The Murine Irak2 Gene Encodes Four Alternatively Spliced Isoforms, Two of Which Are Inhibitory

The interleukin-1 receptor-associated kinases (IRAKs) are important downstream signaling components of Toll-like receptors (TLRs). To date, four mammalian IRAKs have been found, namely IRAK-1, IRAK-2, IRAK-4, and IRAK-M. Herein, we show a detailed analysis of the genomic region encompassing the murine Irak2 gene and the molecular cloning of four isoforms of Irak2 (designated Irak2a, Irak2b, Irak2c, and Irak2d) generated by alternative splicing at the 5′-end of the gene. This alternative splicing has direct effects on the expression of the N-terminal death domain and/or inter-domain. No evidence of similar alternative splicing was found for the human IRAK2 gene. When overexpressed, Irak2a and Irak2b potentiated NF-κB activation by lipopolysaccharide. Importantly, Irak2c and Irak2d were inhibitory. The promoter for Irak2c differed from that of the other Irak2 isoforms in that it contained putative NF-κB binding sites. Lipopolysaccharide induced the expression of Irak2c, indicating a possible negative feedback effect on the signaling pathway. Alternative splicing of the Irak2 gene in mice will therefore generate agonistic or antagonistic Irak2 isoforms, which is likely to have consequences for the regulation of TLR signaling. These observations identify another distinguishing feature between mice and humans in the TLR system that is likely to be due to differences in the selective pressure imposed by pathogens on each species during evolution. The interleukin-1 receptor-associated kinases (IRAKs) are important downstream signaling components of Toll-like receptors (TLRs). To date, four mammalian IRAKs have been found, namely IRAK-1, IRAK-2, IRAK-4, and IRAK-M. Herein, we show a detailed analysis of the genomic region encompassing the murine Irak2 gene and the molecular cloning of four isoforms of Irak2 (designated Irak2a, Irak2b, Irak2c, and Irak2d) generated by alternative splicing at the 5′-end of the gene. This alternative splicing has direct effects on the expression of the N-terminal death domain and/or inter-domain. No evidence of similar alternative splicing was found for the human IRAK2 gene. When overexpressed, Irak2a and Irak2b potentiated NF-κB activation by lipopolysaccharide. Importantly, Irak2c and Irak2d were inhibitory. The promoter for Irak2c differed from that of the other Irak2 isoforms in that it contained putative NF-κB binding sites. Lipopolysaccharide induced the expression of Irak2c, indicating a possible negative feedback effect on the signaling pathway. Alternative splicing of the Irak2 gene in mice will therefore generate agonistic or antagonistic Irak2 isoforms, which is likely to have consequences for the regulation of TLR signaling. These observations identify another distinguishing feature between mice and humans in the TLR system that is likely to be due to differences in the selective pressure imposed by pathogens on each species during evolution. The Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; aa, amino acid(s); DD, death domain; IRAK, interleukin-1 receptor-associated kinase; LPS, lipopolysaccharide; MEF, murine embryonic fibroblast; MyD88, myeloid differentiation factor 88; nt, nucleotide(s); RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; UTR, untranslated region. are a family of molecules tailored to respond to microbial pathogens, with particular TLRs able to recognize and bind to specific pathogen-associated molecular patterns. Once activated, TLRs recruit cytoplasmic adapter molecules such as myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like protein (Mal; also known as TIRAP), TIR domain-containing adaptor protein inducing interferon-β (TRIF; also termed TICAM-1), and TRIF-related adaptor molecule (TRAM; also termed TIRP or TICAM-2), which, in turn, initiate signaling cascades that result in biological responses geared toward the elimination of pathogens during infection (reviewed in Refs. 1Dunne A. O'Neill L. Science's STKE. 2003; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/171/re3)PubMed Google Scholar and 2O'Neill L. Fitzgerald K. Bowie A. Trends Immunol. 