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• W1497648141 abstract "The transcription factor NF-κB (nuclear factor-κB) is neutralized in nonstimulated cells through cytoplasmic retention by IκB inhibitors. In mammalian cells, two major forms of IκB proteins, IκBα and IκBβ, have been identified. Upon treatment with a large variety of inducers, IκBα and IκBβ are proteolytically degraded, resulting in NF-κB translocation into the nucleus. Recent observations suggest that phosphorylation of serines 32 and 36 and subsequent ubiquitination of lysines 21 and 22 of IκBα control its signal-induced degradation. In this study we provide evidence that critical residues in the NH2-terminal region of IκBβ (serines 19 and 23) as well as its COOH-terminal PEST region control IκBβ proteolysis. However Lys-9, the unique lysine residue in the NH2-terminal region of IκBβ, is not absolutely required for its degradation. We also demonstrate that following stimulation, an underphosphorylated nondegradable form of IκBβ accumulates. Surprisingly, our data suggest that unlike IκBα, IκBβ is constitutively phosphorylated on one or two of the critical NH2-terminal serine residues. Thus, phosphorylation of these sites is necessary for degradation but does not necessarily constitute the signal-induced event that targets the molecule for proteolysis. The transcription factor NF-κB (nuclear factor-κB) is neutralized in nonstimulated cells through cytoplasmic retention by IκB inhibitors. In mammalian cells, two major forms of IκB proteins, IκBα and IκBβ, have been identified. Upon treatment with a large variety of inducers, IκBα and IκBβ are proteolytically degraded, resulting in NF-κB translocation into the nucleus. Recent observations suggest that phosphorylation of serines 32 and 36 and subsequent ubiquitination of lysines 21 and 22 of IκBα control its signal-induced degradation. In this study we provide evidence that critical residues in the NH2-terminal region of IκBβ (serines 19 and 23) as well as its COOH-terminal PEST region control IκBβ proteolysis. However Lys-9, the unique lysine residue in the NH2-terminal region of IκBβ, is not absolutely required for its degradation. We also demonstrate that following stimulation, an underphosphorylated nondegradable form of IκBβ accumulates. Surprisingly, our data suggest that unlike IκBα, IκBβ is constitutively phosphorylated on one or two of the critical NH2-terminal serine residues. Thus, phosphorylation of these sites is necessary for degradation but does not necessarily constitute the signal-induced event that targets the molecule for proteolysis. INTRODUCTIONThe transcription factor NF-κB 1The abbreviations used are: NF-κBnuclear factor-κBTNFtumor necrosis factorILinterleukinLPSlipopolysaccharidePMAphorbol 12-myristate 13-acetateHAhemagglutininwtwild typeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineALLNN-acetyl-Leu-Leu-norleucinal. plays a central role in the regulation of genes implicated in the immune response and in inflammatory processes. NF-κB is composed of homo- and heterodimeric complexes of members of the Rel/NF-κB family of polypeptides. In vertebrates, this family comprises p50, p65 (RelA), c-Rel, p52, and RelB.In resting cells, NF-κB is cytosolic, but the nuclear translocation of this factor can be induced by multiple stimuli that act at different levels in the cell. Some, like TNF-α, IL-1, LPS, or antibodies against the T cell receptor-CD3 complex, act on an extracellular receptor, whereas others, like PMA and double-stranded RNA, activate intracellular second messengers (for review, see Refs. 1Israël A. Trends Genet. 1995; 11: 203-205Google Scholar and 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Google Scholar).The molecular mechanism responsible for the cytosolic retention of NF-κB involves its association with the inhibitory ankyrin repeat-containing members of the IκB family of proteins. This family of inhibitors is mainly represented by IκBα and IκBβ (3Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Google Scholar, 4Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar) but also includes IκBγ, Bcl-3, p105, and p100 (for review, see Ref. 5Gilmore T.D. Morin P.J. Trends Genet. 1993; 9: 427-433Google Scholar). p105 and p100, which are also the precursors of the p50 and p52 subunits of NF-κB, function as IκB proteins through association with p50, c-Rel or p65 (6Rice N.R. MacKichan M.L. Israël A. Cell. 1992; 46: 243-253Google Scholar, 7Mercurio F. Didonato J.A. Rosette C. Karin M. Genes Dev. 1993; 7: 705-718Google Scholar, 8Scheinman R.I. Beg A.A. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 6089-6101Google Scholar). Among the different IκBs, IκBα and IκBβ play a major role in the regulation of NF-κB. These two proteins have been originally identified by partial purification (3Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Google Scholar, 4Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar). A cDNA clone has been isolated which encodes a 36-kDa protein called MAD-3, which appears to be identical to IκBα (9Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson J.A. Mondal K. Ralph P. Baldwin A.J. Cell. 1991; 65: 1281-1289Google Scholar). The cloning of IκBβ cDNA is more recent, and this molecule has thus been characterized less thoroughly (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar).Unlike IκBβ, the IκBα gene is positively regulated by NF-κB and glucocorticoids (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar, 11Auphan N. Didonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Google Scholar, 12De Martin R. Vanhove B. Cheng Q. Hofer E. Csizmadia V. Winkler H. Bach F.H. EMBO J. 1993; 12: 2773-2779Google Scholar, 13LeBail O. Schmidt-Ullrich R. Israël A. EMBO J. 1993; 12: 5043-5049Google Scholar, 14Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin A.S. Science. 1995; 270: 283-286Google Scholar). IκBα and IκBβ associate with p50-p65 heterodimers and prevent the nuclear translocation of these complexes by masking their nuclear localization sequence. These two molecules are structurally similar as they contain multiple ankyrin repeats and a COOH-terminal PEST domain, a sequence known to be highly correlated to rapid protein turnover. The PEST domain of IκBα is involved in its degradation (15Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X.X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Google Scholar, 16Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Google Scholar, 17Rodriguez M.S. Michalopoulos I. Arenzana S.F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Google Scholar, 18Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Google Scholar, 19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar) and in its inhibition of the DNA binding activity of NF-κB (20Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Google Scholar).Under the effect of a stimulus, IκBα becomes phosphorylated and is subsequently degraded, allowing NF-κB to translocate into the nucleus. The use of protease inhibitors has shed some light on the proteases responsible for IκBα degradation (21Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut L.I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Google Scholar, 22Didonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Google Scholar, 23Finco T.S. Beg A.A. Baldwin Jr., A.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11884-11888Google Scholar, 24Lin Y.C. Brown K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 552-556Google Scholar, 25Miyamoto S. Maki M. Schmitt M.J. Hatanaka M. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12740-12744Google Scholar, 26Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Google Scholar, 27Traenckner E.B.M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Google Scholar). In the presence of an NF-κB inducer, proteasome inhibitors stabilize a phosphorylated form of IκBα characterized by a slow electrophoretic mobility. The observation that this retarded form is still associated with NF-κB invalidates the former hypothesis that IκBα phosphorylation induces its dissociation from NF-κB.Recently, the sites of phosphorylation of IκBα have been identified as two closely spaced serines at positions 32 and 36 in the NH2-terminal part of the protein (15Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X.X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Google Scholar, 16Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Google Scholar, 17Rodriguez M.S. Michalopoulos I. Arenzana S.F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Google Scholar, 18Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Google Scholar, 19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar, 28Sun S.