MULTIPLE SIGNALING PATHWAYS CONTROL THE ACTIVATION OF THE CEF-4 9E3 CYTOKINE GENE BY PP60(V-SRC)/

The CEF-4/9E3 cytokine gene is expressed aberrantly in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus. The expressio n of CEF-4 is dependent on both transcriptional and post-transcription al mechanisms of regulation. The characterization of the promoter regi on indicated that three distinct regulatory elements corresponding to an AP-1 binding site (or TRE), a PRDII/kappa B domain, and a CAAT box are involved in the activation by pp60(v-src). In this report we inves tigate the signaling pathways controlling the expression of the TRE an d PRDII domain. The expression of a dominant negative mutant of p21(ra s) reduced the activity of both elements. In contrast a similar mutant of c-Raf-1 affected modestly the activation dependent on the TRE but not PRDII. The stress-activated protein kinase (SAPK)/Jun N-terminal k inase (JNK) pathway was important for the activity of PRDII and the TR E but was not markedly stimulated by pp60(v-src). The addition of calp hostin C and the inhibition of protein kinase C (PKC) diminished the a ccumulation of the CEF-4 mRNA and reduced the activity of a TRE-contro lled promoter. Likewise, the depletion of PHC by chronic treatment wit h phorbol esters inhibited the activation of the TRE. Rous sarcoma vir us-transformed CEF treated with calphostin C were also flatter, did no t display a high degree of criss-crossing, and appeared morphologicall y normal. Hence PKC was important for the activation of AP-1 and the m orphological transformation of CEF. The constitutive expression of CEF -4 was correlated with transformation only when dependent on the TRE. This was not true for PRDII, which was the only element required for t he constitutive activation to the CEF-4 promoter in nontransformed cel ls treated chronically with phorbol esters.