2003; 24: 286-290Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Critical to the TLR signaling cascade are the interleukin-1 receptor-associated kinases (IRAKs). The first human IRAK to be cloned was IRAK1 (3Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (773) Google Scholar), followed by IRAK2 (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar), IRAK-M (5Wesche 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 (340) Google Scholar), and IRAK4 (6Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (543) Google Scholar). The IRAKs share sequence homology to the Drosophila melanogaster protein kinase Pelle, and all contain a death domain (DD), which is used for protein-protein interactions with the DDs of other molecules. For example, IRAK2 uses its DD to mediate its interaction with MyD88 (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar). The IRAKs also have putative kinase domains, although IRAK1 has dispensable kinase activity because interleukin-1-induced NF-κB activation could still be driven by a kinase-inactive mutant (7Li 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 addition, both IRAK2 and IRAK-M are catalytically inactive due to the absence of certain key residues within their putative kinase domains (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar, 5Wesche 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 (340) Google Scholar). Adding further complexity to the signaling cascades initiated by the TLRs are the recent findings that some of the genes encoding components of TLR signaling are alternatively spliced, thus generating multiple isoforms. One example is the murine MyD88 gene, which encodes a full-length MyD88 (MyD88L) and a shorter form (MyD88S) generated by the splicing out of exon 3; the removal of this exon causes the deletion in the mature polypeptide of the intermediate domain (8Janssens S. Burns K. Tschopp J. Beyaert R. Curr. Biol. 2002; 12: 467-471Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Both forms of MyD88 are differentially expressed and exhibit differences in their ability to induce NF-κB activation and IRAK phosphorylation, with MyD88S being inhibitory (8Janssens S. Burns K. Tschopp J. Beyaert R. Curr. Biol. 2002; 12: 467-471Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). MyD88S can, however, mediate the activation of c-Jun N-terminal kinase (9Janssens S. Burns K. Vercammen E. Tschopp J. Beya FEBS Lett. 2003; 548: 103-107Crossref PubMed Scopus (151) Google Scholar). Another example is the human IRAK1 gene, which encodes two isoforms generated by the differential usage of a splice acceptor site within exon 12 (10Jensen L.E. Whitehead A.S. J. Biol. Chem. 2001; 276: 29037-29044Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In contrast to the full-length isoform (designated IRAK1a), the slightly shorter isoform (IRAK1b) is kinase-inactive and displays no change in its protein levels following interleukin-1 stimulation (10Jensen L.E. Whitehead A.S. J. Biol. Chem. 2001; 276: 29037-29044Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Here, we report the identification and annotation of the murine Irak2 gene, which generates four alternatively spliced isoforms of Irak2 that contain various deletions of the N-terminal third of the mature protein. Of these, Irak2a and Irak2b enhance the activity of an NF-κB reporter, whereas Irak2c and Irak2d are inhibitory. Our results therefore reveal a level of control of TLR signaling that involves differential splicing of murine Irak2. Sequence Analysis—The complete nucleotide sequence of the murine Irak2 gene and flanking regions was obtained from the National Center for Biotechnology Information (NCBI) mouse genomic data base (contig NT_039353 found at www.ncbi.nlm.nih.gov), following BLAST analysis with the human IRAK2 cDNA (GenBank™ accession number AF026273). Other sequences used in this manuscript that were also obtained from the GenBank™ were the human IRAK2 genomic locus (contig NT_005927) and Irak2 cDNA (accession number AJ440756). Transcription factor binding site searches were performed using TFSEARCH (molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html) (11Heinemeyer T. Wingender E. Reuter I. Hermjakob H. Kel A. Kel. O. Ignatieva E. Ananko E. Podkolodnaya O. Kolpakov F.A. Podkolodny N.L. Kolchanov N.A Nucleic Acids Res. 1998; 26: 362-367Crossref PubMed Scopus (1325) Google Scholar) and MatInspector Release Professional (genomatix.gsf.de/mat_fam) (12Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2424) Google Scholar). Global alignment of the human and mouse genomic sequences of conserved synteny was performed with the program AVID 2N. Bray, A. Fabrikant, J. Lord, J. Schwartz, I. Dubchak, and L. Pachter, unpublished work. using a window size of 100 bp and a conservation level of 70%; the results were viewed with the program VISTA (www-gsd.lbl.gov/vista/) (13Bray N. Dubchak I. Pachter L. Genome Res. 2003; 13: 97Crossref PubMed Scopus (365) Google Scholar, 14Dubchak I. Brudno M. Loots G.G. Pachter L. Mayor C. Rubin E.M. Frazer K.A. Genome Res. 2000; 10: 1304-1306Crossref PubMed Scopus (272) Google Scholar, 15Mayor C. Brudno M. Schwartz J.R. Poliakov A. Rubin E.M. Frazer K.A. Pachter L.S. Dubchak I. Bioinformatics. 