title: Transposon-based mutagenesis identifies short RIP1 as an activator of NFkappaB.
abstract: We used a vector based on the Sleeping Beauty transposon to search for constitutive activators of NFkappaB in cultured cells. Dominant mutations were produced by random insertion of a tetracycline-regulated promoter, which provided robust and exceptionally well-regulated expression of downstream genes. The ability to regulate the mutant phenotype was used to attribute the latter to the insertional event. In one such mutant, the promoter was inserted in the middle of the gene encoding receptor-interacting protein kinase 1 (RIP1). The protein encoded by the hybrid transcript lacks the putative kinase domain of RIP1, but potently stimulates NFkappaB, AP-1 and Ets-1 activity. Similarly to TNFalpha treatment, expression of the short RIP1 was toxic to cells that failed to upregulate NFkappaB. The effects of short RIP1 did not require endogenous RIP1 or cytokine treatment and coincided with reduced responsiveness to TNFalpha. Additional evidence indicates that a similar short RIP1 could be produced naturally from the ripk1 locus. Interestingly, elevated expression of short RIP1 resulted in the loss of full length RIP1 from cells, pointing to a novel mechanism through which the abundance of RIP1 and the status of the related signaling cascades may be regulated.
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Maupali
authors.initials: M
authors.lastname: Dasgupta
authors.forename: Mukesh K
authors.initials: MK
authors.lastname: Agarwal
authors.forename: Patrick
authors.initials: P
authors.lastname: Varley
authors.forename: Tao
authors.initials: T
authors.lastname: Lu
authors.forename: George R
authors.initials: GR
authors.lastname: Stark
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Kandel
mesh_terms: D015152:Blotting, Northern; D002460:Cell Line; D002999:Clone Cells; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D016254:Mutagenesis, Insertional / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D013752:Tetracycline / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D020033:Protein Isoforms; D012333:RNA, Messenger; D018626:Retroelements; D014157:Transcription Factors; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D013752:Tetracycline; D004318:Doxycycline