-C. Maggirwar S.B. Harhaj E. J. Biol. Chem. 1995; 270: 18347-18351Google Scholar). Mutation of these two serines to nonphosphorylatable residues prevents IκBα degradation, suggesting that their phosphorylation is a prerequisite for degradation. In addition, an in vitro study has also shown that only the hyperphosphorylated form of IκBα is degraded (29Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben-Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Google Scholar). It has been reported that degradation of IκBα is triggered by ubiquitination (30Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Google Scholar). Ubiquitination occurs primarily on two adjacent lysines (Lys-21 and Lys-22) (31Baldi L. Brown K. Franzoso G. Siebenlist U. J. Biol. Chem. 1996; 271: 376-379Google Scholar, 32Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Google Scholar).In thymocytes, IκBα is the main inhibitor of NF-κB, and the disruption of IκBα gene by homologous recombination results in a constitutively elevated level of nuclear NF-κB (33Beg A.A. Sha W.C. Bronson R.T. Baltimore D. Genes Dev. 1995; 9: 2736-2746Google Scholar). In contrast to hematopoietic cells, IκBα−/− embryonic fibroblasts behave as wild type cells because of the major role of the IκBβ molecule.A recent study concludes that TNF-α or PMA induces degradation of IκBα but not of IκBβ, suggesting that these two proteins are regulated differentially (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar). The mechanisms responsible for the differential behavior of the two IκB molecules remain unresolved. We provide here some clues as to why these two molecules are regulated differentially. We first show that IκBβ degradation is blocked by proteasome inhibitors, suggesting an involvement of the ubiquitin-proteasome pathway, similar to what has been described for IκBα. These similarities are confirmed by the identification of two critical sites of phosphorylation (Ser-19 and Ser-23) whose mutations decrease the rate of signal-induced degradation of IκBβ. However, IκBβ contains only one lysine NH2-terminal to ankyrin repeats, and its mutation does not prevent the signal-induced degradation of the mutant protein. We also demonstrate that IκBβ preexists as two electrophoretically different variants: the major, slow migrating form, is degraded following stimulation; the minor, faster migrating form accumulates. Our study also suggests that, unlike IκBα, IκBβ is phosphorylated on Ser-19 and/or Ser-23 in noninduced cells. Therefore, these results suggest that IκBβ might differs from IκBα in that the critical event that targets IκBβ for degradation is not the induced phosphorylation of the two conserved serine residues located in the NH2-terminal region of the molecule.DISCUSSIONRecent data have provided some insight into the mechanisms leading to IκBα degradation (15Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X.X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Google Scholar, 16Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Google Scholar, 17Rodriguez M.S. Michalopoulos I. Arenzana S.F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Google Scholar, 18Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Google Scholar, 19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar, 21Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut L.I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Google Scholar, 30Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Google Scholar, 31Baldi L. Brown K. Franzoso G. Siebenlist U. J. Biol. Chem. 1996; 271: 376-379Google Scholar, 32Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Google Scholar, 37Schägger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Google Scholar). The signal-induced degradation of this molecule is dependent upon the presence of an intact COOH-terminal PEST region as well as the induced phosphorylation of serine residues 32 and 36. This phosphorylation probably targets the molecule for ubiquitination on multiple residues, lysines 21 and 22 playing a central role (31Baldi L. Brown K. Franzoso G. Siebenlist U. J. Biol. Chem. 1996; 271: 376-379Google Scholar, 32Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Google Scholar). This in turn targets the molecule for degradation by the 26 S proteasome (30Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Google Scholar), which takes place in the absence of dissociation between the inhibitor and NF-κB.The IκBβ molecule was originally purified and shown to be inactivated in vitro by dephosphorylation, in contrast to the situation observed with IκBα (38Link E. Kerr L.D. Schreck R. Zabel U. Verma I. Baeuerle P.A. J. Biol. Chem. 1992; 267: 239-246Google Scholar). The recent cloning of IκBβ (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar) demonstrated that the protein contained, like IκBα, a COOH-terminal PEST region, several ankyrin repeats, and an NH2-terminal region with two serines (at positions 19 and 23) in an environment similar to IκBα. A unique lysine residue was present in this region, at position 9. However, some differences could be observed between the two molecules following stimulation. First, although PMA, TNF, LPS, and