2000; 16: 1046-1047Crossref PubMed Scopus (794) Google Scholar). The identification of transcribed nucleotide sequences and repeat sequences in the genomic sequence was performed using the NIX application The of putative was out using and amino were performed using were using C.J. L. N. A. L. M. A. P. Bioinformatics. 2002; PubMed Scopus Google Scholar). and of cDNA and cDNA were generated with or reverse to the using of from murine embryonic or or from of human or and were out to the using 100 of of and of in a of were as J. 2002; PubMed Google Scholar). To specific were with and further with using of and of J. 2002; PubMed Google Scholar). were by and to and their was by with an were cloned and in a and and were in with and The human embryonic with was a from Fitzgerald of and was in as with the of Lipopolysaccharide from was obtained from and used at The was obtained from and the was from The of the was a from R. The human was obtained from was from with for and using to the To each of was of and the were at for was to of and at for following The was by at for to of to for further at for The were with of and in of cDNA was generated from using reverse to the To of was of of and the was at for and on for a further To this was of of of and of reverse for or of for the was at for and at for from cDNA was performed on a in a of each and reverse of cDNA or and of were for and at for for and for were cloned and For reverse transcribed generated from the following was used of of of of each and reverse cDNA or of and of for were for and at for for and for followed by a at for For gene were used for an of and of were out using a murine to the were at for with a Irak2 cDNA to of the Irak2 gene. were at with and to for at of Irak2 of Irak2a, Irak2b, Irak2c, and Irak2d were by from a murine cDNA using the Irak2a, and and as a reverse was out on a using the following of of of each of and and of The following were for at for for and for Irak2 were generated to at their using the and contained and at their and were with their and were at in complete and at the of To of was of following and a at RT, the was to of for a further at RT, to each of the This was performed in for each The of the of 100 of of reporter, and of Irak2 expression and the was to were at for or with at for been by and for all was were in of for at and each of the was in for and activity using a activity was as a of activity by the between were using of the Irak2 identify the murine of the human IRAK2 gene, a BLAST of the murine genomic data base using the human IRAK2 cDNA sequence of (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar) was region of homology was found on at This putative gene was murine or because genes on other this gene was therefore likely to be Irak2. To a complete annotation of the putative Irak2 gene, of murine genomic encompassing this gene was in using the NIX of which are able to identify putative and within the genomic sequence to be in the NIX were able to identify the murine Irak2 gene and the of the of the of the Irak2 gene with the of and This was by and analysis and alignment of the cDNA sequence the murine genomic The putative Irak2 gene of murine genomic sequence and is transcribed toward the of of the putative exon of Irak2 the murine gene GenBank™ accession number a gene of transcribed in the as Irak2 of Irak2 another gene with the with homology to human a gene encoding a putative (GenBank™ accession number This murine also to be transcribed in the as and Irak2. The human of all murine genes are found in the on transcribed toward the further of murine transcribed in the was a putative encoding the murine known as the the the and the GenBank™ accession of Irak2 Irak2 cDNA sequence were and within two regions of murine genomic sequence with homology to the human IRAK2 were also at of the putative gene and to as cDNA as amplification from a cDNA using two of and bp which differential splicing of the murine Irak2 gene. No were from that were the result of genomic of it was that the cDNA (designated was by of the putative Irak2 gene. The cDNA sequence also the thus of the of the The cDNA a sequence by exon was to the this cDNA therefore to be a of Irak2 have this cDNA To further the genomic of identify its and and identify Irak2 was performed using murine and cDNA with similar results and was first performed using in and and of and bp were the to Irak2a, and the to the exon of bp was also this out to be also the putative exon and two at and of the Irak2a by were and that generated further Irak2 cDNA of the cDNA generated the bp of exon we this isoform cDNA were also found to contain an Irak2 cDNA sequence similar to that of Irak2a a sequence by exon this isoform has been To the of the Irak2c and Irak2d was performed using a cDNA and and for and for The Irak2c and Irak2d generated and were and the results