Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1 S138E ) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1 S138E expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1 S138E to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1 S138A is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.The iron regulatory proteins IRP1 and IRP2 are involved in the coordinate posttranscriptional regulation of cellular iron metabolism by binding to mRNA iron-responsive elements (IREs). These are hairpin structures within the 5Ј or 3Ј untranslated regions of a growing family of mRNAs that encode proteins of iron uptake, storage, utilization, and transport, as well as energy metabolism (8,18). Among the best-characterized IRE-containing mRNAs are those encoding the transferrin receptor, which plays a critical role in cellular iron uptake, and ferritin, a protein for intracellular iron storage. IRE-IRP interactions largely account for the reciprocal control of the transferrin receptor and ferritin expression in response to iron perturbations (25).IRP1 and IRP2 are homologous to mitochondrial aconitase (5,8,23), an enzyme of the citric acid cycle, and in fact, IRP1 is its cytosolic counterpart. The active site of aconitase contains a cubane [4Fe-4S] cluster (1). In IRP1, this cluster is assembled within iron-replete cells and prevents IRE binding. However, iron starvation, nitrogen monoxide (NO), and extracellular H 2 O 2 (5, 23) trigger the cluster's destabilization. The resulting switch to apo-IRP1 is associated with the loss of aconitase and acquisition of IRE-binding activity. The mechanism for IRP2 regulation is distinct and does not involve iron-sulfur cluster biochemistry. While IRP2 is stable in iron-starved and hypoxic cells, it undergoes proteasomal degradation in the presence of iron, oxygen (16), or the nitrosonium cation (NO ϩ ) (19). It has been proposed that the activities of IRP1 and IRP2 may also be regulated by phosphorylation (8). The physiological significance of this finding remains elusive. Both IRP1 and IRP2 can be subjected to phosphorylation by protein kinase C (PKC) (9, 27). The rate of IRP1 phosphorylation by PKC is negatively affected by the presence of the iron-sulfur cluster (26). Ex...

A computerized DA improves informed decision making in patients with early-stage PTC who are considering adjuvant RAI treatment. DAs are useful for patients facing decisions subject to medical evidence uncertainty.