IL-1 all induce degradation of IκBα, only LPS and IL-1 induced degradation of IκBβ in 70Z/3 cells (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar). Second, the IκBα molecule is resynthesized rapidly following degradation, partly because the promoter of the IκBα gene is positively regulated by NF-κB. On the contrary, the IκBβ molecule is not resynthesized before the stimulus has ceased. This suggests that the promoter of the IκBβ gene does not respond to NF-κB. However, a more recent study seems to indicate that in different cell types the IκBβ molecule is also degraded in response to TNF-α (33Beg A.A. Sha W.C. Bronson R.T. Baltimore D. Genes Dev. 1995; 9: 2736-2746Google Scholar).Therefore, to get some insight into the mechanisms responsible for these different behaviors, we carried out a systematic analysis of the events leading to IκBβ degradation. In contrast to previous work (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar), we found that IκBβ could be degraded in response to TNF-α and PMA in various murine cell lines (Fig. 2). However, the EL-4 T cell line shows a different susceptibility to TNF-α since only IκBα is degraded. This observation raised some important questions concerning the signaling pathway leading to the loss of IκBα and IκBβ. If the two inhibitors were targeted for proteolysis by the same pathway, they would be affected equally in response to a given stimulus. It is however possible that the weakness of the signal via the TNF-α receptor in this cell line could account for the phenomenon observed, since IκBβ can indeed be degraded by other stimuli (IL-1 and PMA) (data not shown).Following treatment of E29.1 murine T cells with TNF-α, two forms of IκBβ become detectable: a major upper form (form I), which is degraded progressively; and a minor lower form (form II), which accumulates progressively and appears to be resistant to degradation in response to TNF-α stimulation. The same kinetics of degradation is observed during treatment of 70Z/3 cells by LPS or PMA. The two forms of IκBβ are both associated with p65. Using cycloheximide, we demonstrated that form II is not derived from form I but requires ongoing protein synthesis to accumulate (Fig. 3A).Treatment of form I with alkaline phosphatase results in a partial conversion into a protein that comigrates with form II, suggesting that form I is a hyperphosphorylated form of form II (data not shown). These data suggest that this hyperphosphorylation might target form I for degradation.Based on the results obtained with IκBα, we asked whether serines 19 and 23 were the sites of phosphorylation responsible for IκBβ proteolysis. In accordance with this model, we observed that mutation of either of these two serines into an alanine abolished IκBβ degradation (Fig. 5). These results are consistent with a recent report (39Didonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Google Scholar). However, the A19 A23 IκBβ mutant still exists as form I and form II, suggesting that form II is not derived from form I by dephosphorylation on Ser-19 and/or Ser-23 (Fig. 5, A and B).The IκBα PEST sequence is constitutively phosphorylated by a highly ubiquitous conserved kinase, casein kinase II. IκBα possesses several consensus sites for casein kinase II, and mutation of one of these sites increased the IκBα half-life (40Barroga C.F. Stevenson J.K. Schwarz E.M. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7637-7641Google Scholar). It has thus been hypothesized that the role of basal phosphorylation is to allow degradation of excess free IκBα by reducing its half-life. This PEST sequence also plays an important role in signal-dependent degradation of IκBα (16Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Google Scholar, 17Rodriguez M.S. Michalopoulos I. Arenzana S.F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Google Scholar, 19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar). As reported for IκBα, we also find that IκBβ-inducible proteolysis requires the PEST sequence present in its COOH-terminal region. However, deletion of the PEST sequence results in the disappearance of form II (Fig. 6). This suggests that form II differs from form I in its level of phosphorylation in the PEST region.In contrast to IκBα, mutation of lysine at position 9 inhibited weakly, if at all, IκBβ degradation, indicating that if this is a site of ubiquitination, its mutation does not prevent ubiquitination at other sites (Fig. 5). Furthermore, no upshift of the molecule could be observed following stimulation in the presence of ALLN, a proteasome inhibitor that prevents degradation of IκBα and IκBβ and allows accumulation of a hyperphosphorylated form of IκBα. However this lack of upshift does not necessary imply a lack of hyperphosphorylation of the molecule, as hyperphosphorylated murine IκBα migrates like its hypophosphorylated counterpart and is identified only by two-dimensional gel electrophoresis3 (19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar). To reveal hyperphosphorylated forms of IκBβ, we performed a two-dimensional gel analysis following various treatments of 70Z/3 cells. We observed that IκBβ preexists as multiple isoforms that most likely correspond, as for IκBα, to differentially phosphorylated forms. Interestingly, the isoforms observed in the presence of LPS and ALLN coincide exactly with those present in untreated cells, suggesting that their net charge does not change following stimulation.From these results we can conclude that serines 19 and 23 are critical determinants of IκBβ degradation but that their phosphorylation status does not seem to change following activation. One intriguing possibility is that they could be constitutively phosphorylated in untreated cells. To test this hypothesis, we compared HA wt ΔPEST and HA A19 A23 ΔPEST proteins, as the number of isoforms should be reduced following deletion of the PEST region. As hypothesized, we observe that mutation of serines 19 and 23 to alanines shifted all isoforms toward more basic pIs. Since these serine to alanines substitutions per se do not result in a modification of calculated pI, the observed difference in pI between the two proteins (0.1 pH unit) suggests that Ser-19 and/or Ser-23 is constitutively phosphorylated. Consequently, we found that the peptide containing serines 19 and 23 was constitutively phosphorylated in wt IκBβ molecules but not when serine 19 and 23 were mutated (Fig. 8). However this result has been obtained in transiently transfected cells as we never obtained enough radioactivity from 32P-labeled cells to carry out the analysis of IκBβ-derived phosphopeptides. The intriguing observation that Ser-19 and/or Ser-23 is constitutively phosphorylated is supported by the the fact that following treatment with calyculin A, a potent inhibitor of serine/threonine phosphatases, IκBα is degraded quickly, whereas the IκBβ molecule is not; but, surprisingly, it dissociates from p65 (Fig. 4). Although this situation is not very physiological, since treatment with calyculin A induces multiple nonspecific phosphorylations (see the multiple bands observed for p65 in Fig. 4, lanes 10-12), it tells us that hyperphosphorylation is unable to induce IκBβ degradation, thus supporting our hypothesis that Ser-19 and/or Ser-23 is constitutively phosphorylated and that this is not sufficient to trigger IκBβ proteolysis. In this context, several speculations can be made concerning the signaling pathways leading to the inducible degradation of IκBα and IκBβ. First, if the critical serine residues of IκBα and IκBβ are phosphorylated by the same kinase, then there should exist a mechanism maintaining serines 32 and 36 of IκBα unphosphorylated in uninduced cells. Since degradation of IκBα is induced by phosphatase inhibitors, it might be that a constitutive phosphatase, which is inactivated upon induction, is involved but does not recognize IκBβ. Alternatively, serines 32 and 36 of IκBα might be masked in resting cells and thus inaccessible to the kinase activity. Finally, the kinase might be constitutively associated with IκBβ but recruited to IκBα upon NF-κB activation. Alternatively, different kinases may phosphorylate the two IκB molecules.Second, if IκBβ is constitutively phosphorylated on Ser-19 and/or Ser-23, the nature of the signal responsible for targeting IκBβ for degradation remains to be identified. Because we have been unable to detect any inducible modification of IκBβ mobility after two-dimensional electrophoresis, we think it is unlikely that other IκBβ-specific phosphorylation events are involved. Alternatively, it might be that degradation per se is induced. For example, enzymes responsible for the ubiquitination of IκBβ could be activated following induction. A possible candidate would be the specific E3 involved. Indeed it has been demonstrated that in the case of E6-AP, activity can be regulated (41Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Google Scholar, 42Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Google Scholar). A change in activity of the 26 S proteasome following induction could also constitute the inducible signal, as has been shown in the case of stimulation by interferon-γ (43Ahn J.Y. Tanahashi N. Akiyama K. Hisamatsu H. Noda C. Tanaka