Nonetheless, SK T phosphorylation was restored upon re expression of either WT or PDK LG . Additionally, the cell size defect noticed in PDK relative to PDK ES cells was also partially reversed upon expression of either PDK allele . We then tested the PP analogues shown in Kinase , as well as additional ones shown in Kinase A for their ability to inhibit PDK signaling in the WT and LG reconstituted ES cells. Two compounds DMB PP and NM PP, emerged as being pretty potent and selective for PDK LG more than PDK WT ES cells. A one particular hour incubation with these compounds inhibited IGF stimulated phosphorylation of PKB T in PDK LG ES cells. Phosphorylation of PKB Akt targets GSK S S, and PRAS T was equally inhibited . These compounds had minimal effects on any of these phosphorylation web pages in PDK WT ES cells at concentrations helpful in PDK LG ES cells. In contrast to , DMB PP and NM PP, many of the other PP analogues that we tested did show some degree of PDK inhibition in PDK WT ES cells in addition to PDK LG ES cells.
Additionally, we noticed that SK T and S S S phosphorylation had been sensitive to phenylalanine hydroxylase inhibitor many of those PP analogues, even in WT PDK ES cells . We also analyzed E BP phosphorylation in WT PDK ES cells in response to these inhibitors. E BP phosphorylation was rarely affected in either cell line , suggesting that mTORC is very likely not the target and that SK itself may be particularly susceptible to this class of PP analogues. Kinase C summarizes the in cell IC values for all compounds and phosphorylation sites tested, and Supplemental Kinase shows representative Western blots from which these data had been calculated. Before examining any potential biological consequences of PDK inhibition, we tested irrespective of whether these compounds had been able to durably inhibit PDK activity.
p38 MAPK Inhibitors Supplemental Kinase shows that at h following administration PDK downstream signaling remained inhibited, as measured by PKB Akt T, GSK S S, and S S S phosphorylation. Interestingly, BX essentially reproducibly triggered increased T phosphorylation at later time points. The explanation for this is not clear but could represent effects of more targets of BX . Phosphorylation of identified and possible PDK targets following long term inhibition of PDK Next, we analyzed the phosphorylation state of additional recognized and potential PDK targets inside the AGC kinase family. Confirming earlier reports, many AGC kinases showed defects in activation loop phosphorylation in PDK ES cells, which includes pRSK, PRK , and a few isoforms of PKC relative to PDK LG ES cells .
Phosphorylation of PKA T relative to total PKA was also slightly decreased in PDK ES cells to PDK LG ES cells. Total levels of many different PKC isoforms were also improved following expression of PDK LG, constant with preceding reports . We then analyzed phosphorylation of PDK substrates following incubation with the PP analogues NM PP and , DMB PP in PDK LG cells.