Tuberin phosphorylation regulates its interaction with hamartin. Two proteins involved in tuberous sclerosis.

Hamartin and tuberin are products of the tumor suppressor genes, TSC1 and TSC2, respectively. When mutated, a characteristic spectrum of tumor-like growths develop resulting in the syndrome of tuberous sclerosis complex. The phenotypes associated with TSC1 and TSC2 mutations are largely indistinguishable suggesting a common biochemical pathway. Indeed, hamartin and tuberin have been shown to interact stably in vitro and in vivo. Factors that regulate their interaction are likely critical to the understanding of disease pathogenesis. In this study, we showed that tuberin is phosphorylated at serine and tyrosine residues in response to serum and other factors, and it undergoes serial phosphorylation that can be detected by differences in electrophoretic mobilities. A disease-related TSC2 mutation (Y1571H) nearly abolished tuberin phosphorylation when stimulated with pervanadate. Expression of this mutant tuberin caused a marked reduction in TSC1-TSC2 interaction compared with wild-type protein and significantly curtailed the growth inhibitory effects of tuberin when overexpressed in COS1 cells, consistent with a loss of function mutation. Examination of a second pathologic mutation, P1675L, revealed a similar relationship between limited phosphorylation and reduced interaction with hamartin. Our data show for the first time that 1) tuberin is phosphorylated at tyrosine and serine residues, 2) TSC1-TSC2 interaction is regulated by tuberin phosphorylation, and 3) defective phosphorylation of tuberin is associated with loss of its tumor suppressor activity. These findings suggest that phosphorylation may be a key regulatory mechanism controlling TSC1-TSC2 function.

Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.

In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.

Quantitative determination of porphyrins in rat and human urine and evaluation of urinary porphyrin profiles during mercury and lead exposures.

Measurement of urinary porphyrin excretion patterns (porphyrin profiles) is useful in the diagnosis and evaluation of diseases and disorders of porphyrin metabolism. However, experimental investigation of such disorders with rodent models has been hampered by the lack of an efficient procedure for the isolation and quantitative evaluation of porphyrins in rodent urine. This article describes an analytic procedure that overcomes the principal difficulties encountered with determination of porphyrins in rodent urine, including the loss of porphyrins during their isolation and interference of porphyrin fluorescence by contaminating materials. The procedure entails application of an acidified urine sample to a preconditioned C-18 preparatory column, preferential separation of essentially all potentially interfering contaminants by sequential phosphate-methanol elution, and selective isolation of porphyrins, which are then separated and quantitated by high-performance liquid chromatography and spectrofluorometric techniques. This method has been used to characterize urinary porphyrin excretion patterns in male rats and to define the distinctive changes in porphyrin profiles associated with prolonged exposure to porphyrinogenic metals. The porphyrin excretion patterns of male and female human subjects are also described. This method is applicable to the investigation of urinary porphyrin profile changes associated with exposure to a wide range of porphyrinogenic chemicals in both animals and human subjects.

Stimulation of porphyrinogen oxidation by mercuric ion. II. Promotion of oxidation from the interaction of mercuric ion, glutathione, and mitochondria-generated hydrogen peroxide.

Previous studies have shown that mercuric ion (Hg2+) reacts with GSH and H2O2 in vitro to form reactive species capable of oxidizing reduced porphyrins (porphyrinogens). This effect is independent of the presence of iron in the reaction mixture. The present studies demonstrate that Hg2+ and GSH can interact in biologically relevant concentrations with H2O2 generated by the mitochondrial electron transport chain to promote oxidation of porphyrinogens via comparable mechanisms. Mitochondria from rat liver or kidney readily oxidize uroporphyrinogen when H2O2 production is stimulated by the presence of a respiratory chain substrate (NADH, succinate) and an electron transport inhibitor (e.g., NaN3). Porphyrinogen oxidation by mitochondria is significantly increased by the addition of Hg2+ and GSH, in a molar ratio of approximately 3:5, to the reaction mixture. Stimulation of porphyrinogen oxidation in the presence of Hg2+ plus GSH increases proportionately with the concentration of mitochondrial protein in the reaction cuvettes but decreases with diminished H2O2 production by the electron transport chain. Studies with reactive oxidant scavengers suggest the participation of reactive oxygen species in Hg plus GSH stimulation of mitochondrial porphyrinogen oxidation. These findings support the hypothesis that Hg2+ and GSH interact with mitochondria-generated H2O2 to promote propagation of reactive oxidants or other free radical species, which, in turn, oxidize reduced porphyrins proximal to mitochondrial membranes. These results suggest a mechanistic explanation for the porphyrinogenic action of mercury compounds, as well as for the oxidative damage to target cell constituents associated with mercury exposure.

Stimulation of porphyrinogen oxidation by mercuric ion. I. Evidence of free radical formation in the presence of thiols and hydrogen peroxide.

The etiology of mercury-induced porphyrinuria was investigated by testing the hypothesis that mercuric ions (Hg2+) promote free radical-mediated oxidation of reduced porphyrins (porphyrinogens) by compromising the antioxidant potential of endogenous thiols, particularly GSH. Studies in vitro demonstrated that porphyrinogens (uroporphyrinogen and coproporphyrinogen) readily undergo H2O2-dependent oxidization in the presence of Fe3(+)-EDTA and that this action is attenuated by GSH at biologically relevant concentrations (0.5-10 mM). At low concentrations, Hg2+ complexes with GSH in a 1:2 molar ratio to decrease the antioxidant effect of GSH. However, at Hg2+ concentrations approaching saturation-complexation with available GSH, stimulation of porphyrinogen oxidation to 2 to 3 times that mediated by the H2O2/Fe3(+)-dependent system alone is observed. Stimulation of porphyrinogen oxidation by Hg2+ plus GSH increases in a dose-related manner with the concentration of H2O2 in the reaction mixture but is independent of the presence of iron. No porphyrinogen oxidation is observed in reaction mixtures containing H2O2 and either Hg2+ or GSH alone or when Hg+ is substituted for Hg2+. Studies with reactive oxidant scavengers and ESR spectroscopy suggest the participation of free radical species in Hg:GSH-mediated porphyrinogen oxidation. A mechanism involving ligand exchange between Hg2+ and GSH, which leads to formation of GS radicals and subsequent propagation of reactive oxygen-based radical species, is proposed. These studies support the view that Hg2+ both compromises the antioxidant potential of GSH and promotes formation of reactive species via thiol complexation. These findings suggest a mechanistic basis underlying the porphyrinogenic as well as tissue-damaging properties of mercuric ions.