Heterogeneity in the electrophoretic mobility of CD5 molecules after phorbol ester stimulation
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Published on: December 12, 2014
F Lozano1, J Alberola-Ila, L Places
1Servei d'Immunologia, Hospital Clínic i Provincial de Barcelona, Spain.
Abstract:
As judged by Western blot analysis, phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBU) induce the rapid and dose-dependent appearance of slower mobility CD5 molecular forms from peripheral blood mononuclear cell (PBMC) and thymus cell lysates. This phenomenon was inhibited by staurosporine, suggesting that it can be mediated by PKC activation. Furthermore, under our experimental conditions, neither Concanavalin A, nor Phytohaemagglutinin P or the calcium ionophore A23187 were able to reproduce the phorbol ester-induced changes in the CD5 electrophoretic mobility. When immunoprecipitated from phorbol ester-stimulated P32 labelled PBMC lysates, the slower mobility of CD5 molecules was associated to important phosphorylation. This special electrophoretic behaviour after phorbol ester-stimulation makes CD5 different from other lymphocyte surface glycoproteins and may have important implications in the elucidation of the biological role of this molecule as discussed below.
Insights
Phorbol esters like PMA and PDBU cause CD5 molecules to change their electrophoretic mobility in immune cells. This change, linked to phosphorylation, suggests a role for protein kinase C in CD5 regulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- The CD5 molecule is a key surface glycoprotein found on lymphocytes.
- Understanding post-translational modifications of CD5 is crucial for elucidating its function.
- Protein kinase C (PKC) is a family of enzymes involved in various cellular processes.
Purpose of the Study:
- To investigate the effect of phorbol esters on the electrophoretic mobility of CD5 molecules.
- To determine the role of protein kinase C (PKC) in mediating these changes.
- To explore the phosphorylation status of CD5 following phorbol ester stimulation.
Main Methods:
- Western blot analysis of peripheral blood mononuclear cell (PBMC) and thymus cell lysates.
- Treatment with phorbol esters (phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate).
- Inhibition studies using staurosporine and stimulation with other agents (Concanavalin A, Phytohaemagglutinin P, A23187).
- Immunoprecipitation and 32P labeling to assess protein phosphorylation.
Main Results:
- Phorbol esters induced a rapid, dose-dependent shift to slower mobility forms of CD5.
- This effect was inhibited by staurosporine, indicating involvement of PKC.
- Other stimuli like Concanavalin A, Phytohaemagglutinin P, and A23187 did not induce similar CD5 mobility changes.
- Slower mobility CD5 forms were significantly phosphorylated in stimulated cells.
Conclusions:
- Phorbol ester stimulation leads to PKC-mediated changes in CD5 electrophoretic mobility.
- These alterations are associated with increased CD5 phosphorylation.
- The unique response of CD5 to phorbol esters highlights its distinct post-translational regulation compared to other lymphocyte surface glycoproteins.
- Further research into these modifications may clarify the biological role of CD5.
