FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2160628268> ?p ?o ?g. }

• W2160628268 endingPage "29044" @default.
• W2160628268 startingPage "29037" @default.
• W2160628268 abstract "Interleukin-1 (IL-1) is a pleiotropic cytokine essential for initiation of the immune response to infections and stress. IL-1 interacts with its type I receptor (IL-1RI) and triggers a number of intracellular signaling cascades leading to activation of transcription factors, transcriptional up-regulation of target genes, and mRNA stabilization. IL-1RI-associated kinase-1 (IRAK1) is a membrane proximal serine-threonine kinase involved in IL-1 signaling that becomes phosphorylated and progressively degraded in response to IL-1 induction. We have identified a novel variant of IRAK1, which we have named IRAK1b, that arises from the use of an alternative 5′-acceptor splice site defined by sequence within exon 12 of IRAK1. IRAK1b mRNA exhibits wide tissue expression and is evolutionarily conserved in both mouse and human. IRAK1b can activate the transcription factor nuclear factor κB and interacts with the IL-1 signaling factors Toll-interacting protein and tumor necrosis factor receptor-associated factor 6. It forms homodimers and heterodimers with the previously described isoform of IRAK1. We show that the IRAK1b protein is kinase-inactive and that, unlike IRAK1, its levels remain constant after IL-1 induction. The presence of an alternative splice variant of IRAK1, which is functionally active and highly stable following IL-1 stimulation, adds further complexity to the control mechanisms that govern IL-1 signaling. Interleukin-1 (IL-1) is a pleiotropic cytokine essential for initiation of the immune response to infections and stress. IL-1 interacts with its type I receptor (IL-1RI) and triggers a number of intracellular signaling cascades leading to activation of transcription factors, transcriptional up-regulation of target genes, and mRNA stabilization. IL-1RI-associated kinase-1 (IRAK1) is a membrane proximal serine-threonine kinase involved in IL-1 signaling that becomes phosphorylated and progressively degraded in response to IL-1 induction. We have identified a novel variant of IRAK1, which we have named IRAK1b, that arises from the use of an alternative 5′-acceptor splice site defined by sequence within exon 12 of IRAK1. IRAK1b mRNA exhibits wide tissue expression and is evolutionarily conserved in both mouse and human. IRAK1b can activate the transcription factor nuclear factor κB and interacts with the IL-1 signaling factors Toll-interacting protein and tumor necrosis factor receptor-associated factor 6. It forms homodimers and heterodimers with the previously described isoform of IRAK1. We show that the IRAK1b protein is kinase-inactive and that, unlike IRAK1, its levels remain constant after IL-1 induction. The presence of an alternative splice variant of IRAK1, which is functionally active and highly stable following IL-1 stimulation, adds further complexity to the control mechanisms that govern IL-1 signaling. interleukin-1 nuclear factor κB activator protein-1 IL-1 receptor type I IL-1RI accessory protein myeloid differentiation factor IL-1RI-associated kinase tumor necrosis factor TNF receptor-associated factor 6 transforming growth factor-β-activated kinase 1 TAK1 binding protein inhibitor of NF-κB Toll-interacting protein kinase-inactive IRAK1a polyacrylamide gel electrophoresis bacterial alkaline phosphatase myelin basic protein reverse transcription-polymerase chain reaction cytomegalovirus phosphate-buffered saline base pair(s) kilobase(s) Infections, tissue injury, and/or stress trigger monocytes and macrophages to produce interleukin-1 (IL-1),1 the cytokine that orchestrates much of the systemic acute phase response, the net effect of which is to neutralize the underlying physiological challenge (reviewed in Ref. 1Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). Systemically, IL-1 stimulates fever, vasodilation, and muscle contractions; at the cellular level it induces cells to adopt an enhanced “host defense” phenotype by