Abstract-Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, I Ks . KCNQ1 and KCNE1 subunits coassemble to form the I Ks channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death.Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum-and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P 2 . Identification of KCNQ1/KCNE1 recycling and its modulation by serum-and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P 2 provides a mechanistic insight into stress-induced acceleration of cardiac repolarization. (Circ Res. 2007;100:686-692.)Key Words: kinase Ⅲ PIP2 Ⅲ RAB Ⅲ trafficking Ⅲ PIKfyve E motional stress activates the sympathetic nervous system 1 and the release of stress hormones such as cortisol via the hypothalamic-pituitary-adrenal (HPA) axis 2 and is a common trigger of sudden cardiac death. 3,4 One of the many genes regulated by cortisol is the serum-and glucocorticoidinducible kinase 1 (SGK1). 5,6 In vitro experiments have shown that SGK1 stimulates I Ks 7 , a repolarizing potassium current conducted by channels composed of KCNQ1 ␣-subunits and KCNE1 ␤-subunits. 8,9 Moreover, a gain-offunction variant of SGK1 is associated with shortening of the QT interval. 10 SGK1-mediated regulation of I Ks might be particularly important in patients with KCNQ1 (Kv7.1, Kv-LQT1) or KCNE1 (minK) mutations that are prone to fatal cardiac arrhythmias triggered by physical and psychological stress. 4 The mechanism responsible for regulation of I Ks channels by SGK1 have remained elusive. SGK1 enhances the abundance of other types of channel protein in the plasma membrane by inhibiting the ubiquitin ligase Nedd4 -2 11 in addition to other mechanisms (summarized by Lang et al 2006 12 ).Other candidate signaling molecules that may affect channel trafficking include RAB family proteins, GTPases involved in vesicle cycling. [13][14][15][16][17][18] RAB5, a monomeric GTPase of the Ras superfamily, has been implicated in the regulation of early steps in the endocytic pathway, whereas the RAB11 GTPase is localized at the trans-Golgi network, post-Golgi vesicles and the recycling endosome. 19 Both RAB5 and RAB11 are expressed in cardiac tissue. 17 Mammalian cells and Xenopus laevis oocytes have been shown to possess and use highly conserved RAB-dependent trafficking pathways. 20,21 Endocytosis by RAB5 and exocytosis by RAB11 have been reported to participate in the regulation of CFTR chloride channels 22 and the glucose transporter GluT4. 15...

Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing PtdIns(3)P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the PtdIns(3)P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised PtdIns(3,4,5)P3 signals via a protein kinase cascade to regulated PtdIns(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

The activation of protein kinase B (or Akt) plays a central role in the stimulation of glucose uptake by insulin. Currently, however, numerous questions remain unanswered regarding the role of this kinase in bringing about this effect. For example, we do not know precisely where in the GLUT4 trafficking pathway this kinase acts. Nor do we know which protein substrates are responsible for mediating the effects of protein kinase B, although two recently identified proteins (AS160 and PIKfyve) may play a role. This paper addresses these important questions by reviewing recent progress in the field.

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The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.