Identification of a C-terminal tripeptide motif involved in the control ofrapid proteasomal degradation of c-Fos proto-oncoprotein during the G(0)-to-S phase transition

c-Fos proto-oncoprotein is rapidly and transiently expressed in cells under going the G(0)-to-S phase transition in response to stimulation for growth by serum. Under these conditions, the rapid decay of the protein occurring after induction is accounted for by efficient recognition and degradation b y the proteasome. PEST motifs are sequences rich in Pro, Glu, Asp, Ser and Thr which have been proposed to constitute protein instability determinants . c-Fos contains three such motifs, one of which comprises the C-terminal 2 0 amino acids and has already been proposed to be the major determinant of c-Fos instability. Using site-directed mutagenesis and an expression system reproducing c-fos gene transient expression in transfected cells, we have analysed the turnover of c-Fos mutants deleted of the various PEST sequence s in synchronized mouse embryo fibroblasts. Our data showed no role for the two internal PEST motifs in c-Fos instability. However, deletion of the C- terminal PEST region led to only a twofold stabilization of the protein. Ta ken together, these data indicate that c-Fos instability during the G0-to-S phase transition is governed by a major non-PEST destabilizer and a C-term inal degradation-accelerating element. Further dissection of c-Fos C-termin al region showed that the degradation-accelerating effect is not contribute d by the whole PEST sequence but by a short PTL tripeptide which cannot be considered as a PEST motif and which can act in the absence of any PEST env ironment. Interestingly, the PTL motif is conserved in other members of the fos multigene family. Nevertheless, its contribution to protein instabilit y is restricted to c-Fos suggesting that the mechanisms whereby the various Fos proteins are broken down are, at least partially, different. MAP kinas es-mediated phosphorylation of two serines close to PTL, which are both pho sphorylated all over the G(0)-to-S phase transition, have been proposed by others to stabilize c-Fos protein significantly. We, however, showed that t he PTL motif does not exert its effect by counteracting a stabilizing effec t of these phosphorylations under our experimental conditions.