Jill L. Thompson

The transcription factor NF-kappa B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein I kappa B alpha maintains NF-kappa B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, I kappa B alpha is rapidly degraded, leading to nuclear translocation of free NF-kappa B. However, NF-kappa B-dependent transcription of the I kappa B alpha gene leads to rapid resynthesis of the I kappa B alpha protein and inhibition of NF-kappa B-dependent transcription. Here we demonstrate a new regulatory function of I kappa B alpha exerted on NF-kappa B in the nuclear compartment. Although normally found in the cytoplasm, I kappa B alpha, newly synthesized in response to tumor necrosis factor or interleukin I, is transported to the nucleus. In the nucleus I kappa B alpha associates with the p50 and p65 subunits of NF-kappa B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of I kappa B alpha correlates with transcription termination of transfected NF-kappa B-dependent luciferase genes. Following the appearance of I kappa B alpha in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-kappa B-I kappa B alpha complexes are cleared from the nucleus.

Transcriptional activation of nuclear factor κB (NF-κB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IκBα. However, NF-κB activation induces rapid resynthesis of IκBα, which is responsible for post-induction repression of transcription. Newly synthesized IκBα translocates to the nucleus, where it dissociates NF-κB from DNA and transports NF-κB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-κB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-κB-dependent transcriptional activation mediated by interleukin 1β or tumor necrosis factor α. This is a consequence of the inhibition of signal-induced degradation of IκBα. Although LMB treatment does not affect the signal transduction pathway leading to IκBα degradation, it blocks IκBα nuclear export. IκBα is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IκBα is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF-κB to be transcriptionally active upon cell stimulation. Transcriptional activation of nuclear factor κB (NF-κB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IκBα. However, NF-κB activation induces rapid resynthesis of IκBα, which is responsible for post-induction repression of transcription. Newly synthesized IκBα translocates to the nucleus, where it dissociates NF-κB from DNA and transports NF-κB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-κB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-κB-dependent transcriptional activation mediated by interleukin 1β or tumor necrosis factor α. This is a consequence of the inhibition of signal-induced degradation of IκBα. Although LMB treatment does not affect the signal transduction pathway leading to IκBα degradation, it blocks IκBα nuclear export. IκBα is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IκBα is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF-κB to be transcriptionally active upon cell stimulation. The NF-κB 1The abbreviations used are:NF-κB, nuclear factor κB; TNF, tumor necrosis factor; IL-1β, interleukin 1β; LMB, leptomycin B; CX, cycloheximide; NES, nuclear export sequence; aa, amino acid(s); IKK, IκB kinase; SLO, streptolysin O; SUMO-1, small ubiquitin-like modifier 1.1The abbreviations used are:NF-κB, nuclear factor κB; TNF, tumor necrosis factor; IL-1β, interleukin 1β; LMB, leptomycin B; CX, cycloheximide; NES, nuclear export sequence; aa, amino acid(s); IKK, IκB kinase; SLO, streptolysin O; SUMO-1, small ubiquitin-like modifier 1./Rel family of transcription factors is implicated in regulation of the expression of a number of cellular genes involved in immune responses, inflammation ,and apoptosis (for recent reviews, see Refs. 1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5542) Google Scholar, 2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar, 3May M.J. Ghosh S. Immunol. Today. 1998; 19: 80-88Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar). In vertebrates, the NF-κB family of proteins is composed of transcriptionally active p65/Rel A (4Nolan G.P. Ghosh S. Liou H.C. Tempst P. Baltimore D. Cell. 1991; 64: 961-969Abstract Full Text PDF PubMed Scopus (417) Google Scholar, 5Ruben S.M. Dillon P.J. Schreck R. Henkel T. Chen C.-H. Maher M. Baeuerle P.A. Rosen C.A. Science. 1991; 251: 1490-1493Crossref PubMed Scopus (289) Google Scholar), c-Rel (6Wilhelmsen K.C. Eggleton K. Temin H.M. J. Virol. 