PHOSPHORYLATION AND HIGH-LEVEL EXPRESSION OF FRA-2 IN V-SRC TRANSFORMED-CELLS - A PATHWAY OF ACTIVATION OF ENDOGENOUS AP-1

Chicken embryo fibroblasts (CEF) transformed with v-src were previousl y reported to revert to normal phenotype after the introduction of dom inant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation, The major chang es in the expression levels of fos and jun family genes induced by v-s rc were the elevation of fra-2 and c-jun transcripts. We show here tha t extensive phosphorylation of the AP-1 component Fra-2 is a major qua litative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are pho sphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-termin al region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significant ly activated in v-src transformed CEF and Fra-2 was not a good substra te for JNKs. fra-2 promoter analysis indicated that this promoter acti vity is elevated in v-src transformed CEP via two AP-1 binding sites a nd CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an activ e one and further that fra-2 expression is autoregulated in response t o the phosphorylation status of its gene product.