BIOCHEMICAL AND FUNCTIONAL-ANALYSIS OF HIGHLY PHOSPHORYLATED FORMS OFC-JUN PROTEIN

We report here that, upon UV irradiation or growth stimulation, endoge nous c-Jun (40 kDa) in chicken embryo fibroblasts (CEF) is converted i nto several forms with apparently higher molecular weights in SDS-poly acrylamide gel electrophoresis (45, 44, 42 kDa), Two of the bands (44 and 45 kDa) were transient after growth stimulation, but were much mor e persistent after UV irradiation. In both cases, the drastic mobility shifts were accompanied with the activation of endogenous JNK activit y but not of MAPK activity, and the bands were shown to represent diff erent phosphorylation states of c-Jun rather than ubiquitinated c-Jun, Biochemical analysis indicated that phosphorylation at Ser(63) and Se r(73) was not sufficient to produce these drastic mobility shifts, whi ch additionally required phosphorylation at Thr(91) and Thr(93). Subst itution of both Ser(63) and Ser(73) with either Ala or Asp had no sign ificant effect on the transforming activity of c-Jun, but the mutants failed to show drastic mobility shifts even after UV irradiation. Thes e results indicate that Ser(63) and Ser(73) are essential for the dras tic mobility shifts and further suggest that the highly phosphorylated forms of c-Jun are not directly involved in cellular transformation. (C) 1998 Federation of European Biochemical Societies.