the of isoforms. the of all putative Irak2 isoforms were by using and for Irak2a, and and and for Irak2c The Irak2a is the cDNA as that O. Res. 2002; PubMed Scopus Google Scholar). of the Irak2d cDNA also a deletion in exon 12 in to the exon For all no were generated using a cDNA To the of the Irak2 gene, was performed using the and in and of was generated which, was found to contain an Irak2 cDNA sequence encoding 12 and four Irak2 cDNA sequences were the murine genomic sequence data base by and and have been GenBank™ the accession and and Alternative of murine Irak2 gene is of and 12 and encodes a full-length protein of amino and Irak2 a kinase domain and a DD the region of Irak2a, and as as their and also encodes the encodes the and we as the and of the the kinase domain; 12 and the and the for all Irak2 in exon Irak2c has its (designated exon that is by a of exon from bp to and the of Irak2 to the M. P. Nucleic Acids Res. PubMed Scopus Google Scholar) The possible are in the Irak2 gene splice and between amino and a between the first and nucleotide and and a between the and third nucleotide of the murine Irak2 acceptor acceptor in a The alternative splicing of Irak2 isoforms is in in Irak2a exon Irak2b and Irak2d are generated by the deletion of and in a exon Irak2d also an alternative splice acceptor site bp exon which causes the deletion of amino (designated the in the The of the is and no to known protein were Irak2c is generated by exon has its specific in its in exon Alternative splicing of Irak2 therefore to of Irak2 as Irak2a putative amino is Irak2b amino the of the Irak2c amino the N-terminal amino and Irak2d amino the DD and the of the nucleotide sequences of the full-length Irak2a cDNA and the Irak2 cDNA by and O. Res. 2002; PubMed Scopus Google Scholar) (GenBank™ accession number nucleotide differences at and is no evidence of being as detailed of generated cDNA sequences from multiple cDNA with genomic and expressed sequence sequences show data that the Irak2 nucleotide sequence generated at by and O. Res. 2002; PubMed Scopus Google Scholar) could be of the amplification of their is a in the Irak2 cDNA that has been by the of multiple cDNA generated a with known cDNA and and of genomic and expressed sequence This at in the cDNA of O. Res. 2002; PubMed Scopus Google Scholar) and results in an of IRAK2 and Irak2 is an alternatively spliced gene, the was that alternatively spliced isoforms also To their we first the putative amino sequences of IRAK2 and IRAK2 and murine Irak2a are conserved amino with their putative death and kinase domains conserved and amino This that the of IRAK2 to domains be in humans and the and the a level of amino and The used by Irak2c is also to a at the in that a similar performed using human and and in exon and exon to to identify possible human and of bp was and a to the IRAK2 cDNA of (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar) was found of To the of alternative splicing of the IRAK2 gene, was performed using to and 12 and of was which was to be full-length IRAK2 cloning and These data that form of human IRAK2 in contrast to the multiple Irak2 isoforms. of human and mouse and genomic sequences in regions of conserved synteny have been in gene expression R. C. Z. Rubin E. Frazer K. Science. 2000; PubMed Scopus Google Scholar). To this we the human and mouse genomic regions and flanking the IRAK2 genes and found that the sequences of the IRAK2 and Irak2 genes were conserved no conserved sequences were between humans and These data that IRAK2 and Irak2 be differentially some by their conserved death and kinase of Irak2 by Irak2a, and and that by Irak2c are by in the mouse This that the expression and regulation of Irak2c a promoter to that of the other Irak2 isoforms. The sequence flanking exon and exon of the Irak2 gene were therefore for the of putative factor binding that as to this gene expression of Irak2 isoforms. of the putative Irak2c promoter of exon multiple factor binding for NF-κB C. S. C. Mol. Cell. Biol. 12: PubMed Google Scholar, M. J. M. PubMed Scopus Google Scholar), and of C. Z. J. 1995; PubMed Scopus Google Scholar), and factor R. P. J. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar) as as an R. N. PubMed Scopus Google Scholar) In the genomic sequence of exon contain of putative binding which that Irak2c be in to such as with the other Irak2 isoforms. of a expression of we a mouse with a cDNA to of the Irak2 gene from a cDNA by using the and These are used by all Irak2 isoforms, that the expression be of Irak2 as a in we the of a in all which is the O. Res. 2002; PubMed Scopus Google Scholar). is that a was the similar of the of Irak2 and the level of the This was in and and in and of was also in the and of this is were with of likely or The expression of Irak2 was found to from that of human IRAK2 which was found to be expressed in the and (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar), in contrast to the expression of Irak2 in murine and as with that in murine These data further that IRAK2 and Irak2 in their protein expression with their could for murine Irak2 protein expression because of the of of an Irak2 on and NF-κB putative to the Irak2 isoforms, we for their ability to the activity of an NF-κB gene or stimulation with in was able to an in activity of in murine as with with the NF-κB reporter, was able to activity and the effect of in this When an Irak2a expression was a similar effect was both on its and on When Irak2b was in the similar results were obtained that the removal of the of the Irak2 has no effect on Irak2 an Irak2c expression was for its ability to NF-κB activity in a of NF-κB activity was that the N-terminal of Irak2 that is in Irak2c the downstream signaling by results were found with Irak2d that the death domain of Irak2 is for Irak2 of the isoforms an effect on with data that the isoforms of Irak2 have with Irak2c and Irak2d being inhibitory. Irak2c we for the ability of murine Irak2 isoforms to be differentially in to LPS, because the putative Irak2c promoter to contain such as used from generated from as have been able to between Irak2 isoforms. in we were able to a cDNA from using the and The levels of Irak2c to to that of the gene following and of toward that This of Irak2c was also the cDNA used was at and that this effect is an of the were also able to a using a and and the reverse the levels of Irak2a cDNA to change following to of the gene the were also Irak2c expression is therefore by and be in a negative feedback on signaling. Here, we report an complexity in TLR signaling by the identification of four isoforms of which we have Irak2a, and The of Irak2 to during the that to the of Irak2 The alternative splicing of the Irak2 gene is the first for genes encoding TLR signaling Both human IRAK1 and MyD88 have been to as multiple isoforms generated by alternative splicing (8Janssens S. Burns K. Tschopp J. Beyaert R. Curr. Biol. 2002; 12: 467-471Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, L.E. Whitehead A.S. J. Biol. Chem. 2001; 276: 29037-29044Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The full-length Irak2a isoform is the murine of IRAK2 and is to the Irak2 O. Res. 2002; PubMed Scopus Google Scholar). The sequence differences are likely due to generated by the used by and the Irak2a and/or Irak2d all four isoforms. The four isoforms of Irak2 are generated by alternative splicing and at an of because of the similar of their and the level of to which were therefore to the Irak2 splice by and the of of an from Irak2 protein The expression of Irak2 that we generated differed from the human IRAK2 expression (4Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (984) Google Scholar). Irak2 with human IRAK2 was in and analysis of the Irak2 isoforms using an NF-κB that the full-length Irak2a was able to This activity to be by the death because the of Irak2 that this domain and Irak2d) as of NF-κB These data that Irak2c and are of signaling induced during in a negative feedback This was further we the regions of the Irak2 as a the regulation of expression of Irak2a and Irak2 are is by Irak2a, and and the of is by This that in to isoforms. This was by the putative factor binding in each region. The putative Irak2c the putative promoter for the other Irak2 isoforms, is to contain binding for in responses such as and of factor and an therefore induce Irak2c, Irak2a and found that was the differential regulation of Irak2 isoforms. is therefore likely that Irak2a and Irak2b mediate signaling by with the of Irak2c during signaling and a on the TLR pathway. This is similar to the of which is generated during signaling and has a negative effect on NF-κB activation (8Janssens S. Burns K. Tschopp J. Beyaert R. Curr. Biol. 2002; 12: 467-471Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, S. Burns K. Vercammen E. Tschopp J. Beya FEBS Lett. 2003; 548: 103-107Crossref PubMed Scopus (151) Google Scholar). Alternative splicing of IRAK2 in The therefore as to mice this level of Other differences have been found in the TLR system between humans and are a for that was found to be in humans C. S. Science. PubMed Scopus Google Scholar), and which is in mice H. H. Kirschning C. S. H. S. Science. PubMed Scopus Google Scholar). of differences during the of the of each species in to selective pressure by therefore be data from mice to humans in the TLR be however, to to human IRAK2 by using the complexity of murine Irak2 as a will the regulation and of the TLR system in