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Protein kinase C (PKC) activation via human leucocyte antigen class II molecules. A novel regulation of PKC activity
C Brick-Ghannam1, F L Huang, N Temime
1Laboratoire d'Immunogénétique Moléculaire, Institut des Cordeliers, Paris, France.
Insights
Anti-human leucocyte antigen (HLA) class II antibodies increase protein kinase C (PKC) activity in B cells. This activation is regulated at the transcriptional level, involving increased PKC mRNA and protein levels.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Protein kinase C (PKC) plays a crucial role in cellular signaling pathways.
- Human leucocyte antigen (HLA) class II molecules are involved in immune responses.
- The regulation of PKC activity by immune stimuli requires further investigation.
Purpose of the Study:
- To investigate the effect of anti-HLA class II antibodies on PKC activity and localization in B cells.
- To elucidate the regulatory mechanisms underlying PKC activation by HLA class II antibodies.
Main Methods:
- Analysis of cytosolic and membrane PKC activities in a lymphoblastoid B cell line.
- Western blot analysis for quantitative assessment of PKC protein levels.
- Northern blot analysis to determine PKC mRNA levels.
- Treatment with transcriptional (actinomycin D) and translational (cycloheximide) inhibitors.
Main Results:
- Anti-HLA class II antibodies increased both cytosolic and membrane PKC activities in a time- and dose-dependent manner.
- PKC activation correlated with a quantitative increase in PKC protein and mRNA levels (PKC alpha and beta).
- Inhibition of transcription or translation abolished the PKC activity increase, indicating transcriptional regulation.
Conclusions:
- HLA class II antibody stimulation leads to increased PKC activity in B cells.
- The observed increase in PKC activity is regulated at the transcriptional level.
- This study reveals a novel mechanism of PKC regulation by HLA class II molecules.
Abstract:
Analysis of intracellular localization of protein kinase C (PKC) in a lymphoblastoid B cell line shows that anti-human leucocyte antigen (HLA) class II antibodies induce an increase of cytosolic and membrane PKC activities. This phenomenon is both time- and dose-dependent. The maximal PKC activation was observed after exposure to 12.5 micrograms/ml antibody for 30 to 45 min. Unlike TPA, no translocation of the cytosolic PKC was observed at any time following exposure to the anti-HLA class II antibodies. We observed a good correlation between the [3H]phorbol dibutyrate binding activity and the enzymatic activity of PKC. Using a panel of antibodies specific for the HLA class II isotypes (DP, DQ, DR), we demonstrated that PKC activation via HLA class II molecules is not restricted to one isotype. We also showed by Western blot analysis that the increased PKC activity correlates with a quantitative increase of PKC. The increase of PKC activity induced by anti-HLA class II antibodies was completely abolished by the treatment with actinomycin D, a transcriptional inhibitor, or cycloheximide, a translational inhibitor. Finally, Northern blot analysis revealed that anti-HLA class II antibodies induce an increase of the PKC alpha and PKC beta mRNAs levels which are significant after 20 min of stimulation and rose to a maximum after 60 min. In summary, our results show that increased PKC activity induced by HLA class II antibody is regulated at the transcriptional level.