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Major vault protein is a substrate of endogenous protein kinases in CHO and PC12 cells
1Biozentrum der Johann-Wolfgang Goethe-Universität, AK Neurochemie, Frankfurt am Main, Germany.
Abstract:
Major vault protein (MVP) is the predominant member of a large cytosolic ribonucleoprotein particle, termed vault. We have previously shown that MVP derived from electric ray electric organ becomes phosphorylated by protein kinase C in vitro and by tyrosine kinase in vivo. Here we show that MVP from two mammalian cell lines (CHO and PC12 cell) becomes highly phosphorylated by endogenous protein kinases in cell-free systems. The susceptibility to protein kinases differs substantially from those observed in MVP derived from electric organ. Phosphorylation of MVP depends on the presence of Mg2+ and can be inhibited by the chelating agent EDTA. Inhibitors of casein kinase II attenuate the phosphorylation of MVP. In contrast to CHO cells, addition of recombinant casein kinase II enhances the phosphorylation of MVP in PC12 cells. Endogenous kinase activity is of particulate nature and copurifies with vault particles. Immuno-affinity purified vaults containing recombinant tagged MVP expressed in CHO cells reveal no autophosphorylation, suggesting that protein kinase activity is not an intrinsic property of vaults. Our results suggest that cell-specific phosphorylation of MVP may play a critical role in vault function.
Insights
Major vault protein (MVP) phosphorylation varies significantly between cell types. This cell-specific protein kinase activity suggests a critical role for MVP in vault function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Major vault protein (MVP) is a key component of cytosolic vault particles.
- Previous research indicated MVP phosphorylation by protein kinase C and tyrosine kinase.
Purpose of the Study:
- To investigate MVP phosphorylation by endogenous protein kinases in mammalian cell lines.
- To compare phosphorylation patterns between electric organ and mammalian MVP.
- To elucidate the role of cell-specific phosphorylation in vault function.
Main Methods:
- Cell-free phosphorylation assays using mammalian cell lines (CHO and PC12).
- Analysis of kinase activity dependence on Mg2+ and inhibition by EDTA.
- Assessment of casein kinase II involvement using inhibitors and recombinant enzyme.
- Copurification of kinase activity with vault particles.
- Investigation of vault autophosphorylation using purified vaults.
Main Results:
- Mammalian MVP (CHO, PC12) undergoes significant phosphorylation by endogenous kinases in cell-free systems.
- Phosphorylation susceptibility differs from electric organ-derived MVP.
- Phosphorylation requires Mg2+ and is sensitive to EDTA.
- Casein kinase II activity influences MVP phosphorylation, with differential effects in CHO and PC12 cells.
- Endogenous kinase activity is particulate and associated with vaults, but vaults do not exhibit autophosphorylation.
Conclusions:
- MVP phosphorylation is cell-specific and mediated by associated kinases, not intrinsic to the vault particle.
- These findings highlight the potential importance of cell-specific MVP phosphorylation in regulating vault function.