eliciting radical changes in their protein expression profiles by modulating mRNA processing and stability, protein translation, and gene transcription (1Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). The latter is achieved by activating transcription factors, e.g. nuclear factor κB (NF-κB) and activator protein-1 (AP-1). Signaling by IL-1 depends on its engagement of the transmembrane IL-1 receptor type I (IL-1RI) and IL-1RI accessory protein (IL-1RAcP) (Ref. 2Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Crossref PubMed Scopus (563) Google Scholar and references therein). Binding of IL-1 to the extracellular domains of IL-1RI and IL-1RAcP is followed by recruitment of the intracellular adapter protein myeloid differentiation factor (MyD88) (3Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (987) Google Scholar, 4Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar, 5Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 6Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar) and a number of kinases to the evolving IL-1·IL-1RI·IL-1RAcP complex. Depending on cell type, these kinases may include IL-1RI-associated kinase-1 (IRAK1), which is expressed in all tissues (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar), IRAK2, which has a narrower cellular distribution (3Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (987) Google Scholar), and IRAK-M, which is mainly restricted to cells of myeloid origin (8Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Phosphatidylinositol 3-kinase has also been implicated as an early component of the IL-1 signaling cascade (9Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). Once established, the cascade progresses through the stepwise activation/recruitment of several additional intermediates, including tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), TRAF6 binding protein, transforming growth factor-β-activated kinase 1 (TAK1), two TAK1 binding proteins (TAB1 and TAB2), and NF-κB-inducing kinase (10Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1123) Google Scholar, 11Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 12Ling L. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9567-9572Crossref PubMed Scopus (53) Google Scholar, 13Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 14Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, 15Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1164) Google Scholar). Finally, the inhibitor of NF-κB (IκB) is phosphorylated by the resulting IκB kinase complex and degraded. This allows NF-κB to translocate to the nucleus where it activates transcription of a wide range of genes that are important for immune function and inflammation (16O'Neill L.A. Greene C. J. Leukocyte. Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar). The branches of the IL-1 signaling pathway that leads to activation of AP-1 and mRNA stabilization may diverge from the NF-κB-activating branch of the cascade at TAK1/TAB1 (Ref. 17Holtmann H. Enninga J. Kälble S. Thiefes A. Dörrie A. Broemer M. Winzen R. Wilhelm A. Ninomiya-Tsuji J. Matsumoto K. Resch K. Kracht M. J. Biol. Chem. 2001; 276: 3508-3516Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar and references therein). However, other studies suggest that the pathways diverge at IRAK1 or even earlier (18Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (144) Google Scholar, 19Greene C. O'Neill L. Biochim. Biophys. Acta. 1999; 1451: 109-121Crossref PubMed Scopus (18) Google Scholar). IRAK1 is a protein of 714 amino acids that has two known functional domains: an N-terminal death domain, which is involved in protein·protein interactions with MyD88 and Toll-interacting protein (Tollip), and a centrally positioned Ser/Thr kinase domain (3Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (987) Google Scholar, 5Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 6Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar,20Trofimova M. Sprenkle A.B. Green M. Sturgill T.W. Goebl M.G. Harrington M.A. J. Biol. Chem. 1996; 271: 17609-17612Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Several