In nmrine plasmacytanas, the c-rtyc gene has frequently been fourd to undergo rearrangement by vi... more In nmrine plasmacytanas, the c-rtyc gene has frequently been fourd to undergo rearrangement by virtue of a T(12;15) chrcarDsce translocation. The inrunoglobulin heavy chain gene switch region (Sa ) constitutes the target for most of these recarbinations particularly in IgA producing plasmacytanas. We sought to identify non-S myc target sites in several IgG producing tumors. The c-iyc target in MPC-f1 (a BALB/c IgG producing plasmacyta) has been cloned, localized to the Igh-C locus and ?iLntified as the Y2a heavy chain gene switch region (S 2a* Furthennore, by Southern blot hybridization, we have detemined that eaS region is the c-myc target in two NZB IgG producing plasmacytanas. e potential relation between Ig class expres ;d and c-nyc translocation target is discussed.
We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha b... more We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha beta+ murine T cell line have undergone secondary V alpha-J alpha rearrangements at the TCR-alpha locus (1). In an effort to examine the molecular mechanism responsible for these V alpha-J alpha replacements, the structures of TCR-alpha cDNA prepared from both the parental and subcloned T cell lines have been determined. Here we report that: 1) the mechanism whereby the secondary rearrangements occur is a precise deletion event that involves germ-line V alpha genes 5' to the preexisting V alpha-J alpha complex joining to J alpha segments 3' of the preexisting complex deleting the region in between, 2) preexisting productive V alpha-J alpha rearrangements of the parental line do not allelically exclude productive and nonproductive secondary rearrangements, 3) both productively rearranged TCR-alpha alleles of the parental cell line can undergo secondary rearrangements, 4) the presen...
M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antige... more M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombinat...
Osteoarthritis (OA) is a multifactorial disease subject to the effects of many genes and environm... more Osteoarthritis (OA) is a multifactorial disease subject to the effects of many genes and environmental factors. Alterations in the normal pattern of chondrocyte gene control in cartilage facilitate the onset and progression of OA. Stable changes in patterns of gene expression, not associated with alterations in DNA sequences, occur through epigenetic changes, including DNA methyla-tion, histone modifications, and alterations in chromatin structure, as well as by microRNA (miRNA)-mediated mechanisms. Moreover, the ability of the host to repair damaged cartilage is reflected in alterations in gene control circuits, suggestive of an epigenetic and miRNA-dependent tug-of-war between tissue homeo-stasis and OA disease pathogenesis. Herein, we summa-rize epigenetic and miRNA-mediated mechanisms impacting on OA progression and in this context offer potential therapeutic strategies for OA treatment. What is OA disease and how does it develop? Idiopathic osteoarthritis (OA) is a late-onset, ...
In plasmacytoma cells producing IgG, IgA, or IgM immunoglobulin heavy chains, the large precursor... more In plasmacytoma cells producing IgG, IgA, or IgM immunoglobulin heavy chains, the large precursors of the heavy chain messenger RNA's contain nucleotide sequences that specify only the expressed class of constant region. This indicates that the switch from one class of heavy chain to another during B cell ontogeny does not occur by altered processing of a complex gene transcript.
Proceedings of the National Academy of Sciences, 1978
The chromosomal locations of the structural genes coding for the constant portions of mouse heavy... more The chromosomal locations of the structural genes coding for the constant portions of mouse heavy (H) and light chain immunoglobulins were studied by molecular hybridization techniques. Complementary DNA probes containing the constant-region sequences of kappa and lambdaI light chain and alpha, gamma2b, and mu heavy chain mRNAs were annealed to a large excess of DNA from a series of eight mouse-human hybrid cell lines that are deficient for various mouse chromosomes. The lines were scored as positive when a high proportion of a probe annealed and negative when an insignificant proportion annealed. Some lines were clearly negative for H and lambda and clearly positive for kappa. Others were positive or intermediate for lambda, positive for kappa and negative for H. Still others, including a line that was selected for the absence of the mouse X chromosome, were positive for all immunoglobulin species. These results demonstrate that the Clambda, Ckappa, and CH genes are located on different autosomes in the mouse. In contrast, the three heavy-chain families exhibited consistently uniform hybridization results, suggesting that the genes for Calpha, Cgamma, and Cmu are located on the same chromosome. A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes.
