When cell viability was quantified at day 44 for BRAFi treated cells, over 90%

It may also be noted from Figure 5B and Figure 5C that ERK2 binds in a different mode abt263 supplier to PAK1 in the pres ence of TQ. ERK2 binding to a different conformation of PAK1 may prevent ERK2 from phosphorylating the Thr212 of PAK1. This could explain why pPAK1Thr212 levels decrease over time in response to TQ. Furthermore, TQ induced pERK12 levels were not re duced by IPA 3 treatment, instead ERK12 activity was further enhanced from 1 h to 6 h when TQ was combined with IPA 3, suggesting the involvement of other upstream kinases in phosphorylating ERK12. If the TQ triggered closer binding between ERK12 and PAK1 is disrupted under IPA 3 this might explain the slight in crease in pPAK1Thr212 levels when TQ is combined with IPA 3. From these findings we propose a functional relation between both phosphorylation sites of PAK1.<br><br> Indeed, T212A mutant induced a significant increase in pPAK1Thr423 levels whereas the hyperphosphorylated オーダー Adriamycin T212E mutant showed a lesser extent of increase. As presented in Additional file 5Table S2, TQ binds in the vicinity of Thr212 and no binding occurs next to the 423 site. Therefore we speculate that the chain reaction orchestrated by TQ starts at Thr212 site, which correlates with the western blot pattern. Fur thermore, T423E resulted in a decrease in ERK12 phos phorylation suggesting an impaired interference of Thr423 residue with the kinase domain of PAK1 under TQ treatment. We analysed the structural changes induced by TQ on PAK1 catalytic site and activation loop. Figure 4G and Figure 4H depict the structural conformation of the catalytic domain of PAK1 in the absence or presence of TQ.<br><br> The kinase domain shows a root mean square deviation of 0. 38accompanied by re arrangements in the main activation loop. Hydrogen bond analysis showed that a higher number of hydrogen bonds are formed in purchase ABT-199 the catalytic loop region in the presence of TQ involving residues Arg388 and Arg421 which are known to interact with Thr423 for the catalytic activity. Finally this results in a disturbed inter action between the Thr423 site and the catalytic kinase do main which inhibits the PAK1 kinase activity and its prosurvival signaling. There are three other lines of evidence for this hypothesisfirst, early TQ induced ERK12 activation is inhibited when PAK1Thr423 is maximally phosphorylated at 24 h.<br><br> Second, when IPA 3 is combined with TQ there is a decrease in Thr423 phos phorylation at early time points accompanied by a signifi cant upregulation of pERK12 levels that confirms an early activation of MEK ERK signaling. The lack of inhib ition of pPAK1Thr423 at 24 h is closely associated with a decrease in prosurvival ERK12 activation and enhanced apoptosis induction at 24 h. This can be explained by an other structural modeling showing that TQ has the ability to effectively bind to the autoregulatory domain of PAK1 preventing IPA 3 ability to interrupt the interaction be tween Cdc42 and pPAK1Thr423. Third, the pPAK1Thr212 upregulation is not as dramatic as the pERK12 activation after combined TQ and IPA 3 treatment reinforcing the inhibitory loop for PAK1 activation. Although pERK12 should disappear completely by the higher level of pPAK1Thr423 at 24 h, it is worth mentioning that other secondary kinase loops could be at play.