Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription.
Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position -14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT.
Department of Tumor Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
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Marek-Bukowiec
Core Research Facility for Health Sciences, University of Cádiz and Puerto Real University Hospital Research Unit, Department of Biomedicine, Biotechnology and Public Health (Immunology), Cadiz, Spain.
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Aguado
Department of Tumor Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
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Miazek
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D006801:Humans; D007438:Introns; D015759:Ionomycin / Q000494:pharmacology; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D008926:Plicamycin / Q000494:pharmacology; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects*; D014635:Valproic Acid / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008565:Membrane Proteins; D010703:Phorbol Esters; D016329:Sp1 Transcription Factor; D015759:Ionomycin; D014635:Valproic Acid; D008926:Plicamycin