studies have shown that the kinase activity is not necessary for IRAK1 to be functional (21Knop J. Martin M.U. FEBS Lett. 1999; 448: 81-85Crossref PubMed Scopus (90) Google Scholar, 22Maschera B. Ray K. Burns K. Volpe F. Biochem. J. 1999; 339: 227-231Crossref PubMed Scopus (79) Google Scholar, 23Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). In un-stimulated cells IRAK1 is associated with Tollip; however, following stimulation with IL-1, its recruitment to the IL-1·IL-1RI·IL-1RAcP complex is facilitated by the interaction between Tollip and IL-1RAcP (24Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google Scholar). IRAK1 becomes phosphorylated, dissociates from Tollip, and is degraded by proteasomes (24Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google Scholar, 25Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Crossref PubMed Scopus (245) Google Scholar). Studies to date indicate that the phosphorylation of IRAK1 is mediated by IRAK1 itself (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, 20Trofimova M. Sprenkle A.B. Green M. Sturgill T.W. Goebl M.G. Harrington M.A. J. Biol. Chem. 1996; 271: 17609-17612Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), although it remains a possibility that additional kinases are also involved (23Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). It has furthermore been suggested, but not yet established, that IL-1-mediated degradation of IRAK1 may be the mechanism whereby cells become desensitized after prolonged exposure to IL-1 (25Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Crossref PubMed Scopus (245) Google Scholar). Because IRAK1 is also involved in the signaling pathway from Toll receptors induced by lipopolysaccharide (see Ref. 26Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar for additional references), it has also been suggested that a similar mechanism plays a role in the desensitization of monocytes and neutrophils to bacterial lipopolysaccharide, which is a feature of sepsis (26Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Alternative splicing is one of the mechanisms whereby an increased complexity in the number of functionally distinct protein products may be generated from a fixed pool of genes in the genome (27Black D.L. Cell. 2000; 103: 367-370Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). We have identified a novel alternatively spliced variant of IRAK1 mRNA. It encodes an IRAK1 protein that has functional activity similar to that of the previously described IRAK1 isoform but is biologically distinct in that it is relatively much more stable after IL-1 activation. The existence of alternative forms of IRAK1 with very different capacities to support IL-1 signaling over time has important implications for the initiation and maintenance of inflammatory processes. Human hepatoma cells (HepG2) and human embryonic kidney cells (293) were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium with 25 mm HEPES and glutamax-1 (l-alanyl-l-glutamine) supplemented with 10% (v/v) fetal calf serum, 1 mm sodium pyruvate, 0.01 mm nonessential amino acids, and 50 µg/ml gentamicin (Life Technologies, Inc., Grand Island, NY). For protein harvesting 106 cells were used per data point. For transfection experiments 2.5 × 105 cells were used per data point. Treatments were performed in duplicate or triplicate. All experiments were repeated at least twice. For transfection experiments, cells were grown to ∼50% confluence and transfected with 2–3 µg of total DNA using FuGene6 (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. In experiments where cells were transfected with varying amounts of expression vectors, the total DNA used in each transfection was held constant by co-transfecting appropriate amounts of empty vector. Cells were treated with medium (control) or IL-1β (10 ng/ml, National Cancer Institute, Frederick, MD) for 4 h. For luciferase reporter assays, cells were lysed and lysates were assayed for luciferase andRenilla-luciferase activity according to the manufacturer's instructions (Dual-Luciferase Reporter Assay System, Promega, Madison, WI). Human tissue total RNA was obtained from CLONTECH Laboratories (Palo Alto, CA). Mouse total RNA was extracted using RNeasy (Qiagen, Santa Clarita, CA) according to the manufacturer's instructions. RNA from tissue culture cells was extracted as described elsewhere (28Jensen L.E. Whitehead A.S. J. Immunol. Meth. 1998; 215: 45-58Crossref PubMed Scopus (21) Google Scholar). Reverse transcription of 1 µg of total RNA was performed at 42 °C using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI), oligo-(dN)6 primer (Amersham Pharmacia Biotech, Piscataway, NJ) and ANTI-RNase RNase inhibitor (Ambion Inc., Austin, TX). Proportional quantitative RT-PCR was performed as described elsewhere (28Jensen L.E. Whitehead A.S. J. Immunol. Meth. 1998; 215: 45-58Crossref PubMed Scopus (21) Google Scholar) using the GC-RICH PCR system (Roche Molecular Biochemicals) with forward (5′-AAAGGAGGCCTCCTATGACC-3′) and reverse (5′-ATGATGCAGAGCTG-3′) primers based on sequence from GenBank™ accession number L76191. PCR products were separated in agarose gels and visualized after ethidium-bromide staining. Mouse cDNAs were amplified with forward (5′-AGAAGAGGCCCCCCATGACC-3′) and reverse (5′-CAGGGATGAGCTGCCCGTGG-3′) primers based on sequence from GenBank™ accession number U56773. TheRenilla-luciferase and NF-κB-responsive luciferase reporter constructs are described elsewhere (29Jensen L.E. Muzio M. Mantovani A. Whitehead A.S. J. Immunol. 2000; 164: 5277-5286Crossref PubMed Scopus (105) Google Scholar). cDNAs were amplified using the GC-RICH PCR system according to the manufacturer's instructions. Primers for amplification of the coding sequence of IRAK1 were derived from GenBank™ accession number L76191. The coding sequences of IRAK1 variants were cloned into the mammalian expression vectors pCI-Neo (Promega), pcDNA4/HisMax (Invitrogen, Carlsbad, CA), and p3XFLAG-CMV-14 (Sigma Chemical Co.). A kinase-inactive IRAK1a (kiIRAK1a) clone was generated containing two point mutations (E248A and I326V) in the kinase domain. IRAK1a mutants IRAK1a(S536A), IRAK1a(S541A), IRAK1a(S536A, S541A), IRAK1a(S536A,S541A,Y515F), IRAK1a(K520A), and IRAK1a(K520A,S536A,S541A) were generated using overlapping primers containing the appropriate point mutations and PCR amplification of fragments of the IRAK1a coding sequence. PCR products were cloned into previously generated IRAK1a expression constructs. Primers for amplification of Tollip and TRAF6 coding sequences were derived from GenBank™ accession numbersAJ242972 and U78798, respectively. cDNAs were cloned into pcDNA4/HisMax. The expression vector p3xFLAG-CMV-BAP encoding FLAG-tagged bacterial alkaline phosphatase (BAP) was obtained from Sigma. Total proteins were extracted on ice in 50 mm HEPES, 150 mmNaCl, 20 mm β-glycerophosphate, 1 mm EDTA, 1 mm benzamidine, 50 mm NaF, and 1 mmNa3VO4, 5 mm para-nitrophenyl phosphate, 2 mm dithiothreitol, 10% (v/v) Protease Inhibitor Mixture (Sigma, St. Louis, MO), 1% Nonidet P-40. Cellular debris was removed by centrifugation at 10,000 × g for 10 min. Total protein extracts were separated in 6% SDS-polyacrylamide gel electrophoresis (PAGE) gels and transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech). Excess protein binding sites were blocked with 5% nonfat milk in PBS. Immunodetection of IRAK1 was performed using polyclonal rabbit anti-IRAK1 (IRAK-1 (H273), Santa Cruz Biotechnology, Santa Cruz, CA), horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Santa Cruz Biotechnology), and enhanced chemiluminescence (ECL Western blotting detection reagents, Amersham Pharmacia Biotech). His-tagged proteins were detected using polyclonal rabbit anti-His-probe-(H-15) (Santa Cruz Biotechnology). FLAG-tagged proteins were immunoprecipitated with ANTI-FLAG M2 (Sigma) monoclonal antibodies and agarose-conjugated protein A (Life Technologies, Inc.) at 4 °C for 4 h. IRAK-1 (H273) and polyclonal rabbit anti-TRAF6 (TRAF6 (H274), Santa Cruz Biotechnology) were used for IRAK1- and TRAF6-specific immunoprecipitations, respectively. Beads were