[PubMed] [Google Scholar] 35. transcription. The adenovirus (Ad) E1B 19,000-molecular-weight (19K) protein functions to protect cells from apoptosis during an Ad lytic infection (8, 47). This antiapoptotic activity of the E1B 19K protein is also essential for its cooperation with Ad E1A proteins to induce full oncogenic transformation of primary rodent cells (8, 47). The E1B 19K oncoprotein is functionally homologous to the mammalian Bcl-2, both of which can act to suppress apoptosis (7, 47, 64, 66). Both Bcl-2 and the E1B 19K protein have been shown to interact with a common set of cellular proteins, including the apoptotic inducer Bax (21), Bak (15), Bik/Nbk (4, 22), and Nip 1, 2, and 3 (5), and these interactions are believed to be important for the biological functions of both proteins. Another important activity of the E1B 19K protein is its ability to regulate transcription. The E1B 19K protein can specifically block tumor suppressor p53-mediated transcriptional repression, Chlorprothixene and this ability of E1B 19K in part underlies its ability to inhibit p53-induced apoptosis (49, 55). E1B 19K can also activate enhancer-dependent transcription (74). In contrast, E1A represses viral and cellular enhancers, and this inhibitory effect is achieved at least in part by targeting the transcriptional cofactor p300 (14). The transcriptional antagonism between E1A and E1B 19K may be important for the maintenance of a balanced transcriptional program that is critical Chlorprothixene for a successful adenoviral infection and for the E1A- and E1B-induced oncogenic transformation. Although it has been known for some time that E1B 19K can function as a transcriptional regulator, the mechanisms by which it regulates transcription are largely unknown. Chlorprothixene In this report, we show that E1B 19K can strongly activate Chlorprothixene transcription mediated by c-Jun, and we investigate the mechanism by which E1B 19K induces c-Jun-dependent transcription. c-Jun belongs to a family of related proteins that includes JunB and JunD. These proteins can heterodimerize with the Fos family of transcription factors to form what is known as the AP-1 transcription factor (1). Previous work has shown that c-Jun-dependent transcription is stimulated by c-Jun N-terminal kinase (JNK), which is also known as stress-activated protein kinase (SAPK) (12, 33). JNK was first characterized as a mitogen-activated protein kinase (MAPK) family member that binds c-Jun and phosphorylates serines 63 and 73 located within the transactivation domain of c-Jun (12, 33). Phosphorylation of these serine sites results in increased transactivation potential of c-Jun (12, 24). The substrates of JNK have now been extended to include other transcription factors, such as ATF-2 (19) and Elk-1 (6). The Rabbit Polyclonal to XRCC5 activity of JNK is regulated by dual phosphorylation on specific threonine and tyrosine residues by MKK4 (otherwise known as SEK1) (13, 40). MKK4, in turn, is activated by the upstream protein kinase MEKK1 (13, 35, 70). Growth factors such as epidermal growth factor (EGF) and activated Ras that lie further upstream of this signaling pathway appear to stimulate MEKK1 and subsequently downstream JNK through GTP-binding proteins such as Cdc42 and Rac1 (10, 43) and Rho (61). In Chlorprothixene this study, we report that Ad infection of human cells results in the activation of JNK during the later stages of infection. Analysis of Ad early-region mutants suggests that E1B proteins are required for JNK activation. Subsequent transfection assays demonstrate that E1B 19K is sufficient to induce JNK activation. This JNK activation is accompanied by a strong induction of c-Jun-dependent transcription. Interestingly, full induction of c-Jun-dependent transcription by E1B 19K is only partially dependent on c-Jun phosphorylation (Ser63 and Ser73) by the activated JNK. We show that the transcriptional cofactor p300 can synergize with E1B 19K to activate c-Jun-mediated transcription, suggesting that p300 may play a role in the full induction of c-Jun-mediated transcription by E1B. To investigate the biochemical mechanisms by which E1B 19K activates c-Jun-dependent transcription via activated JNK, we analyzed the effect of known upstream JNK-activating kinases. Our results show that activation of c-Jun-dependent transcription by E1B 19K involves MEKK1 and MKK4, two JNK upstream activating kinases. However, Ras is not involved, suggesting that E1B 19K activates JNK in a manner that is different from that of growth factors. The kinase dependence of E1B 19K in activating c-Jun-dependent transcription is in contrast to.