Inhibition of NF-AT signal transduction events by a dominant-negative form of calcineurin.
An inhibitory, "dominant-negative," form of the calcineurin catalytic (A) subunit was prepared, which lacks the calmodulin-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block calcineurin-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleukin (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced NF-AT activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt calcineurin-mediated signal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits.
Muramatsu
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Section on Immunology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852, USA.
Kincaid
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RL
D000818:Animals; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases