title: Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells.
abstract: Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.
authors.affiliation: Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: N
authors.initials: N
authors.lastname: Hama
authors.forename: F
authors.initials: F
authors.lastname: Paliogianni
authors.forename: B J
authors.initials: BJ
authors.lastname: Fessler
authors.forename: D T
authors.initials: DT
authors.lastname: Boumpas
mesh_terms: D019703:Calcineurin; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology; D015536:Down-Regulation; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D015847:Interleukin-4 / Q000096:biosynthesis / Q000235:genetics*; D008213:Lymphocyte Activation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D015847:Interleukin-4; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases