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Phosphorylation is a regulatory mechanism in apolipoprotein B mRNA editing
Z Chen1, T L Eggerman, A P Patterson
1National Heart, Lung and Blood Institute, National Institutes of Health, 6000 Executive Boulevard, Suite 302, Bethesda, MD 20892, USA.
Abstract:
The editing of apolipoprotein B (apoB) mRNA is under tissue-specific, developmental and metabolic regulation. We found that multiple protein kinase inhibitors or activators increased apoB mRNA editing up to 2.5-fold in Caco-2 cells and 3-8-fold in McA7777 and FAO rat cells respectively. The phosphorylation-agent-induced modulation is independent of the apolipoprotein B editing catalytic subunit 1 (APOBEC-1) and of apoB mRNA expression levels, indicating the involvement of a protein modification, such as phosphorylation, regulating the cellular editing of apoB mRNA. Transient expression of protein kinase C-θ more than doubled apoB mRNA editing in FAO cells. Chronic exposure to ethanol, a treatment known to increase the expression of protein kinases and to change protein phosphorylation status, increased apoB mRNA editing in FAO cells up to 2.5-fold without increasing the mRNA abundance of APOBEC-1. The elimination of potential phosphorylation sites 47 and 72 of human APOBEC-1 decreased its activity to approx. one-eighth of control levels by a Ser(47)-->Ala mutation, but more than doubled the activity by a Ser(72)-->Ala mutation. The activity modulation was reversed by a Ser-->Asp mutation at sites 47 and 72, which introduced a phosphorylation-like carbonic acid group. Both human APOBEC-1 dephosphorylated by alkaline phosphase and the Ser(47,72)-to-alanine double mutant protein demonstrated a shifted isoelectric focusing pattern compared with the wild type, indicating phosphorylation at these sites. Taken together, these results suggest that phosphorylation might be an important mechanism in the regulation of apoB mRNA editing.
Insights
Protein phosphorylation regulates apolipoprotein B (apoB) mRNA editing. Kinase modulators and ethanol treatment increased apoB mRNA editing, suggesting phosphorylation controls this process independently of APOBEC-1 levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Gene Regulation
Background:
- Apolipoprotein B (apoB) mRNA editing is a crucial post-transcriptional modification.
- This process is known to be regulated by tissue-specific, developmental, and metabolic factors.
Purpose of the Study:
- To investigate the role of protein phosphorylation in regulating apoB mRNA editing.
- To determine if protein kinase activity influences apoB mRNA editing efficiency.
Main Methods:
- Utilized cell culture models (Caco-2, McA7777, FAO rat cells).
- Administered protein kinase inhibitors/activators and ethanol.
- Assessed apoB mRNA editing levels and APOBEC-1 expression.
- Performed site-directed mutagenesis on human APOBEC-1 to analyze phosphorylation sites (Ser47, Ser72).
- Used isoelectric focusing to detect protein phosphorylation.
Main Results:
- Protein kinase modulators significantly increased apoB mRNA editing in various cell lines.
- Ethanol exposure also elevated apoB mRNA editing without altering APOBEC-1 mRNA levels.
- Mutations at Ser47 and Ser72 of APOBEC-1 affected its editing activity, with Ser47 phosphorylation decreasing and Ser72 phosphorylation increasing activity.
- Phosphorylation-mimicking mutations reversed these effects.
- Isoelectric focusing confirmed phosphorylation at Ser47 and Ser72.
Conclusions:
- Protein phosphorylation is a key regulatory mechanism for apoB mRNA editing.
- Kinase activity and specific phosphorylation sites on APOBEC-1 play critical roles in modulating editing efficiency.