washed four times with lysis buffer. Beads were resuspended in SDS-PAGE sample buffer, and proteins were separated in NuPAGE 4–12% Bis-Tris gels (SDS-PAGE, Invitrogen). Transfected cell monolayers were rinsed twice with PBS and incubated with methionine-deficient Dulbecco's modified Eagle's medium for 1 h prior to addition of 15 µCi of [35S]methionine (Amersham Pharmacia Biotech). After labeling for 1 h, medium was removed and cells were rinsed twice with PBS prior to the addition of methionine-sufficient Dulbecco's modified Eagle's medium with or without 10 ng/ml IL-1β. At appropriate times cells were lysed in the presence of 50 mm4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone (NLVS, proteasome inhibitor, Calbiochem, San Diego, CA), and proteins were immunoprecipitated with ANTI-FLAG M2 as described above. Following SDS-PAGE and transfer to nitrocellulose, 35S-labeled proteins were quantified using STORM and ImageQuaNT technologies (Molecular Dynamics Inc., Sunnyvale, CA). Washed immunoprecipitates were resuspended in kinase buffer (20 mm Tris, pH 7.6, 1 mmdithiothreitol, 20 mm MgCl2, 20 mmβ-glycerophosphate, 1 mm EDTA, 1 mmNa3VO4, 50 mm NaF, 20 mm para-nitrophenylphosphate, 10% (v/v) Protease Inhibitor Mixture (Sigma), and 5 µM ATP). To each sample 2 µg of myelin basic protein (MBP, Sigma) was added. Each sample was divided into two aliquots, to one of which 10 µCi of [γ-32P]ATP (Amersham Pharmacia Biotech) was added. Samples were incubated at 37 °C for 30 min. Reactions were stopped by addition of SDS-PAGE sample buffer and boiling for 3 min. Proteins were separated in 4–12% Bis-Tris SDS-PAGE gels. Proteins were detected after Western blotting or autoradiography. Computer analyses were performed through the Baylor College of Medicine search launcher available at searchlauncher.bcm.tmc.edu. The programs HSPL (prediction of splice sites in human DNA sequences) and FEXH (prediction of internal, 5′- and 3′-exons in human DNA sequences) were used to predict splice sites (30Solovyev V.V. Salamov A.A. Lawrence C.B. Nucleic Acids Res. 1994; 22: 5156-5163Crossref PubMed Scopus (281) Google Scholar) in the IRAK1 sequence. IRAK1 is an essential component of the IL-1 signaling cascade. It has been suggested that degradation of IRAK1 is responsible for desensitization of cellular targets to IL-1 and lipopolysaccharide after prolonged exposure to these agents (25Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Crossref PubMed Scopus (245) Google Scholar, 26Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). To test this hypothesis we generated a range of IRAK1 expression vectors. Primers for amplification of IRAK1 sequences were designed from GenBank™ accession number L76191, and the entire coding sequence was amplified by PCR using proofreading DNA polymerases. The resulting cDNA was cloned into the mammalian expression vector pCI-Neo. Sequencing of four individual clones revealed that two contained an identical deletion of 90 bp (sequence was submitted to GenBank™ under accession numberAF346607). To determine whether these clones represent an alternatively spliced variant of IRAK1 mRNA, we aligned their sequences with that of the known IRAK1 gene sequence (GenBank™ accession number U52112). The 90 bp, which were missing in two of our clones, aligned perfectly with the first 90 bp of IRAK1 exon 12 (Fig.1 A). We then performed computer analysis to predict the likely locations of the exon splice sites involving IRAK1 exons 11–13. The program HSPL (prediction of splice sites in human DNA sequences) predicted one 5′-acceptor site at position +1 of exon 12 and a second 5′-acceptor site at position +91 of exon 12 (Fig. 1 A). The FEXH (prediction of internal, 5′- and 3′-exons in human DNA sequences) program only predicted the 5′-acceptor site at position +91. These data strongly support the possibility of alternative splicing in exon 12 of the IRAK1 gene. Alternative splicing of exon 12 at position +1 or +91 would result in the previously described form of IRAK1 (hereafter referred to as IRAK1a for clarity) and a shorter