induced CYP expression, further supporting the view that PKC is the kinase involved in this process. However, the effects of TCDD on PKC and inositol phosphate metabolism in primary cultures of rat hepatocytes exhibit markedly different profiles from phorbol esters, suggesting that these tumor promoters modulate hepatocyte signal transduction via distinct mechanisms (Wolfle et al., 1993). Thus, it remains to be determined with certainty whether PKC is involved in phosphorylation of the ligand/AhR complex or whether phosphorylation of the AhR occurs subsequent to ligand binding and transformation. This issue was addressed by Schafer et al. (1993), who examined the role of PKC on the functionality of the hepatic cytosolic Ah receptor. In these experiments, two nonspecific PKC inhibitors, H7 and staurosporine, and one specific PKC inhibitor, calphostin c, were employed in assays of Ah receptor transformation and DNA binding. AhR transformation and DNA binding occurred in hepatic cytosol despite the absence of detectable kinase activity. Functional PKC activity was not required for ligand-dependent transformation or DNA binding of the Ah receptor complex. An interesting finding in their studies was that staurosporine, a nonspecific kinase inhibitor, formed an AhR/DNA complex that co-migrates with that produced by TCDD and can effectively compete with TCDD for DNA binding. Although some controversy remains, it appears that phosphorylation events occur early in the process of AhR complex formation in the absence of ligand binding. Phosphorylation may influence the stability of the AhR protein and/or DNA binding.
The biological significance of TCDD-induced changes in protein phosphorylation remains to be defined. TCDD modulates PKC as well as other kinases in somatic cells at low concentrations, and these effects can influence the physiology of the cells. For instance, a dose-dependent increase in tyrosine phosphorylation of five hepatic intracellular proteins has been reported in TCDD-treated C57BL/6J female mice (Ma et al., 1992). TCDD induces a rapid rise in protein phosphorylation activities in the extranuclear fraction (i.e., cytosol and cellular membranes) of the adipose tissue from male guinea pigs (Enan and Matsumura, 1994). This effect occurs both in vivo and in vitro and is not abolished by actinomycin D, an inhibitor of transcription. However, inhibition of protein synthesis by cycloheximide partially suppresses the effect of TCDD. These responses correlate with a quick rise in ras GTP binding activity, as well as phosphorylation of nuclear c-myc protein. In view of the lack of inhibition by actinomycin D and the short time required for TCDD to induce phosphorylation, it has been suggested that stimulation of protein phosphorylation activities by TCDD is not mediated via a transcriptional process. In support of this interpretation