ansduced with increasing amounts of adenovirus e pressing eIF5A1 or a mutant of eIF5A1 selleck that cannot be hypusinated, and analyzed by immunoblot for effects on MAPK SAPK signaling pathways. A dose dependent increase in e pression of eIF5A1 was observed after infection with increasing amounts of either Ad eIF5A1 or Ad Inhibitors,Modulators,Libraries eIF5A1K50A. To determine whether the high levels of eIF5A1 produced by adenovirus resulted in increased levels of hypusine modified eIF5A1, two dimensional gel electrophoresis of adenovirus infected A549 cells was performed. Hypusination ensues almost immediately following translation of eIF5A1 and, conse quently, the majority of eIF5A1 present in untreated healthy cells is hypusinated. Treatment with the DHS inhibitor GC7, which inhibits the first enzymatic step in the conversion of lysine to hypusine, results in ac cumulation of unhypusinated eIF5A1.
A549 cells infected with Ad eIF5A1 and Ad eIF5A1K50A both e hibited a substantial Inhibitors,Modulators,Libraries increase in the relative abundance of unhypusinated eIF5A1, suggesting that the accu mulation of newly translated eIF5A1 generated by adeno virus overwhelmed the catalytic functions of DHH and DOHH. Ad eIF5A1 and Ad eIF5A1K50A infection of A549 cells did not deplete hypusine eIF5A1 levels, indicating that the consequences of eIF5A1 and eIF5A1K50A over e pression are due to accu mulation of non modified eIF5A1 and not to depletion of hypusine eIF5A levels. EIF5A1 and eIF5A1K50A over e pression both resulted in dose dependent phosphorylation of ERK, p38 MAPK and JNK at sites associated with increased kinase activity.
Inhibitors,Modulators,Libraries A clear dose dependent increase in phos phorylation of p38 in response to Inhibitors,Modulators,Libraries increasing Ad eIF5A1 e pression was observed. Although e pres sion of phosphorylated ERK decreases at the highest Ad eIF5A1 e pression level, there is a trend towards in creased e pression of phosphorylated ERK with increasing viral dose. Phosphorylation of p90RSK, a kinase that is phosphorylated and activated by ERK, was also observed in response to Ad eIF5A1 and Ad eIF5A1K50A, indicating increased ERK activity. An increase in phosphorylated p38 and a decrease in phos phorylated JNK were observed when Ad eIF5A1K50A infected cells were treated with the MAPK kinase inhibitor U1026, indicating that ERK negatively and positively regulates p38 and JNK, respectively, in A549 cells.
Phosphorylation Brefeldin_A at serine 63 of the transcription factor c Jun, a component of the acti vating protein 1 transcriptional comple was ob served in response to Ad eIF5A1 infection, which is consistent with activation of SAPK JNK in response to eIF5A1. Ad eIF5A1 induces MEK dependent activation and phosphorylation of the p53 tumor suppressor protein A549 cells have been reported to have a functional p53 tumor suppressor protein. E pression of eIF5A1 has previously been correlated to p53 levels in lung cancer cells, and in this study a dose dependent increase in not p53 was observed in response to Ad eIF5A1 and Ad eIF5A1K50A infection in A549 cells. Phosphoryla