The transcription factor NF-B is retained in the cytoplasm by its interaction with the inhibitory subunit known as IB. Signal-induced serine phosphorylation and subsequent ubiquitination of IB␣ target it for degradation by the 26 S proteasome. Recently, pervanadate, a protein-tyrosine phosphatase inhibitor, was shown to block the degradation of IB␣, thus inhibiting NF-B activation. We investigated the mechanism by which pervanadate inhibits the degradation of IB␣. Western blot analysis of IB␣

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... om tumor necrosis factor-treated cells revealed a slower migrating IB␣ species that was subsequently degraded. However, pervanadate-treated cells also revealed a slower migrating species of IB␣ that appeared in a time-and dose-dependent manner and was not degraded by tumor necrosis factor. The slower migrating species of IB␣ from pervanadatetreated cells was tyrosine-phosphorylated as revealed by cross-reactivity with anti-phosphotyrosine antibodies, by the ability of the specific tyrosine phosphatase PTP1B to dephosphorylate it, and by phosphoamino acid analysis of IB␣ immunoprecipitated from 32 P-labeled cells. By site-specific mutagenesis and deletion analysis, we identified Tyr-42 on IB␣ as the phosphoacceptor site. Furthermore, in an in vitro reconstitution system, tyrosine-phosphorylated IB␣ was protected from degradation. Our results demonstrate that inducible phosphorylation and degradation of IB␣ are negatively regulated by phosphorylation at Tyr-42, thus preventing NF-B activation. The transcription factor NF-B regulates the expression of many genes that play essential roles in immune and inflammatory responses including the type I human immunodeficiency virus (1-4). Like all members of the Rel/NF-B transcription factor family, NF-B has the unique property of being sequestered in its inactive state in the cytoplasm by a noncovalent association with inhibitory proteins called IB (4). In mammalian species, at least seven structural homologs of IB have been identified (4), but only the IB␣ form has been extensively studied. Recently, IB␣-deficient mice have been generated; they exhibit constitutive NF-B activation, severe runting, dermatitis, extensive granulopoiesis, and neonatal death (5, 6). However, tumor necrosis factor (TNF), 1 which initiates the degradation of IB␣, causes sustained NF-B activation in embryonic fibroblasts from these knockout mice, suggesting that IB␣ is necessary for the postinduction repression of NF-B activity (5). Indeed, IB␣ controls the activation of NF-B by masking the nuclear localization signal located on the p50-p65 heterodimer of NF-B (7). In response to a wide variety of stimuli besides TNF, IB␣ undergoes degradation, allowing the p50-p65 heterodimer to migrate to the nucleus (8, 9). Since protein synthesis is not required for activation of this transcription factor, induction of target genes can occur within minutes of extracellular stimulus. Site-specific mutagenesis and peptide mapping have revealed that inducible phosphorylation of IB␣ occurs at both serines 32 and 36 (10 -12). Although all known activators of NF-B induce phosphorylation of IB␣, leading to its degradation, how phosphorylation makes IB␣ susceptible to degradation is not understood (4). Recently, specific proteasome inhibitors have been shown to block the inducible degradation of IB␣, leading to the accumulation of a phosphorylated form of IB␣ while still bound to NF-B (12-18). These findings suggest that induced phosphorylation of IB␣ is needed but not sufficient for its degradation by the proteasome. Ubiquitination of lysines 21 and 22 (11, 19) follows inducible serine phosphorylation, leading to degradation of IB␣ by the 26 S proteasome (8, 9) . The protein kinase involved in the inducible phosphorylation of IB␣ has been shown to be part of a 700-kDa complex (20), but the molecular identity of this kinase remains elusive. Recently, reports from our laboratory (21) and another (22) demonstrated that TNF-mediated NF-B activation is completely abolished by the protein-tyrosine phosphatase inhibitor pervanadate (PV), which blocks the degradation of IB␣. In this report, we examined the mechanism by which PV blocks the degradation of IB␣. We demonstrate that inhibition of protein-tyrosine phosphatase activity by PV results in sitespecific tyrosine phosphorylation of IB␣, which prevents its inducible phosphorylation and degradation and hence inhibits the activation of NF-B. EXPERIMENTAL PROCEDURES Materials-The cell lines employed in this study included ML-1a, a human myelomonoblastic leukemia cell line kindly provided by Dr. Ken Takeda (Showa University, Showa, Japan). U937, a human histiocytic lymphoma cell line, and HeLa, an epithelial carcinoma cell line, were obtained from the American Type Culture Collection (Rockville, MD). ML-1a and U937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. HeLa cells were cultured in minimum Eagle's medium supplemented with 10% fetal