K. Chung C.H. Shibmara N. Willy P.J. Mott J.D. Slaughter C.A. DeMartino G.N. FEBS Lett. 1995; 366: 37-42Google Scholar). INTRODUCTIONThe transcription factor NF-κB 1The abbreviations used are: NF-κBnuclear factor-κBTNFtumor necrosis factorILinterleukinLPSlipopolysaccharidePMAphorbol 12-myristate 13-acetateHAhemagglutininwtwild typeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineALLNN-acetyl-Leu-Leu-norleucinal. plays a central role in the regulation of genes implicated in the immune response and in inflammatory processes. NF-κB is composed of homo- and heterodimeric complexes of members of the Rel/NF-κB family of polypeptides. In vertebrates, this family comprises p50, p65 (RelA), c-Rel, p52, and RelB.In resting cells, NF-κB is cytosolic, but the nuclear translocation of this factor can be induced by multiple stimuli that act at different levels in the cell. Some, like TNF-α, IL-1, LPS, or antibodies against the T cell receptor-CD3 complex, act on an extracellular receptor, whereas others, like PMA and double-stranded RNA, activate intracellular second messengers (for review, see Refs. 1Israël A. Trends Genet. 1995; 11: 203-205Google Scholar and 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Google Scholar).The molecular mechanism responsible for the cytosolic retention of NF-κB involves its association with the inhibitory ankyrin repeat-containing members of the IκB family of proteins. This family of inhibitors is mainly represented by IκBα and IκBβ (3Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Google Scholar, 4Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar) but also includes IκBγ, Bcl-3, p105, and p100 (for review, see Ref. 5Gilmore T.D. Morin P.J. Trends Genet. 1993; 9: 427-433Google Scholar). p105 and p100, which are also the precursors of the p50 and p52 subunits of NF-κB, function as IκB proteins through association with p50, c-Rel or p65 (6Rice N.R. MacKichan M.L. Israël A. Cell. 1992; 46: 243-253Google Scholar, 7Mercurio F. Didonato J.A. Rosette C. Karin M. Genes Dev. 1993; 7: 705-718Google Scholar, 8Scheinman R.I. Beg A.A. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 6089-6101Google Scholar). Among the different IκBs, IκBα and IκBβ play a major role in the regulation of NF-κB. These two proteins have been originally identified by partial purification (3Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Google Scholar, 4Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar). A cDNA clone has been isolated which encodes a 36-kDa protein called MAD-3, which appears to be identical to IκBα (9Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson J.A. Mondal K. Ralph P. Baldwin A.J. Cell. 1991; 65: 1281-1289Google Scholar). The cloning of IκBβ cDNA is more recent, and this molecule has thus been characterized less thoroughly (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar).Unlike IκBβ, the IκBα gene is positively regulated by NF-κB and glucocorticoids (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar, 11Auphan N. Didonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Google Scholar, 12De Martin R. Vanhove B. Cheng Q. Hofer E. Csizmadia V. Winkler H. Bach F.H. EMBO J. 1993; 12: 2773-2779Google Scholar, 13LeBail O. Schmidt-Ullrich R. Israël A. EMBO J. 1993; 12: 5043-5049Google Scholar, 14Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin A.S. Science. 1995; 270: 283-286Google Scholar). IκBα and IκBβ associate with p50-p65 heterodimers and prevent the nuclear translocation of these complexes by masking their nuclear localization sequence. These two molecules are structurally similar as they contain multiple ankyrin repeats and a COOH-terminal PEST domain, a sequence known to be highly correlated to rapid protein turnover. The PEST domain of IκBα is involved in its degradation (15Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X.X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Google Scholar, 16Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Google Scholar, 17Rodriguez M.S. Michalopoulos I. Arenzana S.F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Google Scholar, 18Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Google Scholar, 19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar) and in its inhibition of the DNA binding activity of NF-κB (20Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Google Scholar).Under the effect of a stimulus, IκBα becomes phosphorylated and is subsequently degraded, allowing NF-κB to translocate into the nucleus. The use of protease inhibitors has shed some light on the proteases responsible for IκBα degradation (21Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut L.I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Google Scholar, 22Didonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Google Scholar, 23Finco T.S. Beg A.A. Baldwin Jr., A.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11884-11888Google Scholar, 24Lin Y.C. Brown K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 552-556Google Scholar, 25Miyamoto S. Maki M. Schmitt M.J. Hatanaka M. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12740-12744Google Scholar, 26Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Google Scholar, 27Traenckner E.B.M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Google Scholar). In the presence of an NF-κB inducer, proteasome inhibitors stabilize a phosphorylated form of IκBα characterized by a slow electrophoretic mobility. The observation that this retarded form is still associated with NF-κB invalidates the former hypothesis that IκBα phosphorylation induces its dissociation from NF-κB.Recently, the sites of phosphorylation of IκBα have been identified as two closely spaced serines at positions 32 and 36 in the NH2-terminal part of the protein (15Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X.X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Google Scholar, 16Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Google Scholar, 17Rodriguez M.S. Michalopoulos I. Arenzana S.F. Hay R.T. Mol. Cell. Biol. 1995; 15: 2413-2419Google Scholar, 18Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Google Scholar, 19Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israël A. Mol. Cell. Biol. 1995; 15: 5339-5345Google Scholar, 28Sun S.-C. Maggirwar S.B. Harhaj E. J. Biol. Chem. 1995; 270: 18347-18351Google Scholar). Mutation of these two serines to nonphosphorylatable residues prevents IκBα degradation, suggesting that their phosphorylation is a prerequisite for degradation. In addition, an in vitro study has also shown that only the hyperphosphorylated form of IκBα is degraded (29Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben-Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Google Scholar). It has been reported that degradation of IκBα is triggered by ubiquitination (30Chen Z.J. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Google Scholar). Ubiquitination occurs primarily on two adjacent lysines (Lys-21 and Lys-22) (31Baldi L. Brown K. Franzoso G. Siebenlist U. J. Biol. Chem. 1996; 271: 376-379Google Scholar, 32Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Google Scholar).In thymocytes, IκBα is the main inhibitor of NF-κB, and the disruption of IκBα gene by homologous recombination results in a constitutively elevated level of nuclear NF-κB (33Beg A.A. Sha W.C. Bronson R.T. Baltimore D. Genes Dev. 1995; 9: 2736-2746Google Scholar). In contrast to hematopoietic cells, IκBα−/− embryonic fibroblasts behave as wild type cells because of the major role of the IκBβ molecule.A recent study concludes that TNF-α or PMA induces degradation of IκBα but not of IκBβ, suggesting that these two proteins are regulated differentially (10Thompson J.E. Phillips R.J. Erdjument-bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Google Scholar). The mechanisms responsible for the differential behavior of the two IκB molecules remain unresolved. We provide here some clues as to why these two molecules are regulated differentially. We first show that IκBβ degradation is blocked by proteasome inhibitors, suggesting an involvement of the ubiquitin-proteasome pathway, similar to what has been described for IκBα. These similarities are confirmed by the identification of two critical sites of phosphorylation (Ser-19 and Ser-23) whose mutations decrease the rate of signal-induced degradation of IκBβ. However, IκBβ contains only one lysine NH2-terminal to ankyrin repeats, and its mutation does not prevent the signal-induced degradation of the mutant protein. We also demonstrate that IκBβ preexists as two electrophoretically different variants: the major, slow migrating form, is degraded following stimulation; the minor, faster migrating form accumulates. Our study also suggests that, unlike IκBα, IκBβ is phosphorylated on Ser-19 and/or Ser-23 in noninduced cells. Therefore, these results suggest that IκBβ might differs from IκBα in that the critical event that targets IκBβ for degradation is not the induced phosphorylation of the two conserved serine residues located in the NH2-terminal region of the molecule." @default.
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• W1497648141 title "Regulation of IκBβ Degradation" @default.
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