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Prothymosin alpha is phosphorylated in proliferating stimulated cells
M G Barcia1, J M Castro, C D Jullien
1Departamento de Bioquimica e Bioloxia Molecular, Facultade de Bioloxia, Universidade de Santiago, Santiago de Compostela, Galicia, Spain.
Insights
Prothymosin alpha phosphorylation is linked to cell proliferation and mitogenic activation. Casein kinase-2 is not responsible for in vivo prothymosin alpha phosphorylation in lymphocytes.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Prothymosin alpha is a polypeptide implicated in cell proliferation.
- Its in vitro phosphorylation by casein kinase-2 is known.
- The in vivo function and regulation of prothymosin alpha remain unclear.
Purpose of the Study:
- To investigate the in vivo phosphorylation of prothymosin alpha.
- To identify the phosphorylation sites and responsible kinases in stimulated lymphocytes.
- To compare phosphorylation patterns in different cell types.
Main Methods:
- Labeling of murine splenic lymphocytes with [32P] orthophosphate following mitogenic stimulation (concanavalin A + interleukin-2).
- Extraction and analysis of phosphorylated prothymosin alpha.
- Structural analysis to determine phosphorylation sites (Thr residues).
- Comparison of in vivo and in vitro (casein kinase-2) phosphorylation.
Main Results:
- [32P]prothymosin alpha was detected in mitogenically stimulated murine splenic lymphocytes.
- Phosphorylation activity increased with stimulation time and was dependent on mitogens.
- In vivo phosphorylation occurred at Thr residues within the first 14 amino acids.
- Casein kinase-2 phosphorylates both Ser and Thr residues in vitro, suggesting it's not the primary in vivo kinase.
- Phosphorylation was also observed in proliferating thymocytes and HeLa cells, with similar sites but lower rates.
Conclusions:
- Prothymosin alpha phosphorylation is associated with cell proliferation and mitogenic activation.
- Casein kinase-2 is unlikely to be the main enzyme responsible for prothymosin alpha phosphorylation in vivo.
- Further research is needed to identify the specific in vivo kinase(s) and fully elucidate prothymosin alpha's role in cell proliferation.
Abstract:
Prothymosin alpha is a widely distributed polypeptide whose function, though unknown, seems to be related to cell proliferation. In vitro, it is a substrate for casein kinase-2. In this work, extracts of mitogenically stimulated murine splenic lymphocytes labeled with [32P] orthophosphate were found to contain [32P]prothymosin alpha. Phosphorylation activity was highly dependent on mitogenic activation with concanavalin A plus interleukin-2. While cells remained viable, phosphorylation increased with stimulation time in the presence of [32P]orthophosphate. Structural analysis showed that prothymosin alpha was phosphorylated at Thr residues located among its first 14 amino acids, whereas its in vitro phosphorylation by casein kinase-2 affects both Ser and Thr residues in this fragment, apparently in similar proportions. Thus, casein kinase-2 seems not to be responsible for the phosphorylation of prothymosin alpha in vivo. Prothymosin alpha was also found to be phosphorylated in proliferating murine thymocytes and HeLa cells; the phosphorylation sites were the same as in splenic lymphocytes, but the rate of phosphorylation was about 5 times lower. In thymocytes and subconfluent HeLa cells, the [32P]prothymosin alpha concentrations of the cytosolic and nuclear fractions were similar; in splenic lymphocytes, [32P]prothymosin alpha was found mostly in cytosol.