We noted that the level of phosphorylated ERK12 increased at 8 hpi, an observation not reported earlier. This is unlikely to be related to any infec tion event selleck chem because phosphorylated ERK12 was similarly elevated Inhibitors,Modulators,Libraries at this time point in the mock infected sample. Our search for additional HAstV1 infection related signaling pathways uncovered evidence for the import ance of PI3K activation. The PI3K inhibitor LY294002 effectively blocked post infection viral capsid expression, whereas the other PI3K inhibitor, wortmannin, was slightly less effective, evidenced by the unusual punctate signal of capsid protein. A possible explanation is that although more potent than LY294002 in inhibiting PI3K activation, wortmannin is only stable for a few minutes in the cellular environment, making the PI3K inhibiting effect of LY294002 more apparent in a treat ment that lasted 24 h.
One possibility consistent with the observed effect of PI3K inhibitors on HAstV1 infection is that they may have led to the inhibition of ERK phosphorylation. PI3K and MAP kinase pathways are known to crosstalk through small GTPases such Inhibitors,Modulators,Libraries as Ras and Raf1. To evaluate this possibility, the phosphorylation Inhibitors,Modulators,Libraries level of ERK in the presence or the absence of a PI3K blocker was analyzed by Western blotting. We found that, unlike U0126, which abolished post infection ERK phosphoryl Inhibitors,Modulators,Libraries ation, LY294002 did not affect their phosphorylation. Thus, the PI3K inhibitor did not exert its effect through an interference with ERK activation, but acted on a distinct, essential process in HAstV1 infection.
We then asked whether known downstream targets of PI3K signaling, such as Akt, play a role in HAstV1 infection. Consistent with PI3K activation in the viral infection and with Akt being a target of activated PI3K, the extent of Akt phosphorylation was greater in the 0. 25 h and 0. 5 h post infection samples than in the corresponding mock infected control. However, treatment Inhibitors,Modulators,Libraries with 10 uM triciribine or with 10 uM MK2206, both of which are known to inhibit Akt activation as well as Akt mediated phosphorylation, had marginal effects on viral capsid expression. Examin ation of the phosphorylation level of Akt in the HAstV1 infected cells incubated with LY294002, wortmannin, triciribine, or MK2206 for 24 h showed that all but triciribine treatment effectively blocked the phosphoryl ation of Akt.
In addition to the Akt mediated cascade, Rac1 is also known to be targeted by PI3K activation. Blocking Rac1 with 50 uM NSC23766, an inhibitor of Rac1 specific GEF, did not interfere with the infection. We also tested for the involvement of other signaling cascades. H89 blocks the activity of protein kinase A by competing for the AZD2281 ATP binding site of PKAs catalytic subunit. Y27632 inhibits Rho associating pro tein kinase.