Important note: Information in this article was accurate in 1998. The state of the art may have changed since the publication date.
Molecular analysis of human T cell leukemogenesis by HTLV-1 Tax protein (Meeting abstract).
Proc Annu Meet Am Assoc Cancer Res; 38:A1075 1997. Unique Identifier : AIDSLINE ICDB/98638075 Petropoulos L; Crepieux P; Lin R; Hiscott J; Molecular Oncology, Lady Davis Inst., McGill University,; Montreal, Canada H3T 1E2
Abstract:
Human T cell Leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL). The oncogenic potential of HTLV-1 resides in the 40 kD viral Tax protein which has been characterized as a strong transactivator of transcription. One of the targets of interaction for the Tax protein is NF-kappaB/Rel, a family of regulatory proteins involved in immunoregulation and cellular growth control. Our recent studies have demonstrated that Tax physically associates with specific NF-kappaB subunits leading to an increase in the on-rate of NF-kappaB DNA binding and an upregulation of NF-kappaB dependent gene transcription. Using protein-protein cross-linking assays, we also showed that Tax increased the dimerization of NF-kappaB p52 and p65 subunits, even in the absence of DNA. HTLV-1 Tax expression also increased phosphorylation and proteolytic degradation of the NF-kappaB inhibitor, IkappaBalpha. Tax was found to physically associate with recombinant IkappaBalpha in vitro by affinity chromatography and in vivo by co-immunoprecipitation; Tax and endogenous IkappaBalpha were co-immunoprecipitated from HTLV-1 infected cells only after stabilization of IkappaBalpha by the addition of proteasome inhibitors that prevented IkappaBalpha degradation. A possible role for Tax in targeting newly synthesized IkappaBalpha to the proteasome for degradation will be discussed. To date, the effectors involved in IkappaBalpha inducible degradation have been poorly characterized. In an attempt to identify regulators of the IkappaBalpha inhibitory activity, we undertook a yeast two-hybrid genetic screen of an LMP-1 transformed B cell library, using the amino-terminal 55 aa of IkappaBalpha (NIK) as bait. Eight independent clones interacting with NIK were identified. Sequencing and database analysis identified the cDNA clones as Dlc-1, a sequence encoding a small 9 kD human homologue of the dynein light chain protein, originally identified in C. reinhardtii. In yeast, Dlc-1 also interacted with the full-length IkappaBalpha protein, but not with N-terminal deleted versions of IkappaBalpha containing ankyrin repeats (ANK) or the C-terminal PEST domain (CIK). Consistently, IkappaBalpha was efficiently pulled down by the GST-Dlc-1 fusion protein in affinity chromatography studies, whereas p65 was unable to displace this interaction, confirming that p65 and Dlc-1 contact different protein motifs of IkappaBalpha. Importantly, in Hela cells, endogenous IkappaBalpha co-immunoprecipitated with overexpressed Dlc-1. Analysis of expression patterns indicates that Dlc-1 is widely expressed in many cell-types. Using a combination of co-transfection, immunofluorescence and confocal microscopy, we have demonstrated that IkappaBalpha and Dlc-1 travel within the cell and under certain experimental conditions may be found in the nuclear or cytoplasmic compartments. Furthermore, confocal microscopy has localized Dlc-1 and a-tubulin to the microtubule organizing center, a perinuclear cytoplasmic organelle from which microtubules radiate. At present, we are exploring the possibility that Dlc-1 may play a role in the nucleocytoplasmic trafficking of IkappaBalpha and associated transcription factors or may represent a docking protein that mediates IkappaBalpha/NF-kappaB interaction with the cytoskeleton. Taken together, these results highlight an intriguing interaction between the IkappaBalpha protein and the human homologue of a member of the dynein family of motor proteins involved in cytoskeletal dynamics in mammalian cells, as well as in lower organisms.
Keywords: Gene Products, tax/*GENETICS Human Leukemia, T-Cell/*GENETICS NF-kappa B/GENETICS Transcription, Genetic ABSTRACT 980228
M9820761
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Important note: Information in this article was accurate in 1998. The state of the art may have changed since the publication date.
