RhoB facilitates c-Myc turnover by supporting efficient nuclear accumulation of GSK-3.

The small GTPase RhoB suppresses cancer in part by limiting cell proliferation. However, the mechanisms it uses to achieve this are poorly understood. Recent studies link RhoB to trafficking of Akt, which through its regulation of glycogen synthase kinase-3 (GSK-3) has an important role in controlling the stability of the c-Myc oncoprotein. c-Myc stabilization may be a root feature of human tumorigenesis as it phenocopies an essential contribution of SV40 small T antigen in human cell transformation. In this study we show that RhoB directs efficient turnover of c-Myc in established or transformed mouse fibroblasts and that the attenuation of RhoB which occurs commonly in human cancer is a sufficient cause to elevate c-Myc levels. Increased levels of c-Myc elicited by RhoB deletion increased the proliferation of nullizygous cells, whereas restoring RhoB in null cells decreased the stability of c-Myc and restrained cell proliferation. Mechanistic analyses indicated that RhoB facilitated nuclear accumulation of GSK-3 and GSK-3-mediated phosphorylation of c-Myc T58, the critical site for ubiquitination and degradation of c-Myc. RhoB deletion restricted nuclear localization of GSK-3, reduced T58 phosphorylation, and stabilized c-Myc. These effects were not associated with changes in phosphorylation or localization of Akt, however, differences were observed in phosphorylation and localization of the GSK-3 regulatory Akt-related kinase, serum- and glucocorticoid-inducible protein kinase (SGK). The ability of RhoB to support GSK-3-dependent turnover of c-Myc offers a mechanism by which RhoB acts to limit the proliferation of neoplastically transformed cells.

Guanylyl cyclase stimulatory coupling to K(Ca) channels.

We coexpressed the human large-conductance, calcium-activated K (K(Ca)) channel (alpha- and beta-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell K(Ca) currents by 21 +/- 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased K(Ca) currents (20 +/- 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of K(Ca) currents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the alpha-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920-14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate K(Ca) channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling.

Molecular cloning and expression characterization of a rice K+ channel beta subunit.

K+ channel proteins native to animal membranes have been shown to be composed of two different types of polypeptides: the pore-forming alpha subunit and the beta subunit which may be involved in either modulation of conductance through the channel, or stabilization and surface expression of the channel complex. Several cDNAs encoding animal K+ channel beta subunits have been recently cloned and sequenced. We report the molecular cloning of a rice plant homolog of these animal beta subunits. The rice cDNA (KOB1) described in this report encodes a 36 kDa polypeptide which shares 45% sequence identity with these animal K+ channel beta subunits. and 72% identity with the only other cloned plant (Arabidopsis thaliana) K+ channel beta subunit (KAB1). The KOB1 translation product was demonstrated to form a tight physical association with a plant K+ channel alpha subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a K+ channel beta subunit. Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein with the flag leaf having the highest titer of KOB1. Leaf sheaths are known to accumulate excess K+ and act as reserve sources of this cation when new growth requires remobilization of K+. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when K+ was lost from older leaves of plants grown on K+-deficient fertilizer, the loss of cellular K+ was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration (in either plants or animals) that changes in cellular K+ status may specifically alter expression of a gene encoding a K+ channel subunit.