form, respectively. We have named this shorter putative splice variant IRAK1b. IRAK1 will be used below as a generic term covering both splice variants. The IRAK1b sequence predicts an in-frame deletion of 30 amino acids (residues 514–543) at the C-terminal end of the kinase domain (Fig. 1 B). IRAK1a has previously been shown to be expressed in a wide range of cell types as an ∼3.5-kb mRNA (7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, 20Trofimova M. Sprenkle A.B. Green M. Sturgill T.W. Goebl M.G. Harrington M.A. J. Biol. Chem. 1996; 271: 17609-17612Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The similarity in size between the IRAK1a and IRAK1b mRNAs precludes their resolution by Northern blot analysis. We therefore chose a RT-PCR approach to identify cell types that express the IRAK1b mRNA. We designed a forward primer corresponding to sequence within IRAK1 exon 11 and a reverse primer corresponding to sequence withinIRAK1 exon 12 downstream of the putative alternative splice site. To test the relative amplification efficiencies of the two IRAK1 cDNAs derived from the alternatively spliced IRAK1 mRNA species, plasmids carrying IRAK1a and IRAK1b coding sequence were co-amplified by PCR, and multiple aliquots were taken for analysis after each of five cycles. Equivalent amplification kinetics during the exponential phase of IRAK1a cDNA and IRAK1b cDNA amplification was confirmed; the ratio of products was also maintained after the plateau phase of amplification had been reached (not shown). Reverse-transcribed total RNA from a range of human tissues was subjected to the above proportional quantitative PCR method. A strong band of 441 bp corresponding to the predicted size of IRAK1a cDNA and a weaker band of ∼350 bp were detected in all samples (Fig.2 A). Because the expected size of the IRAK1b cDNA is 351 bp, we concluded that IRAK1b mRNA is co-expressed with IRAK1a mRNA in all tissues tested. The ratio of cDNA products derived from the IRAK1a to IRAK1b mRNAs was similar in all samples. We next wished to establish whether the putative IRAK1b protein could be detected in protein extracts from HepG2 cells. Cells were transfected with pCI-Neo expression plasmids encoding either IRAK1a or IRAK1b. Because the former becomes hyperphosphorylated when overexpressed and consequently undergoes a shift in electrophoretic mobility (5Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 7Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, 8Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 20Trofimova M. Sprenkle A.B. Green M. Sturgill T.W. Goebl M.G. Harrington M.A. J. Biol. Chem. 1996; 271: 17609-17612Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) (see below), we used a kinase-inactive variant (kiIRAK1a) for this transfection series. Proteins were extracted from both transfected and untransfected HepG2 cells and IRAK1 proteins detected by immunoblotting. Single bands of 76 and 73 kDa were detected in cell extracts from the kiIRAK1a- and IRAK1b-transfected cells, respectively (Fig. 2 B). These sizes are similar to those predicted for the two isoforms based on their polypeptide sequences. Two bands with mobilities identical to those observed in cells transfected with kiIRAK1a and IRAK1b were also observed in extracts from untransfected cells (Fig. 2 B), suggesting that HepG2 cells endogenously synthesize both IRAK1a (upper band) and IRAK1b (lower band) proteins. The above results indicate that IRAK1b is an alternatively spliced variant of IRAK1 that is expressed at the mRNA level, and probably at the protein level, in a wide range of tissues. The mouse IRAK1a mRNA sequence (IRAK1/mPLK, GenBank™ accession number U56773) was aligned with that of the human IRAK1 mRNA. The two splice sites for IRAK1a and IRAK1b are perfectly conserved in the mouse sequence (not shown). Intriguingly, three amino acid residues, which are present in the region of the human IRAK1 that is spliced out of human IRAK1b, are absent from the mouse gene (Fig. 1 B). A proportional quantitative RT-PCR method similar to the one described above for human IRAK1a