Abstract
Sensitive to Apoptosis Gene (SAG), a RING component of SCF E3 ubiquitin ligase, was shown to be phosphorylated by protein kinase CK2 at the Thr10 residue. It is, however, unknown whether this phosphorylation is stress-responsive or whether the phosphorylation changes its E3 ubiquitin ligase activity. To address these, we made a specific antibody against the phosphor-SAGThr10. Transient transfection experiment showed that SAG was phosphorylated at Thr10 which can be significantly inhibited by TBB, a relatively specific inhibitor of protein kinase CK2. To determine whether this SAG phosphorylation is stress-responsive, we defined a chemical-hypoxia condition in which SAG and CK2 were both induced. Under this condition, we failed to detect SAG phosphorylation at Thr10, which was readily detected, however, in the presence of MG132, a proteasome inhibitor, suggesting that the phosphorylated SAG has undergone a rapid degradation. To further define this, we made two SAG mutants, SAG-T10A which abolishes the SAG phosphorylation and SAG-T10E, which mimics the constitutive SAG phosphorylation. The half-life study revealed that indeed, SAG-T10E has a much shorter protein half-life (2 h), as compared to wild-type SAG (10 h). Again, rapid degradation of SAG-T10E in cells can be blocked by MG132. Thus, it appears that CK2-induced SAG phosphorylation at Thr10 regulates its stability through a proteasome-dependent pathway. Immunocytochemistry study showed that SAG as well as its phosphorylation mutants, was mainly localized in nucleus and lightly in cytoplasm. Hypoxia condition did not change their sub-cellular localization. Finally, an in vitro ubiqutination assay showed that SAG mutation at Thr10 did not change its E3 ligase activity when complexed with cullin-1. These studies suggested that CK2 might regulate SAG-SCF E3 ligase activity through modulating SAG’s stability, rather than its enzymatic activity directly.
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Abbreviations
- CHX:
-
cycloheximide
- CK2:
-
protein kinase CK2
- ROC1:
-
Regulator of Cullin-1
- Rbx1:
-
RING box protein-1
- SAG:
-
Sensitive to Apoptosis Gene
- SCF:
-
Skp1, cullin, F-box protein complex
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Acknowledgments
This work was supported by NIH grants R01-CA111554 and R21-CA116982 (YS), and by V.A. Medical Research Fund and NIH grant CA-15062 (KA).
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He, H., Tan, M., Pamarthy, D. et al. CK2 phosphorylation of SAG at Thr10 regulates SAG stability, but not its E3 ligase activity . Mol Cell Biochem 295, 179–188 (2007). https://doi.org/10.1007/s11010-006-9287-3
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DOI: https://doi.org/10.1007/s11010-006-9287-3