... Nature 261, 159 - 162 (13 May 1976); doi:10.1038/261159a0. Effect of ribothymidine in specifi... more ... Nature 261, 159 - 162 (13 May 1976); doi:10.1038/261159a0. Effect of ribothymidine in specific eukaryotic tRNAs on their efficiency in in vitro protein synthesis. KENNETH B. MARCU * & BERNARD S. DUDOCK. ... 5 and KBM, D. Marcu, and BSD, manuscript in preparation). ...
Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by som... more Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10 −3 of yeast cells survived low stringency selection and expressed β-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.
The nuclear precursors of the immunoglobulin messenger RNAs of MPC-11 cells were characterized wi... more The nuclear precursors of the immunoglobulin messenger RNAs of MPC-11 cells were characterized with respect to size, amount per cell and extent of polyadenylation. These cells produce three Ig mRNAs: a 1.8 kb component coding for a gamma2b heavy chain (H mRNA), a 1.2 kb mRNA coding for a k light chain (L mRNA) and a 0.8 kb mRNA coding for the constant region portion of the k light chain (Lf mRNA). To identify the pre-mRNAs without ambiguity, we constructed recombinant DNA plasmids containing H and L cDNA sequences, and used the cloned cDNAs as hybridization probes for analysis of steady state nuclear RNA and in DNA excess hybridization experiments with pulse-labeled nuclear RNA. The nuclear molecules containing Ig sequences consist of an 11 kb component (H1), which we believe to be the primary transcript of the H gene, 5.3 kb (L1), and 3.3 kb (L2) components, which seem to be primary transcripts of the L and L1 genes, components corresponding to mature size H, L and Lf mRNAs, and several intermediate-sized components which include the processing derivatives. The precursor role of these nuclear molecules was established by studies of their labeling kinetics and by appropriate pulse-chase experiments. All the pre-mRNA species including H1, L1 and L2 contain poly(A), thus suggesting that polyadenylation is an early event in the processing of these mRNAs. The MPC-11 cell contains about 30,000 and 40,000 cytoplasmic H and L mRNA molecules, respectively, which must be produced within one cell generation (approximately 24 hr). In comparison, the nucleus contains about 100-150 molecules of total pre-mRNA and only about 10-15 molecules of presumptive primary transcripts for each of these Ig species. These values indicate very rapid transcription rates (greater than 20 transcripts per min) and exceptionally fast processing rates (approximately 0.5 min for the primary transcripts and approximately 5 min for overall nuclear processing) for the Ig mRNAs. Thus rapid transcription and processing, together with high cytoplasmic stability, account for the high abundance of Ig mRNAs in the myeloma cell.
... to dimethylsulfoxide (DMSO) but early molecular events associated with inducer-mediatedcommit... more ... to dimethylsulfoxide (DMSO) but early molecular events associated with inducer-mediatedcommitment re-main ... Cell specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth ... 19 ZULLO, J. N., COCHRAN, B. H., HUANG, A. S. & STILES, CD (1986 ...
Methods. MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction ... more Methods. MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and -catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage.
Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alph... more Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-␣), induces nuclear NF-B expression. TNF-␣ signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-␣ receptor tail, leading to the sequential activation of the downstream NF-B-inducing kinase (NIK) and IB-specific kinases (IKK␣ and IKK). When activated, IKK␣ and IKK directly phosphorylate the two N-terminal regulatory serines within IB␣, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-B complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKK␣. Such a NIK-T559A mutant also dominantly interferes with TNF-␣ induction of NF-B. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKK␣; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-␣-mediated induction of NF-B and alone is sufficient to disrupt the physical association of NIK and IKK␣. Together, these findings provide new insights into the molecular basis for TNF-␣ signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.