and IRAK1b mRNA splice variants was developed for the mouse putative IRAK1 mRNA splice variants. Both cDNA products could be amplified from total RNA extracted from mouse liver, kidney, and testis (Fig. 2 C). The retention of the IRAK1b alternative splice variant in two evolutionarily distant mammalian species suggests that it has an important physiological function. PELLE, the Drosophila homologue of IRAK1, contains death and kinase domains that are similar to those present in IRAK1; however, the PELLE peptide sequence ends immediately after the kinase domain and the full-length protein is only 501 amino acid residues long (31Shelton C.A. Wasserman S.A. Cell. 1993; 72: 515-525Abstract Full Text PDF PubMed Scopus (185) Google Scholar). PELLE therefore does not contain the C-terminal region, which is subject to alternative splicing in mammalian IRAK1. The presence of this domain in both human and mouse IRAK1 suggests that it serves functions, as yet undefined, that are specific to mammals. In this context it is intriguing that the C-terminal domain incorporates potential further structural diversity via the use of alternative splicing to yield distinct protein products that may themselves have differentiated functions. To determine if IRAK1b is capable of activating NF-κB, HepG2 cells were co-transfected with an NF-κB luciferase reporter, a control luciferase-Renilla reporter, and increasing amounts of IRAK1a or IRAK1b expression constructs. The level of NF-κB activation was evaluated after 24 h by assessment of luciferase/Renilla reporter ratios. Both IRAK1a and IRAK1b result in a concentration-dependent activation of NF-κB (Fig. 3). IRAK1a appears to be more efficient at activating NF-κB than IRAK1b, because ∼20 times less IRAK1a than IRAK1b expression construct is needed to induce equivalent levels of NF-κB activation. The proportional difference in NF-κB activation between IRAK1a and IRAK1b is similar to that previously reported between IRAK1a and either IRAK2 or IRAK-M (8Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar), i.e.IRAK1b appears to be as efficient as IRAK2 and IRAK-M at activating NF-κB. The lower efficiency of NF-κB activation by IRAK1b relative to that effected by IRAK1a prompted us to investigate if there are measurable differences in the interactions of IRAK1a and IRAK1b with the downstream signaling component TRAF6. His-tagged IRAK1 proteins were expressed either alone or with His-tagged TRAF6 in 293 cells. TRAF6 was immunoprecipitated from cellular extracts with a TRAF6-specific antibody, and co-immunoprecipitated proteins wer" @default.
• W2160628268 created "2016-06-24" @default.
• W2160628268 creator A5002091882 @default.
• W2160628268 creator A5014073749 @default.
• W2160628268 date "2001-08-01" @default.
• W2160628268 modified "2023-09-29" @default.
• W2160628268 title "IRAK1b, a Novel Alternative Splice Variant of Interleukin-1 Receptor-associated Kinase (IRAK), Mediates Interleukin-1 Signaling and Has Prolonged Stability" @default.
• W2160628268 cites W1501181174 @default.
• W2160628268 cites W1515994079 @default.
• W2160628268 cites W1549464896 @default.
• W2160628268 cites W1575383081 @default.
• W2160628268 cites W1925157258 @default.
• W2160628268 cites W1965971815 @default.
• W2160628268 cites W1969501356 @default.
• W2160628268 cites W1977835740 @default.
• W2160628268 cites W1981194416 @default.
• W2160628268 cites W1988217385 @default.
• W2160628268 cites W1990720057 @default.
• W2160628268 cites W1997865069 @default.
• W2160628268 cites W1998585620 @default.
• W2160628268 cites W2001361260 @default.
• W2160628268 cites W2004986546 @default.
• W2160628268 cites W2017883909 @default.
• W2160628268 cites W2035217892 @default.
• W2160628268 cites W2039109795 @default.
• W2160628268 cites W2048618925 @default.
• W2160628268 cites W2064558057 @default.
• W2160628268 cites W2067680656 @default.
• W2160628268 cites W2067684810 @default.
• W2160628268 cites W2078136106 @default.
• W2160628268 cites W2089938437 @default.
• W2160628268 cites W2114269227 @default.
• W2160628268 cites W2116390865 @default.
