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Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells
D A Fruman1, S Y Pai, S J Burakoff
1Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Abstract:
The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.
Insights
A mutation in calcineurin A alpha disrupts its autoinhibition, leading to altered calcium signaling in T-lymphocytes. This finding highlights the role of a specific aspartic acid in calcineurin regulation and T-cell activation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Calcineurin is a phosphatase crucial for calcium-dependent T-lymphocyte activation.
- Understanding calcineurin's regulatory mechanisms is key to T-cell signaling research.
Purpose of the Study:
- To identify and characterize a mutation in the calcineurin A alpha gene in EL4 T-lymphoma cells.
- To investigate the functional impact of this mutation on calcineurin activity and T-cell signaling.
Main Methods:
- Missense mutation identification in calcineurin A alpha gene.
- Peptide inhibition studies comparing wild-type and mutant calcineurin.
- Analysis of T-lymphoma cell activation via calcium ionophore and phorbol esters.
- Gene transfection experiments in Jurkat T cells.
Main Results:
- A mutation changed a conserved aspartic acid to asparagine in the calcineurin A alpha autoinhibitory domain.
- The mutation significantly reduced calcineurin phosphatase activity inhibition.
- Mutant calcineurin expression led to aberrant T-cell activation by suboptimal stimuli.
- The aberrant signaling phenotype was transferable to other T-cell lines.
Conclusions:
- A conserved aspartic acid residue is critical for calcineurin autoinhibition.
- Mutation of this residue impairs autoinhibition, causing aberrant calcium-dependent T-cell signaling.
- This study provides insights into calcineurin's role in T-lymphocyte activation pathways.