1984; 52: 172-182Crossref PubMed Google Scholar), or Rel B (7Ryseck R.P. Bull P. Takamiya M. Bours V. Siebenlist U. Dobrzanski P. Bravo R. Mol. Cell. Biol. 1992; 12: 674-684Crossref PubMed Scopus (274) Google Scholar) and transcriptionally silent p50/NF-κB1 (8Ghosh S. Gifford A.M. Riviere L.R. Tempst P. Nolan G.P. Baltimore D. Cell. 1990; 62: 1019-1029Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 9Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israël A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (601) Google Scholar) or p52/NF-κB2 (10Bours V. Burd P.R. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar, 11Neri A. Chang C.C. Lombardi L. Salina M. Corradini P. Maiolo A.T. Chaganti R.S.K. Dalla-Favera R. Cell. 1991; 67: 1075-1087Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 12Schmid R.M. Perkins N.D. Duckett C.S. Andrews P.C. Nabel G.J. Nature. 1991; 352: 733-736Crossref PubMed Scopus (274) Google Scholar). All NF-κB proteins share a conserved region known as the Rel homology domain that contains the nuclear signal as as the and DNA The NF-κB by is composed of and NF-κB transcriptional is by IκB proteins that of with IκB not the nuclear of and leading to cytoplasmic NF-κB DNA activity. IκBα S. S.M. A. K. P. A.S. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar), U. Baeuerle P.A. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar), A. J. PubMed Scopus Google Scholar), and Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). the of and that in known as V. Kourilsky P. A. J. 1991; PubMed Scopus Google Scholar, J. A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, H.C. Nolan G.P. Ghosh S. T. Baltimore D. J. 1992; PubMed Scopus Google Scholar) and F. J. M. DNA Biol. 1992; PubMed Scopus Google Scholar, F. M. PubMed Scopus Google Scholar), is in signal a region of and a region a domain that is to the domain by a and is by Mol. Cell. Biol. PubMed Google Scholar). and for the signal-induced degradation of IκBα K. L. L. L. S. Siebenlist U. Mol. Cell. Biol. PubMed Google Scholar, M. M. M.J. A. D. F. PubMed Scopus Google Scholar). signal IκBα is and S.M. Mol. Cell. Biol. PubMed Google Scholar, K. S. L. Siebenlist U. Science. PubMed Scopus Google Scholar, Henkel T. S. Baeuerle P.A. J. 14: PubMed Scopus Google Scholar, J. F. J. Ghosh S. M. Mol. Cell. Biol. 1996; PubMed Google Scholar, M. J. F. F. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) by the IκB M. M. Nature. PubMed Scopus Google Scholar, F. A. M. M. A. A. Science. PubMed Scopus Google Scholar, M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar, M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). IκBα is and Chen T. U. S. A. PubMed Scopus Google Scholar, L. Brown K. Siebenlist U. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. D. F. F. 1996; 12: Google Scholar), which the for degradation by the IκBα is NF-κB to the nucleus, where it induces the transcription of genes that of its inhibitor, synthesized IκBα is accumulated in the cytoplasm in the nucleus, where it NF-κB-dependent transcription. This is by inhibition of the and export of NF-κB to the cytoplasm F. J. F. D. Mol. Cell. Biol. PubMed Google Scholar). The of IκBα is by a nuclear export present in its region F. P. M. D. J. PubMed Google Scholar) and to the in proteins the protein and the protein A U. J. R. Cell. Full Text PDF PubMed Scopus Google Scholar, C.C. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). that and to rapid and active export from the nucleus to the The nuclear protein known as as the M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, K. C.S. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). to the family in it homology in the domain with from this family M. J. S. A. D. J. J. PubMed Scopus Google Scholar). The of is by the of in its that this is the nuclear and dissociates in the cytoplasm to by M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In to be the cellular of the leptomycin B that inhibits protein export in and in M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, Biol. Full Text PDF PubMed Scopus Google the present study, LMB was used as a tool to inhibit nuclear protein export and evaluate the consequences for regulation of NF-κB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-κB-dependent transcriptional activation mediated by or This is a consequence of the inhibition of signal-induced degradation of IκBα. Although LMB treatment does not affect the signal transduction pathway leading to IκBα degradation, it blocks IκBα nuclear export. IκBα is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IκBα is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF-κB to be transcriptionally active upon cell indicate that inhibition of IκBα nuclear export not the post-induction repression of NF-κB-dependent transcription the activation of NF-κB upon cell stimulation. nuclear IκBα to be resistant to signal-induced phosphorylation and degradation, and this results in nuclear of transcriptionally export of IκBα and proteins that the nucleus and the cytoplasm is mediated by a that is by M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, K. C.S. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In nuclear export of IκBα was by the LMB by its with the F. Science. PubMed Scopus Google Scholar). Although the inhibition of by LMB is it was to the that LMB be with the signal transduction that to NF-κB this used a protein the amino and of IκBα. this protein is in it is to to the nucleus signal-induced degradation in to as and M. M. M.J. A. D. F. PubMed Scopus Google Scholar). degradation of the protein was by LMB that LMB does not inhibit the signal transduction pathway that to IκBα degradation. is that LMB does not inhibit transcription in a the of the was by the of LMB The from is that in signal-induced phosphorylation and degradation of IκBα in the to this is that essential of the signal transduction pathway that to IκBα phosphorylation to the that the IκBα and present in a that as NF-κB P.A. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar, M. M. U. S. A. 1998; PubMed Scopus Google Scholar) and proteins as S. R. F. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). to be if this be the The be that the signal to the nucleus, that the nuclear IκBα is in to to this of IκBα to a that is by the that IκBα is by the small ubiquitin-like protein Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). this protein is to the that used for of IκBα, this the of IκBα resistant to signal-induced degradation. Although the known proteins that for in the nucleus or involved in nuclear M. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar), to the that nuclear IκBα in the of LMB is resistant to signal-induced phosphorylation it is by is that IκBα with a nuclear protein that the region in IκBα and thus it from signal-induced is a for the transcription of essential NF-κB-dependent transcription of genes does not as a consequence of IκBα the of a NF-κB-dependent in S. it that the cell a to for transcription of NF-κB-dependent This the of IκBα, which a of NF-κB that to the in the nucleus, NF-κB NF-κB-dependent that of IκBα. to the IκBα and the IκBα is to the nucleus, where it with NF-κB and dissociates the of the of a in IκBα F. P. M. D. J. PubMed Google Scholar), by which nuclear export F. Science. PubMed Scopus Google Scholar). this is a the cell the NF-κB transcriptional by the which IκBα is The of this of the cells to as or In this cytoplasmic IκBα is and a of NF-κB is the nucleus to transcription of NF-κB-dependent However, the is used to the F. J. F. D. Mol. Cell. Biol. PubMed Google Scholar, F. P. M. D. J. PubMed Google Scholar). A to to the level of the In this the of for degradation, and of this the to the of of transcription of the and the synthesized protein the nucleus, where it transcriptional contains a NES, and this is used to export the to the cytoplasm the pathway that is used for nuclear export of IκBα. of export that nuclear export of is to the degradation of J. M. T. J. 1998; PubMed Scopus Google Scholar). In the of IκBα and degradation in the in However, the nucleus and the cytoplasm A. S. J. 1996; PubMed Scopus Google Scholar, D. J. Biol. Google Scholar), that nuclear and cytoplasmic The to the cell of to and transcription is that sensitive to and a to The NF-κB 1The abbreviations used are:NF-κB, nuclear factor κB; TNF, tumor necrosis factor; IL-1β, interleukin 1β; LMB, leptomycin B; CX, cycloheximide; NES, nuclear export sequence; aa, amino acid(s); IKK, IκB kinase; SLO, streptolysin O; SUMO-1, small ubiquitin-like modifier 1.1The abbreviations used are:NF-κB, nuclear factor κB; TNF, tumor necrosis factor; IL-1β, interleukin 1β; LMB, leptomycin B; CX, cycloheximide; NES, nuclear export sequence; aa, amino acid(s); IKK, IκB kinase; SLO, streptolysin O; SUMO-1, small ubiquitin-like modifier 1./Rel family of transcription factors is implicated in regulation of the expression of a number of cellular genes involved in immune responses, inflammation ,and apoptosis (for recent reviews, see Refs. 1Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5542) Google Scholar, 2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar, 3May M.J. Ghosh S. Immunol. Today. 1998; 19: 80-88Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar). In vertebrates, the NF-κB family of proteins is composed of transcriptionally active p65/Rel A (4Nolan G.P. Ghosh S. Liou H.C. Tempst P. Baltimore D. Cell. 1991; 64: 961-969Abstract Full Text PDF PubMed Scopus (417) Google Scholar, 5Ruben S.M. Dillon P.J. Schreck R. Henkel T. Chen C.-H. Maher M. Baeuerle P.A. Rosen C.A. Science. 1991; 251: 1490-1493Crossref PubMed Scopus (289) Google Scholar), c-Rel (6Wilhelmsen K.C. Eggleton K. Temin H.M. J. Virol. 1984; 52: 172-182Crossref PubMed Google Scholar), or Rel B (7Ryseck R.P. Bull P. Takamiya M. Bours V. Siebenlist U. Dobrzanski P. Bravo R. Mol. Cell. Biol. 1992; 12: 674-684Crossref PubMed Scopus (274) Google Scholar) and transcriptionally silent p50/NF-κB1 (8Ghosh S. Gifford A.M. Riviere L.R. Tempst P. Nolan G.P. Baltimore D. Cell. 1990; 62: 1019-1029Abstract Full Text PDF PubMed Scopus (588) Google Scholar, 9Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israël A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (601) Google Scholar) or p52/NF-κB2 (10Bours V. Burd P.R. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar, 11Neri A. Chang C.C. Lombardi L. Salina M. Corradini P. Maiolo A.T. Chaganti R.S.K. Dalla-Favera R. Cell. 1991; 67: 1075-1087Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 12Schmid R.M. Perkins N.D. Duckett C.S. Andrews P.C. Nabel G.J. Nature. 1991; 352: 733-736Crossref PubMed Scopus (274) Google Scholar). All NF-κB proteins share a conserved region known as the Rel homology domain that contains the nuclear signal as as the and DNA The NF-κB by is composed of and NF-κB transcriptional is by IκB proteins that of with IκB not the nuclear of and leading to cytoplasmic NF-κB DNA activity. IκBα S. S.M. A. K. P. A.S. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar), U. Baeuerle P.A. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar), A. J. PubMed Scopus Google Scholar), and Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). the of and that in known as V. Kourilsky P. A. J. 1991; PubMed Scopus Google Scholar, J. A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, H.C. Nolan G.P. Ghosh S. T. Baltimore D. J. 1992; PubMed Scopus Google Scholar) and F. J. M. DNA Biol. 1992; PubMed Scopus Google Scholar, F. M. PubMed Scopus Google Scholar), IκBα is in signal a region of and a region a domain that is to the domain by a and is by Mol. Cell. Biol. PubMed Google Scholar). and for the signal-induced degradation of IκBα K. L. L. L. S. Siebenlist U. Mol. Cell. Biol. PubMed Google Scholar, M. M. M.J. A. D. F. PubMed Scopus Google Scholar). signal IκBα is and S.M. Mol. Cell. Biol. PubMed Google Scholar, K. S. L. Siebenlist U. Science. PubMed Scopus Google Scholar, Henkel T. S. Baeuerle P.A. J. 14: PubMed Scopus Google Scholar, J. F. J. Ghosh S. M. Mol. Cell. Biol. 1996; PubMed Google Scholar, M. J. F. F. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) by the IκB M. M. Nature. PubMed Scopus Google Scholar, F. A. M. M. A. A. Science. PubMed Scopus Google Scholar, M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar, M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). IκBα is and Chen T. U. S. A. PubMed Scopus Google Scholar, L. Brown K. Siebenlist U. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. D. F. F. 1996; 12: Google Scholar), which the for degradation by the IκBα is NF-κB to the nucleus, where it induces the transcription of genes that of its inhibitor, IκBα. Newly synthesized IκBα is accumulated in the cytoplasm in the nucleus, where it NF-κB-dependent transcription. This is by inhibition of the and export of NF-κB to the cytoplasm F. J. F. D. Mol. Cell. Biol. PubMed Google Scholar). The of IκBα is by a nuclear export present in its region F. P. M. D. J. PubMed Google Scholar) and to the in proteins the protein and the protein A U. J. R. Cell. Full Text PDF PubMed Scopus Google Scholar, C.C. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). that and to rapid and active export from the nucleus to the The nuclear protein known as as the M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, K. C.S. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). to the family in it homology in the domain with from this family M. J. S. A. D. J. J. PubMed Scopus Google Scholar). The of is by the of in its that this is the nuclear and dissociates in the cytoplasm to by M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In to be the cellular of the leptomycin B that inhibits protein export in and in M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, Biol. Full Text PDF PubMed Scopus Google Scholar). In the present study, LMB was used as a tool to inhibit nuclear protein export and evaluate the consequences for regulation of NF-κB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-κB-dependent transcriptional activation mediated by or This is a consequence of the inhibition of signal-induced degradation of IκBα. Although LMB treatment does not affect the signal transduction pathway leading to IκBα degradation, it blocks IκBα nuclear export. IκBα is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IκBα is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF-κB to be transcriptionally active upon cell stimulation. indicate that inhibition of IκBα nuclear export not the post-induction repression of NF-κB-dependent transcription the activation of NF-κB upon cell stimulation. nuclear IκBα to be resistant to signal-induced phosphorylation and degradation, and this results in nuclear of transcriptionally export of IκBα and proteins that the nucleus and the cytoplasm is mediated by a that is by M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, K. C.S. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In nuclear export of IκBα was by the LMB by its with the F. Science. PubMed Scopus Google Scholar). Although the inhibition of by LMB is it was to the that LMB be with the signal transduction that to NF-κB this used a protein the amino and of IκBα. this protein is in it is to to the nucleus signal-induced degradation in to as and M. M. M.J. A. D. F. PubMed Scopus Google Scholar). degradation of the protein was by LMB that LMB does not inhibit the signal transduction pathway that to IκBα degradation. is that LMB does not inhibit transcription in a the of the was by the of LMB The from is that in signal-induced phosphorylation and degradation of IκBα in the to this is that essential of the signal transduction pathway that to IκBα phosphorylation to the that the IκBα and present in a that as NF-κB P.A. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar, M. M. U. S. A. 1998; PubMed Scopus Google Scholar) and proteins as S. R. F. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). to be if this be the The be that the signal to the nucleus, that the nuclear IκBα is in to to this of IκBα to a that is by the that IκBα is by the small ubiquitin-like protein Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). this protein is to the that used for of IκBα, this the of IκBα resistant to signal-induced degradation. Although the known proteins that for in the nucleus or involved in nuclear M. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar), to the that nuclear IκBα in the of LMB is resistant to signal-induced phosphorylation it is by is that IκBα with a nuclear protein that the region in IκBα and thus it from signal-induced is a for the transcription of essential NF-κB-dependent transcription of genes does not as a consequence of IκBα the of a NF-κB-dependent in S. it that the cell a to for transcription of NF-κB-dependent This the of IκBα, which a of NF-κB that to the in the nucleus, NF-κB NF-κB-dependent that of IκBα. to the IκBα and the IκBα is to the nucleus, where it with NF-κB and dissociates the of the of a in IκBα F. P. M. D. J. PubMed Google Scholar), by which nuclear export F. Science. PubMed Scopus Google Scholar). this is a the cell the NF-κB transcriptional by the which IκBα is The of this of the cells to as or In this cytoplasmic IκBα is and a of NF-κB is the nucleus to transcription of NF-κB-dependent However, the is used to the F. J. F. D. Mol. Cell. Biol. PubMed Google Scholar, F. P. M. D. J. PubMed Google Scholar). A to to the level of the In this the of for degradation, and of this the to the of of transcription of the and the synthesized protein the nucleus, where it transcriptional contains a NES, and this is used to export the to the cytoplasm the pathway that is used for nuclear export of IκBα. of export that nuclear export of is to the degradation of J. M. T. J. 1998; PubMed Scopus Google Scholar). In the of IκBα and degradation in the in However, the nucleus and the cytoplasm A. S. J. 1996; PubMed Scopus Google Scholar, D. J. Biol. Google Scholar), that nuclear and cytoplasmic The to the cell of to and transcription is that sensitive to and a to The indicate that inhibition of IκBα nuclear export not the post-induction repression of NF-κB-dependent transcription the activation of NF-κB upon cell stimulation. nuclear IκBα to be resistant to signal-induced phosphorylation and degradation, and this results in nuclear of transcriptionally export of IκBα and proteins that the nucleus and the cytoplasm is mediated by a that is by M. M. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. T. M. M. M. Nature. PubMed Scopus Google Scholar, F. Science. PubMed Scopus Google Scholar, K. C.S. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In nuclear export of IκBα was by the LMB by its with the F. Science. PubMed Scopus Google Scholar). Although the inhibition of by LMB is it was to the that LMB be with the signal transduction that to NF-κB this used a protein the amino and of IκBα. this protein is in it is to to the nucleus signal-induced degradation in to as and M. M. M.J. A. D. F. PubMed Scopus Google Scholar). degradation of the protein was by LMB that LMB does not inhibit the signal transduction pathway that to IκBα degradation. is that LMB does not inhibit transcription in a the of the was by the of LMB The from is that in signal-induced phosphorylation and degradation of IκBα in the to this is that essential of the signal transduction pathway that to IκBα phosphorylation to the that the IκBα and present in a that as NF-κB P.A. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar, M. M. U. S. A. 1998; PubMed Scopus Google Scholar) and proteins as S. R. F. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). to be if this be the The be that the signal to the nucleus, that the nuclear IκBα is in to to this of IκBα to a that is by the that IκBα is by the small ubiquitin-like protein Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). this protein is to the that used for of IκBα, this the of IκBα resistant to signal-induced degradation. Although the known proteins that for in the nucleus or involved in nuclear M. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar), to the that nuclear IκBα in the of LMB is resistant to signal-induced phosphorylation it is by is that IκBα with a nuclear protein that the region in IκBα and thus it from signal-induced In is a for the transcription of essential NF-κB-dependent transcription of genes does not as a consequence of IκBα the of a NF-κB-dependent in S. it that the cell a to for transcription of NF-κB-dependent This the of IκBα, which a of NF-κB that to the in the nucleus, NF-κB NF-κB-dependent that of IκBα. to the IκBα and the IκBα is to the nucleus, where it with NF-κB and dissociates the of the of a in IκBα F. P. M. D. J. PubMed Google Scholar), by which nuclear export F. Science. PubMed Scopus Google Scholar). this is a the cell the NF-κB transcriptional by the which IκBα is The of this of the cells to as or In this cytoplasmic IκBα is and a of NF-κB is the nucleus to transcription of NF-κB-dependent However, the is used to the F. J. F. D. Mol. Cell. Biol. PubMed Google Scholar, F. P. M. D. J. PubMed Google Scholar). A to to the level of the In this the of for degradation, and of this the to the of of transcription of the and the synthesized protein the nucleus, where it transcriptional contains a NES, and this is used to export the to the cytoplasm the pathway that is used for nuclear export of IκBα. of export that nuclear export of is to the degradation of J. M. T. J. 1998; PubMed Scopus Google Scholar). In the of IκBα and degradation in the in However, the nucleus and the cytoplasm A. S. J. 1996; PubMed Scopus Google Scholar, D. J. Biol. Google Scholar), that nuclear and cytoplasmic The to the cell of to and transcription is that sensitive to and a to for IκB for with the S. for the SLO, and for the

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.

Agonist-activated Ca2+ entry plays a critical role in Ca2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca2+ release-activated Ca2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.

Recent studies have indicated a critical role for STIM (stromal interacting molecule) proteins in the regulation of the store-operated mode of receptor-activated Ca2+ entry. Current models emphasize the role of STIM located in the endoplasmic reticulum membrane, where a Ca2+-binding EF-hand domain within the N-terminal of the protein lies within the lumen and is thought to represent the sensor for the depletion of intracellular Ca2+ stores. Dissociation of Ca2+ from this domain induces the aggregation of STIM to regions of the ER immediately adjacent to the plasma membrane where it acts to regulate the activity of store-operated Ca2+ channels. However, the possible effects of STIM on other modes of receptor-activated Ca2+ entry have not been examined. Here we show that STIM1 also regulates the arachidonic-acid-regulated Ca2+-selective (ARC) channels - receptor-activated Ca2+ entry channels whose activation is entirely independent of store depletion. Regulation of the ARC channels by STIM1 does not involve dissociation of Ca2+ from the EF-hand, or any translocation of STIM1. Instead, a critical role of STIM1 resident in the plasma membrane is indicated. Thus, exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1 inhibits ARC channel activity without significantly affecting the store-operated channels. A similar specific inhibition of the ARC channels is seen in cells expressing a STIM1 construct in which the N-linked glycosylation sites essential for the constitutive cell surface expression of STIM1, were mutated. We conclude that, in contrast to store-operated channels, regulation of ARC channels by STIM1 depends exclusively on the pool of STIM1 constitutively residing in the plasma membrane. These data demonstrate that STIM1 is a more universal regulator of Ca2+ entry pathways than previously thought, and appears to have multiple modes of action.