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Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells
1Department, and Beirne B. Carter Center for Immunology Research, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Abstract:
Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.
Insights
Protein kinase C (PKC) isozymes PKCeta and PKCtheta have distinct roles in T cell activation. PKCeta influences cytoskeletal organization, while PKCtheta regulates cell cycle progression following phorbol ester treatment.
Area of Science:
- Immunology
- Cell Biology
Background:
- Sensitive EL4 mouse thymoma cells (s-EL4) exhibit growth inhibition, adherence, and cytokine production upon phorbol ester stimulation.
- These cells express multiple phorbol ester-sensitive protein kinase C (PKC) isozymes, necessitating clarification of individual functions.
- Previous research indicated higher expression of PKCeta and PKCtheta in s-EL4 cells compared to resistant EL4 (r-EL4) cells.
Purpose of the Study:
- To investigate the specific roles of PKCeta and PKCtheta in EL4 T cell activation.
- To elucidate the functional differences between PKCeta and PKCtheta in response to phorbol ester treatment.
Main Methods:
- Transient expression of wild-type and constitutively active PKCeta and PKCtheta isozymes in s-EL4 and r-EL4 cells using a Sindbis virus system.
- Assessment of cellular morphology, cytoskeletal structure, and cell cycle progression following isozyme expression and/or phorbol ester treatment.
Main Results:
- Constitutively active PKCeta expression altered cell morphology and cytoskeletal structure, mimicking phorbol ester effects, suggesting a role in cytoskeletal organization.
- PKCeta expression did not affect phorbol ester-induced cell cycle inhibition.
- PKCtheta expression, but not PKCeta, rescued cell cycle progression inhibited by prolonged phorbol ester treatment, indicating a role in cell cycle regulation.
Conclusions:
- PKCeta and PKCtheta play differential roles in T cell activation.
- PKCeta is implicated in regulating cytoskeletal organization in response to phorbol esters.
- PKCtheta is involved in modulating T cell cycle progression during prolonged phorbol ester exposure.