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Phosphorylation of the secreted, free alpha subunit of human chorionic gonadotropin
Abstract:
Phosphorylation of secretory proteins is an uncommon event. In this manuscript, the phosphorylation of human chorionic gonadotropin, a glycoprotein hormone secreted by the JAR choriocarcinoma cell line, is described. Labeling of JAR cells with 32PO4 indicates that both the intracellular and the secreted forms of the free alpha subunit are phosphorylated. Although the secreted alpha beta dimer also incorporates 32PO4, there is little detectable phosphorylation of the intracellular precursors of alpha beta dimer, suggesting that dimer phosphorylation occurs as a late event in post-translational processing. In addition, phorbol 12-myristate 13-acetate markedly stimulates the phosphorylation of both intracellular and secreted forms of free alpha subunit and to a lesser extent of secreted alpha beta dimer. In vitro assays, using homogenates of JAR cells as a source of protein kinase activity, indicate that the uncombined alpha subunit is preferentially phosphorylated. The phosphorylation sites are on serine and threonine residues in the alpha subunit.
Insights
This study reveals that the alpha subunit of human chorionic gonadotropin (hCG) is phosphorylated in JAR cells. This phosphorylation, occurring on serine and threonine residues, is enhanced by phorbol ester treatment.
Area of Science:
- Biochemistry
- Cell Biology
- Endocrinology
Background:
- Phosphorylation of secretory proteins is a rare post-translational modification.
- Human chorionic gonadotropin (hCG) is a glycoprotein hormone with alpha and beta subunits.
Purpose of the Study:
- To investigate the phosphorylation of hCG in the JAR choriocarcinoma cell line.
- To identify the subunits and conditions affecting hCG phosphorylation.
Main Methods:
- JAR cells were labeled with 32PO4 to trace phosphorylation.
- In vitro kinase assays were performed using JAR cell homogenates.
- Phorbol 12-myristate 13-acetate was used to stimulate phosphorylation.
Main Results:
- Both intracellular and secreted forms of the free alpha subunit of hCG are phosphorylated.
- Phosphorylation of the alpha beta dimer occurs late in post-translational processing.
- Phorbol ester significantly increases phosphorylation of the free alpha subunit and, to a lesser extent, the alpha beta dimer.
- In vitro assays show preferential phosphorylation of the uncombined alpha subunit.
- Phosphorylation occurs on serine and threonine residues of the alpha subunit.
Conclusions:
- The alpha subunit of hCG undergoes significant phosphorylation in JAR cells.
- Phosphorylation of the alpha subunit is stimulated by phorbol ester.
- Dimerization and secretion represent late stages in the post-translational modification of hCG, with phosphorylation occurring at multiple stages.