Page 1. Volume 5 Number 9 September 1978 Nucleic Acids Research A characterization of mRNA activi... more Page 1. Volume 5 Number 9 September 1978 Nucleic Acids Research A characterization of mRNA activities and their sequence complexities in Trypanosoma brucei: partial purification and properties of the VSSA mRNA Richard ...
Five novel murine plasma cell (PC) tumors with chromosome translocations 350-500 bp 5' of the fir... more Five novel murine plasma cell (PC) tumors with chromosome translocations 350-500 bp 5' of the first c-myc exon are described. The t(12;15)s of TEPC 1194, ABPC 33 and TEPC 1165 position the intact c-myc locus 5' of the CA, C72a and C, IgCH genes resepctively. In ABPC 17, the IgH enhancer element and adjacent switch (S) sequences were found 5' of the first c-myc exon while this enhancer is associated with the reciprocal products of the TEPC 1194, ABPC 33 and TEPC 1033 translocations. Quantitative Si nuclease analyses demonstrate that the ratios of transcription from the two c-myc promoters (P1 and P2) are increased 4to 7-fold in these five tumors. With the exception of TEPC 1165, (which contains a small deletion in exon 1), such increases in P1:P2 ratios appear to be manifested by a reduction in P2 usage in comparison to other tumors without such promoter shifts. A survey of 27 additional PC and non-PC B lymphoid tumors and cell lines revealed that myc promoter shifts of this magnitude are unique to PC tumors with 5'-proximal translocations. We propose that (i) these clustered breakpoints identify a normal c-myc regulatory element located at least 350 bp 5' of c-myc exon 1; (ii) the loss or disruption of this cis-acting upstream element and the linkage of c-myc to the IgCH locus would result in abnormal expression of this oncogene in these as well as most other PC tumors.
In nmrine plasmacytanas, the c-rtyc gene has frequently been fourd to undergo rearrangement by vi... more In nmrine plasmacytanas, the c-rtyc gene has frequently been fourd to undergo rearrangement by virtue of a T(12;15) chrcarDsce translocation. The inrunoglobulin heavy chain gene switch region (Sa ) constitutes the target for most of these recarbinations particularly in IgA producing plasmacytanas. We sought to identify non-S myc target sites in several IgG producing tumors. The c-iyc target in MPC-f1 (a BALB/c IgG producing plasmacyta) has been cloned, localized to the Igh-C locus and ?iLntified as the Y2a heavy chain gene switch region (S 2a* Furthennore, by Southern blot hybridization, we have detemined that eaS region is the c-myc target in two NZB IgG producing plasmacytanas. e potential relation between Ig class expres ;d and c-nyc translocation target is discussed.
We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha b... more We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha beta+ murine T cell line have undergone secondary V alpha-J alpha rearrangements at the TCR-alpha locus (1). In an effort to examine the molecular mechanism responsible for these V alpha-J alpha replacements, the structures of TCR-alpha cDNA prepared from both the parental and subcloned T cell lines have been determined. Here we report that: 1) the mechanism whereby the secondary rearrangements occur is a precise deletion event that involves germ-line V alpha genes 5' to the preexisting V alpha-J alpha complex joining to J alpha segments 3' of the preexisting complex deleting the region in between, 2) preexisting productive V alpha-J alpha rearrangements of the parental line do not allelically exclude productive and nonproductive secondary rearrangements, 3) both productively rearranged TCR-alpha alleles of the parental cell line can undergo secondary rearrangements, 4) the presen...
M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antige... more M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombinat...
