Functional characterization of APOBEC-1 complementation factor phosphorylation sites.
ApoB mRNA editing involves site-specific deamination of cytidine 6666 producing an in-frame translation stop codon. Editing minimally requires APOBEC-1 and APOBEC-1 complementation factor (ACF). Metabolic stimulation of apoB mRNA editing in hepatocytes is associated with serine phosphorylation of ACF localized to editing competent, nuclear 27S editosomes. We demonstrate that activation of protein kinase C (PKC) stimulated editing and enhanced ACF phosphorylation in rat primary hepatocytes. Conversely, activation of protein kinase A (PKA) had no effect on editing. Recombinant PKC efficiently phosphorylated purified ACF64 protein in vitro, whereas PKA did not. Mutagenesis of predicted PKC phosphorylation sites S154 and S368 to alanine inhibited ethanol-stimulated induction of editing suggesting that these sites function in the metabolic regulation of editing. Consistent with this interpretation, substitution of S154 and S368 with aspartic acid stimulated editing to levels comparable to ethanol treatment in control McArdle RH7777 cells. These data suggest that phosphorylation of ACF by PKC may be a key regulatory mechanism of apoB mRNA editing in rat hepatocytes.
Lehmann
David M
DM
Environmental Health Sciences Center, Department of Toxicology, University of Rochester, Rochester, NY 14642, USA.
Galloway
Chad A
CA
MacElrevey
Celeste
C
Sowden
Mark P
MP
Wedekind
Joseph E
JE
Smith
Harold C
HC
D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics; D002478:Cells, Cultured; D004789:Enzyme Activation; D022781:Hepatocytes / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment
C511990:APOBEC-1 complementation factor, rat; D001055:Apolipoproteins B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012333:RNA, Messenger; D011494:Protein Kinases