• W2160628268 cites W2117807589 @default.
• W2160628268 cites W2119218166 @default.
• W2160628268 cites W2141150979 @default.
• W2160628268 cites W2188779778 @default.
• W2160628268 cites W4297590147 @default.
• W2160628268 doi "https://doi.org/10.1074/jbc.m103815200" @default.
• W2160628268 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11397809" @default.
• W2160628268 hasPublicationYear "2001" @default.
• W2160628268 type Work @default.
• W2160628268 sameAs 2160628268 @default.
• W2160628268 citedByCount "75" @default.
• W2160628268 countsByYear W21606282682012 @default.
• W2160628268 countsByYear W21606282682013 @default.
• W2160628268 countsByYear W21606282682014 @default.
• W2160628268 countsByYear W21606282682015 @default.
• W2160628268 countsByYear W21606282682016 @default.
• W2160628268 countsByYear W21606282682017 @default.
• W2160628268 countsByYear W21606282682018 @default.
• W2160628268 countsByYear W21606282682019 @default.
• W2160628268 countsByYear W21606282682020 @default.
• W2160628268 countsByYear W21606282682021 @default.
• W2160628268 countsByYear W21606282682022 @default.
• W2160628268 countsByYear W21606282682023 @default.
• W2160628268 crossrefType "journal-article" @default.
• W2160628268 hasAuthorship W2160628268A5002091882 @default.
• W2160628268 hasAuthorship W2160628268A5014073749 @default.
• W2160628268 hasBestOaLocation W21606282681 @default.
• W2160628268 hasConcept C104317684 @default.
• W2160628268 hasConcept C105580179 @default.
• W2160628268 hasConcept C170493617 @default.
• W2160628268 hasConcept C185592680 @default.
• W2160628268 hasConcept C194583182 @default.
• W2160628268 hasConcept C203014093 @default.
• W2160628268 hasConcept C2777977768 @default.
• W2160628268 hasConcept C2778690821 @default.
• W2160628268 hasConcept C2780989783 @default.
• W2160628268 hasConcept C54355233 @default.
• W2160628268 hasConcept C62478195 @default.
• W2160628268 hasConcept C74172505 @default.
• W2160628268 hasConcept C86803240 @default.
• W2160628268 hasConcept C95444343 @default.
• W2160628268 hasConceptScore W2160628268C104317684 @default.
• W2160628268 hasConceptScore W2160628268C105580179 @default.
• W2160628268 hasConceptScore W2160628268C170493617 @default.
• W2160628268 hasConceptScore W2160628268C185592680 @default.
• W2160628268 hasConceptScore W2160628268C194583182 @default.
• W2160628268 hasConceptScore W2160628268C203014093 @default.
• W2160628268 hasConceptScore W2160628268C2777977768 @default.
• W2160628268 hasConceptScore W2160628268C2778690821 @default.
• W2160628268 hasConceptScore W2160628268C2780989783 @default.
• W2160628268 hasConceptScore W2160628268C54355233 @default.
• W2160628268 hasConceptScore W2160628268C62478195 @default.
• W2160628268 hasConceptScore W2160628268C74172505 @default.
• W2160628268 hasConceptScore W2160628268C86803240 @default.
• W2160628268 hasConceptScore W2160628268C95444343 @default.
• W2160628268 hasIssue "31" @default.
• W2160628268 hasLocation W21606282681 @default.
• W2160628268 hasOpenAccess W2160628268 @default.
• W2160628268 hasPrimaryLocation W21606282681 @default.
• W2160628268 hasRelatedWork W2001319824 @default.
• W2160628268 hasRelatedWork W2008389025 @default.
• W2160628268 hasRelatedWork W2038617558 @default.
• W2160628268 hasRelatedWork W2067501355 @default.
• W2160628268 hasRelatedWork W2093378391 @default.
• W2160628268 hasRelatedWork W2136641749 @default.

• next