Osteoarthritis (OA) is a multifactorial disease subject to the effects of many genes and environm... more Osteoarthritis (OA) is a multifactorial disease subject to the effects of many genes and environmental factors. Alterations in the normal pattern of chondrocyte gene control in cartilage facilitate the onset and progression of OA. Stable changes in patterns of gene expression, not associated with alterations in DNA sequences, occur through epigenetic changes, including DNA methyla-tion, histone modifications, and alterations in chromatin structure, as well as by microRNA (miRNA)-mediated mechanisms. Moreover, the ability of the host to repair damaged cartilage is reflected in alterations in gene control circuits, suggestive of an epigenetic and miRNA-dependent tug-of-war between tissue homeo-stasis and OA disease pathogenesis. Herein, we summa-rize epigenetic and miRNA-mediated mechanisms impacting on OA progression and in this context offer potential therapeutic strategies for OA treatment. What is OA disease and how does it develop? Idiopathic osteoarthritis (OA) is a late-onset, ...
In plasmacytoma cells producing IgG, IgA, or IgM immunoglobulin heavy chains, the large precursor... more In plasmacytoma cells producing IgG, IgA, or IgM immunoglobulin heavy chains, the large precursors of the heavy chain messenger RNA's contain nucleotide sequences that specify only the expressed class of constant region. This indicates that the switch from one class of heavy chain to another during B cell ontogeny does not occur by altered processing of a complex gene transcript.
Proceedings of the National Academy of Sciences, 1978
The chromosomal locations of the structural genes coding for the constant portions of mouse heavy... more The chromosomal locations of the structural genes coding for the constant portions of mouse heavy (H) and light chain immunoglobulins were studied by molecular hybridization techniques. Complementary DNA probes containing the constant-region sequences of kappa and lambdaI light chain and alpha, gamma2b, and mu heavy chain mRNAs were annealed to a large excess of DNA from a series of eight mouse-human hybrid cell lines that are deficient for various mouse chromosomes. The lines were scored as positive when a high proportion of a probe annealed and negative when an insignificant proportion annealed. Some lines were clearly negative for H and lambda and clearly positive for kappa. Others were positive or intermediate for lambda, positive for kappa and negative for H. Still others, including a line that was selected for the absence of the mouse X chromosome, were positive for all immunoglobulin species. These results demonstrate that the Clambda, Ckappa, and CH genes are located on different autosomes in the mouse. In contrast, the three heavy-chain families exhibited consistently uniform hybridization results, suggesting that the genes for Calpha, Cgamma, and Cmu are located on the same chromosome. A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes.
... Nature 261, 159 - 162 (13 May 1976); doi:10.1038/261159a0. Effect of ribothymidine in specifi... more ... Nature 261, 159 - 162 (13 May 1976); doi:10.1038/261159a0. Effect of ribothymidine in specific eukaryotic tRNAs on their efficiency in in vitro protein synthesis. KENNETH B. MARCU * & BERNARD S. DUDOCK. ... 5 and KBM, D. Marcu, and BSD, manuscript in preparation). ...
Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by som... more Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10 −3 of yeast cells survived low stringency selection and expressed β-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.
The nuclear precursors of the immunoglobulin messenger RNAs of MPC-11 cells were characterized wi... more The nuclear precursors of the immunoglobulin messenger RNAs of MPC-11 cells were characterized with respect to size, amount per cell and extent of polyadenylation. These cells produce three Ig mRNAs: a 1.8 kb component coding for a gamma2b heavy chain (H mRNA), a 1.2 kb mRNA coding for a k light chain (L mRNA) and a 0.8 kb mRNA coding for the constant region portion of the k light chain (Lf mRNA). To identify the pre-mRNAs without ambiguity, we constructed recombinant DNA plasmids containing H and L cDNA sequences, and used the cloned cDNAs as hybridization probes for analysis of steady state nuclear RNA and in DNA excess hybridization experiments with pulse-labeled nuclear RNA. The nuclear molecules containing Ig sequences consist of an 11 kb component (H1), which we believe to be the primary transcript of the H gene, 5.3 kb (L1), and 3.3 kb (L2) components, which seem to be primary transcripts of the L and L1 genes, components corresponding to mature size H, L and Lf mRNAs, and several intermediate-sized components which include the processing derivatives. The precursor role of these nuclear molecules was established by studies of their labeling kinetics and by appropriate pulse-chase experiments. All the pre-mRNA species including H1, L1 and L2 contain poly(A), thus suggesting that polyadenylation is an early event in the processing of these mRNAs. The MPC-11 cell contains about 30,000 and 40,000 cytoplasmic H and L mRNA molecules, respectively, which must be produced within one cell generation (approximately 24 hr). In comparison, the nucleus contains about 100-150 molecules of total pre-mRNA and only about 10-15 molecules of presumptive primary transcripts for each of these Ig species. These values indicate very rapid transcription rates (greater than 20 transcripts per min) and exceptionally fast processing rates (approximately 0.5 min for the primary transcripts and approximately 5 min for overall nuclear processing) for the Ig mRNAs. Thus rapid transcription and processing, together with high cytoplasmic stability, account for the high abundance of Ig mRNAs in the myeloma cell.
... to dimethylsulfoxide (DMSO) but early molecular events associated with inducer-mediatedcommit... more ... to dimethylsulfoxide (DMSO) but early molecular events associated with inducer-mediatedcommitment re-main ... Cell specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth ... 19 ZULLO, J. N., COCHRAN, B. H., HUANG, A. S. & STILES, CD (1986 ...
Methods. MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction ... more Methods. MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and -catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage.
Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alph... more Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-␣), induces nuclear NF-B expression. TNF-␣ signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-␣ receptor tail, leading to the sequential activation of the downstream NF-B-inducing kinase (NIK) and IB-specific kinases (IKK␣ and IKK). When activated, IKK␣ and IKK directly phosphorylate the two N-terminal regulatory serines within IB␣, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-B complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKK␣. Such a NIK-T559A mutant also dominantly interferes with TNF-␣ induction of NF-B. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKK␣; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-␣-mediated induction of NF-B and alone is sufficient to disrupt the physical association of NIK and IKK␣. Together, these findings provide new insights into the molecular basis for TNF-␣ signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.
Page 1. Volume 5 Number 9 September 1978 Nucleic Acids Research A characterization of mRNA activi... more Page 1. Volume 5 Number 9 September 1978 Nucleic Acids Research A characterization of mRNA activities and their sequence complexities in Trypanosoma brucei: partial purification and properties of the VSSA mRNA Richard ...
Five novel murine plasma cell (PC) tumors with chromosome translocations 350-500 bp 5' of the fir... more Five novel murine plasma cell (PC) tumors with chromosome translocations 350-500 bp 5' of the first c-myc exon are described. The t(12;15)s of TEPC 1194, ABPC 33 and TEPC 1165 position the intact c-myc locus 5' of the CA, C72a and C, IgCH genes resepctively. In ABPC 17, the IgH enhancer element and adjacent switch (S) sequences were found 5' of the first c-myc exon while this enhancer is associated with the reciprocal products of the TEPC 1194, ABPC 33 and TEPC 1033 translocations. Quantitative Si nuclease analyses demonstrate that the ratios of transcription from the two c-myc promoters (P1 and P2) are increased 4to 7-fold in these five tumors. With the exception of TEPC 1165, (which contains a small deletion in exon 1), such increases in P1:P2 ratios appear to be manifested by a reduction in P2 usage in comparison to other tumors without such promoter shifts. A survey of 27 additional PC and non-PC B lymphoid tumors and cell lines revealed that myc promoter shifts of this magnitude are unique to PC tumors with 5'-proximal translocations. We propose that (i) these clustered breakpoints identify a normal c-myc regulatory element located at least 350 bp 5' of c-myc exon 1; (ii) the loss or disruption of this cis-acting upstream element and the linkage of c-myc to the IgCH locus would result in abnormal expression of this oncogene in these as well as most other PC tumors.
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Papers by Kenneth Marcu