Ligand-dependent ubiquitination of Smad3 is regulated by casein kinase 1 gamma 2, an inhibitor of TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) elicits a variety of cellular activities primarily through a signaling cascade mediated by two key transcription factors, Smad2 and Smad3. Numerous regulatory mechanisms exist to control the activity of Smad3, thereby modulating the strength and specificity of TGF-beta responses. In search for potential regulators of Smad3 through a yeast two-hybrid screen, we identified casein kinase 1 gamma 2 (CKIgamma2) as a novel Smad3-interacting protein. In mammalian cells, CKIgamma2 selectively and constitutively binds Smad3 but not Smad1, -2 or -4. Functionally, CKIgamma2 inhibits Smad3-mediated TGF-beta responses including induction of target genes and cell growth arrest, and this inhibition is dependent on CKIgamma2 kinase activity. Mechanistically, CKIgamma2 does not affect the basal levels of Smad proteins or activity of the receptors. Rather, CKIgamma2 preferentially promotes the ubiquitination and degradation of activated Smad3 through direct phosphorylation of its MH2 domain at Ser418. Importantly, mutation of Ser418 to alanine or aspartic acid causes an increase or decrease of Smad3 activity, respectively, in the presence of TGF-beta. CKIgamma2 is the first kinase known to mark activated Smad3 for destruction. Given its negative function in TGF-beta signaling and its reported overexpression in human cancers, CKIgamma2 may act as an oncoprotein during tumorigenesis.

Effects of ionization mode on charge-site-remote and related fragmentation reactions of long-chain quaternary ammonium ions.

Comparison of collisionally activated fragment spectra of long-chain quaternary ammonium ions, formed by liquid-assisted secondary ion mass spectrometry (LSIMS) and electrospray ionization (ESI), shows the latter are dominated by radical cations while the former yield mainly even-electron charge-site-remote (CSR) fragments, similar to the report for different precursors by Cheng et al., J. Am. Soc. Mass Spectrom. 1998, 9, 840. Here, mixed-site fragmentation products (formal loss of a radical directly bonded to the nitrogen plus a radical derived from the long chain) are of comparable importance for both ionization techniques. These observations are difficult to understand if the CSR ions are formed by a concerted rearrangement-elimination reaction, since precollision internal energies of the ESI ions are much lower than those of the ions from LSIMS. Alternatively, if one discards the concerted mechanism for high-energy CA, and assumes that the even-electron fragments are predominantly formed via homolytic bond cleavage, the colder radical cations from ESI survive to the detector while the more energized counterparts from LSIMS preferentially lose a hydrogen atom to yield the CSR ions, as proposed by Wysocki and Ross (Int. J. Mass Spectrom. Ion Processes 1991, 104, 179). The present work also attempts to reconcile discrepancies involving critical energies and known structures for neutral fragments.

Low levels of cadmium chloride alter the immunoprecipitation of corneal cadherin-complex proteins.

The effect of cadmium chloride on the immunoprecipitation of cadherin and the associated adherens junctional proteins, alpha- and beta-catenin, was examined in isolated bullfrog (Rana catesbeiana) corneas utilizing Western blot and enhanced chemoluminescent techniques. Application of either 1.0 microM or 75.0 microM CdCl2 to the corneal endothelium for 2 h markedly decreased the immunoprecipitation of cadherins as compared to paired control corneas. Immunoprecipitation of alpha-catenin was increased in response to both doses of CdCl2, while the immunoprecipitation of beta-catenin was little changed by either cadmium dose. There is accumulating evidence that cadmium may increase epithelial paracellular permeability by interfering with cadherin complex activity at intercellular junctions. The present study suggests that inorganic cadmium in low micromolar concentrations may decrease the integrity of the corneal endothelium, at least in part through a similar mechanism involving disruption of junctional cadherin complex function.

Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein.

Saquinavir, a peptidomimetic HIV protease inhibitor, has been shown to be effective in reducing patient viral load and reducing mortality. In this report we investigated whether saquinavir is a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), which may reduce the effective intracellular concentration of the drug. G185 cells, which highly express P-gp, are resistant to saquinavir-mediated cytotoxicity, and co-administration of cyclosporine reversed this resistance. Saquinavir and saquinavir mesylate inhibited basolateral to apical transport of the fluorescent dye rhodamine 123 in a polarized epithelial transport assay, a result that suggests competition of these drugs for the P-gp transporter. Finally, we measured specific, directional transport of saquinavir and saquinavir mesylate in an epithelial monolayer model. Transport in the basolateral to apical direction was 3-fold greater than apical to basolateral flux for both saquinavir and saquinavir mesylate and was blocked by co-incubation with the established P-gp reversal agents cyclosporine and verapamil. These data provide evidence that saquinavir is a substrate for the P-gp transporter and suggest that this protein may affect intracellular accumulation of the drug and contribute to its poor oral bioavailability.