The mechanisms involved in the regulation of the epithelial sodium channel (ENaC) via the cAMP pathway are not yet completely understood. The aim of the present study was to investigate cAMP-mediated ENaC regulation in Xenopus laevis oocytes heterologously expressing the three subunits (␣␤␥) of rat ENaC and to determine the ENaC regions important for mediating the stimulatory effect of cAMP. In oocytes treated for about 24 h with 1 mM 3-isobutyl-1-methylxanthine (IBMX) and 1 M forskolin (FSK) so as to increase intracellular cAMP, the amiloride-sensitive whole cell current (⌬I Ami ) was on average 10-fold larger than ⌬I Ami in matched control oocytes. This effect on ⌬I Ami was paralleled by an increase in ENaC surface expression caused by a reduced rate of ENaC retrieval. In addition, IBMX/FSK also enhanced ENaC open probability from about 0.2 to 0.5. The stimulatory effect of IBMX/FSK was dependent on the presence of intact PY motifs in the C termini of the channel. Mutagenesis of putative protein kinase A and CK-2 consensus motifs in the cytosolic domains of the channel did not reveal critical sites involved in mediating the stimulatory effect of IBMX/FSK. In contrast, site-directed mutagenesis of two putative ERK-consensus motifs (T613A in ␤ENaC and T623A in ␥ENaC) largely reduced the stimulatory effect of IBMX/FSK. Phosphorylation of these ERK sites has previously been reported to enhance the interaction of ENaC and Nedd4 (Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R., and Garty, H. (2002) J. Biol. Chem. 277, 13539 -13547). Using co-expression experiments we demonstrated that mutating the two ERK sites attenuates the inhibitory effect of Nedd4-2 on ENaC currents. We conclude that an increase in intracellular cAMP favors the dephosphorylation of the two ERK sites, which reduces channel retrieval and increases P O by modulating ENaC/Nedd4 interaction. This defines a novel regulatory pathway likely to be relevant for cAMP-induced stimulation of ENaC in vivo.The apically localized amiloride-sensitive epithelial sodium channel (ENaC) 2 plays a critical role in fluid and electrolyte homeostasis and is widely expressed in absorptive epithelia such as the aldosterone-sensitive distal nephron, the distal colon, respiratory epithelia, sweat and salivary ducts (1, 2). ENaC is composed of three homologous subunits (␣, ␤, and ␥) that contain two transmembrane domains, a large extracellular loop, and short intracellular amino and carboxyl termini. The functional importance of a highly conserved PPXY sequence (the PY motif) in the carboxyl terminus of each subunit was recognized in Liddle syndrome (3). The PY motifs mediate ENaC binding to WW domains of the ubiquitin ligase Nedd4/Nedd4-2. This binding of Nedd4/Nedd4-2 to ENaC leads to ubiquitination of lysine residues in the N terminus of the channel and to channel internalization and proteasomal degradation (4, 5). In Liddle syndrome, mutations and/or deletions of the PY motif in ␤ or ␥ ENaC reduce the Nedd4/Nedd4-2-mediated endocytic retrieval of ENaC ...

No abstract

Treatment of M1 myeloid leukemia cells with leukemia inhibitory factor (LIF) causes activation of transcription factors Stat1, Stat3 and Stat5a (signal transducers and activators of transcription). DNA-binding of Stat proteins was detectable for extended periods of time in LIF-treated M1 cells, which simultaneously underwent terminal differentiation. The relative composition of Stat factors in the protein-DNA complexes changed during time. Whereas Stat3 was activated up to 36 h during treatment with LIF, Stat5a was activated only short-termed. Similarly, high expression of the immediate early gene CIS (cytokine-inducible SH2-containing protein), a known target gene of Stat5 in hematopoietic cells, occurred only during the onset of differentiation. This suggests a role of Stat5a in the early phase of LIF-induced differentiation and growth arrest of M1 cells.

No abstract