Polyomavirus middle-sized tumor antigen modulates c-Jun phosphorylation and transcriptional activity

Polyomavirus middle-sized tumor antigen (MT) increases the expression of c-jun through a phorbol 12-O-tetradecanoate 13-acetate response element in the c-jun promoter. To investigate the cellular signaling pathways affected by MT, we studied the role of the c-Ras and Raf-1 proteins in MT-induced transactivation of c-jun and cell transformation. There was an increase in GTP complexed to Ras in MT-expressing cells, indicating an increase in Ras activity. Coexpression of dominant inhibitory mutants of Ha-ras and raf-1 with MT inhibited MT-mediated transactivation and focus formation. Studies of the phosphorylation of c-Jun showed that MT expression increased the phosphorylation of Ser-63 and Ser-73 in the transactivation domain and decreased the phosphorylation of a peptide containing Ser-243, Ser-249, and Thr-231 in the DNA binding domain. MT increased the transcriptional activating ability of c-Jun but failed to increase the transcriptional activating ability of c-Jun mutants with Ser-63 and Ser-73 changed to nonphosphorylatable Ala, indicating that MT modulates c-Jun activity through phosphorylation. The dominant inhibitory mutants of Ha-ras and raf-1 interfered with the ability of MT to activate c-Jun. The results indicate that MT induces a phosphorylation cascade through the activation of c-Ras and Raf-1 and that c-Jun is one of the downstream targets that may cause changes in gene expression leading to cell transformation.