KKb and although it exhibits non speci ficity at higher concentrations, it does not affect the activity of LKB1, the further information other primary AMPKK. In the presence of STO 609, PAR2 induded AMPK phosphory lation was blocked. In fact, although STO 609 treatment did not significantly decrease baseline pAMPK levels, we observed a mild decrease in AMPK phosphorylation below baseline levels upon PAR2 stimulation. These data suggest that PAR2 is capable of inhibiting as well as promoting AMPK phosphorylation, an obser vation that is consistent with previous studies in which we demonstrated that a number of Gaq Ca2 dependent signaling pathways are opposed by b arrestins and vice versa. We conclude that PAR2 stimulated AMPK activation requires the activity of CAMKKb and may be opposed by a separate Inhibitors,Modulators,Libraries PAR2 stimulated pathway.

We address whether this inhibitory pathway is mediated by b arrestins, similar to what has been observed for other proteins in the next section. The other kinase capable of activating AMPK is LKB 1, a tumor suppressor, which is activated by STRAD and Inhibitors,Modulators,Libraries STE 20 related kinases and which potentiates Inhibitors,Modulators,Libraries the effect of AMP on AMPK activity. Transfection of siRNA to LKB 1 reduced LKB 1 protein by 70%, and resulted in a 50% decrease in PAR2 stimulated AMPK phosphorylation. We next measured AMP and ATP levels in cells treated with or without 2fAP for 0 120 minutes by liquid chromatography tandem mass spectrometry. PAR2 increased AMP ATP ratios at 120 minutes and to a lesser extent at 5 minutes. We conclude that LKB 1 also contributes to AMPK phosphorylation downstream of PAR2, which may involve increased AMP ATP ratios observed in response to PAR2 activation.

Because CAMKKb signaling downstream PAR2 is better understood, and the effect of CAMKKb inhibition on Inhibitors,Modulators,Libraries PAR2 stimulated AMPK phos phorylation was more pronounced than that of LKB1, the remainder of these studies will focus on the CAMKKb arm of this signaling pathway. b arrestin 2 inhibits PAR2 stimulated AMPK activation In light of studies suggesting that PAR2 induced, Ca2 dependent activation of other enzymes is inhibited by b arrestins, we hypothesized that b arrestins might be capable of inhibiting the PAR2 stimulated increase in AMPK phosphorylation. We examined AMPK phos phorylation in mouse embryonic fibroblasts from wild type mice, b arrestin double knockout mice, or from MEFbarrDKO transfected with either b arrestin 1 or b arrestin 2.

These transfected MEFs have been previously characterized and found to express levels of either b arrestin 1 or 2 similar to those expressed Brefeldin_A in the wild type cells, and avoid the possible complications of com pensatory mechanisms that may be present selleck products in either b arrestin 1 or b arrestin 2 knockout mice. In wtMEF, no significant increase in AMPK phosphoryla tion was observed upon PAR2 activation, consistent with the higher levels of b arrestins present in MEFs com pared with NIH3T3 cells. However, in MEF barrDKO, and in MEFDKO barr1, PAR2 promoted a 2 2. 5 fold increase