Cyclic AMP responsive element (CRE)‐binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133‐independent activation. One study identified a kinase, salt‐inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB‐specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1‐defective HeLa cells to further elucidate TORC‐dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE‐dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 n m staurosporine in LKB1‐positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1‐phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1‐defective cells or in staurosporine‐treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.
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Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein.Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry.Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking.Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.
Abstract Single-walled carbon nanotubes (SWNTs) were well dispersed in both water and organic solvent by the use of fullerodendron as a dispersant. A C60 moiety at the focal point of dendron plays a crucial role in the dispersing process, because dendron having an anthracene unit at the focal point can not disperse SWNTs in THF. The dispersions of SWNTs were characterized by UV–vis–NIR spectroscopy, Raman spectroscopy, SEM, HRTEM, and AFM.
