Biochemical and functional analysis of highly phosphorylated forms of c-Jun protein

We report here that, upon UV irradiation or growth stimulation, endogenous c-Jun (40 kDa) in chicken embryo fibroblasts (CEF) is converted into several forms with apparently higher molecular weights in SDS-polyacrylamide gel electrophoresis (45, 44, 42 kDa), Two of the bands (44 and 45 kDa) were transient after growth stimulation, but were much more persistent after UV irradiation. In both cases, the drastic mobility shifts were accompanied with the activation of endogenous JNK activity but not of MAPK activity, and the bands were shown to represent different phosphorylation states of c-Jun rather than ubiquitinated c-Jun, Biochemical analysis indicated that phosphorylation at Ser(63) and Ser(73) was not sufficient to produce these drastic mobility shifts, which additionally required phosphorylation at Thr(91) and Thr(93). Substitution of both Ser(63) and Ser(73) with either Ala or Asp had no significant effect on the transforming activity of c-Jun, but the mutants failed to show drastic mobility shifts even after UV irradiation. These results indicate that Ser(63) and Ser(73) are essential for the drastic 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.