mRNA editing is a regulated, nuclear process that requires the assembly of multi-protein editosomes. Reconstitution assays have identified the essential protein factors as the cytidine deaminase APOBEC-1 and the auxiliary factor ACF. Data presented in this report demonstrate that ACF is a phosphoprotein and that phosphoACF is restricted to nuclear 27S editosomes. ACF was phosphorylated on one or more serine residues under basal media conditions and the proportion of total cellular ACF that became phosphorylated increased upon ethanol or insulin treatment, stimuli both known to enhance editing activity. This suggests that ACF phosphorylation is metabolically regulated and a part of the mechanism for activating apoB
mRNA editing regardless of whether APOBEC-1 expression is increased [insulin treatment, (17
)] or not [ethanol treatment, (21
Recombinant APOBEC-1 and ACF purified from Escherichia coli
, baculovirus or prepared from in vitro
translation extracts can edit apoB
RNA in vitro
suggesting that phosphorylation is not essential for editosome assembly and editing activity. However, we argue that the efficiency of recombinant APOBEC-1 alone with recombinant ACF is entirely dependent on input protein concentration and the reaction has a very poor catalytic turnover, capable of only attomolar RNA substrate editing per hour (3
). This is in sharp contrast to the highly efficient endogenous hepatic or intestinal cell editing activity. In fact, editing activity in vivo
is regulated in a species- and tissue-specific manner, and inducible during development and in response to metabolic and hormonal perturbations (14
) [and reviewed in (1
)]. In this context, our data suggested that phosphorylation of ACF optimized and/or stabilized the functional interaction with APOBEC-1 in the nucleus leading to efficient editing activity. This mechanism explained how apoB
mRNA editing activity can be activated metabolically or during development using pre-existing editing factors.
Under basal conditions, inhibition of PP1 activity resulted in the nuclear retention and increased recovery of phosphoACF and increased apoB
mRNA editing activity, suggesting that ACF phosphorylation/dephosphorylation contributes to the modulation of editosome assembly and editing activity. Given that not all nuclear ACF is phosphorylated ( and ) and that not all ACF is assembled in 27S editosomes [ and (12
)] our data suggest that at any given time not all of the cellular ACF is involved in editing. This implies that there is a pool of ACF that can be used to rapidly modulate editosome assembly upon metabolic or hormonal stimuli or that ACF has additional roles in the cell.
In addition to editosome structure and function, ACF plays an important role in the cellular regulation of apoB
mRNA editing through its trafficking activity between the cytoplasm and the nucleus (24
). Although the site of apoB
mRNA editing is within the cell nucleus (2
) and takes place during or immediately after pre-mRNA splicing (2
), APOBEC-1 and ACF are distributed in both the nucleus and cytoplasm (9
). The data presented here suggest that nuclear retention/import of ACF was increased in ethanol or insulin treated hepatocytes through ACF phosphorylation. The mechanism for regulating APOBEC-1 and ACF trafficking are unknown, and the dependence of each protein’s trafficking on ACF–APOBEC-1 complex formation is controversial (25
). Data from our laboratory suggest that APOBEC-1 has strong cytoplasmic retention signals, and that its nuclear import is mediated by interactions with ACF (25
). We report that ACF and APOBEC-1 are present in both the cytoplasm and nucleus of editing competent cells, but that they only co-immunopurify from nuclear extracts. Our data suggest that nuclear retention/import of ACF is increased in ethanol or insulin treated hepatocytes through modulation of ACF phosphorylation state. We propose a model in which phosphorylation of ACF results in its nuclear accumulation and enhances or stabilizes APOBEC-1 nuclear retention and ACF binding, leading to increased editing activity. In support of this model, phosphatase treatment of cell extracts was associated with reduced co-immunoprecipitation of APOBEC-1 with ACF and reduced editing activity.
A small proportion of nuclear ACF was phosphorylated in non-stimulated hepatocytes (0.1 nM insulin) () which increased several-fold upon insulin and ethanol stimulation (). These data suggested that a low level turnover of ACF phosphorylation maybe required to maintain basal apoB
mRNA editing activity. Significantly, PP1 inhibition stimulated editing activity even under basal media conditions. The turnover of editing complexes has been suggested from studies that demonstrated nucleocytoplasmic shuttling of ACF and APOBEC-1 (26
) and from in vitro
studies of editosome assembly (12
). Addition of ethanol (or its catabolite, acetaldehyde) or chemicals affecting protein kinases and phosphatases to nuclear extracts did not affect in vitro
editing activity or ACF phosphorylation (data not shown). These data indicate that intact cell signal transduction cascades are required for the regulation of ACF phosphorylation and apoB
mRNA editing. The identification of PP1 as a candidate phosphatase involved in regulating phosphate turnover on ACF is relevant, as high levels of PP1 are present in rat hepatocyte nuclei (52
) of which 90% was associated with chromatin (53
). Nuclear ACF is also associated with chromatin (24
) placing it theoretically within the general domain of nuclear PP1. The ability of phosphoACF to serve as substrate for PP1 remains to be formally addressed.
The lack of labeled ACF in the cytoplasm also suggests that ACF is dephosphorylated prior to or during nuclear export. These data suggest an interesting hypothesis: if phosphorylation of ACF is restricted to the nucleus and associated with enhanced editing activity, then dephosphorylation of ACF might regulate its nuclear export. Given that ACF binds to both unedited and edited apoB
) and that dephosphorylated ACF binds to apoB
RNA. ACF is likely to remain bound to apoB
mRNA and co-export to the cytoplasm following apoB
mRNA editing and ACF dephosphorylation. In this scenario, the regulation of ACF dephosphorylation would modulate apoB
mRNA export to the cytoplasm in addition to protecting edited apoB
mRNA from nonsense mediated decay (NMD) (31
Under basal conditions, 2D gel electrophoresis analyses suggested that ACF contained 2–3 phosphates. 2D phosphoamino acid analyses indicated serines were the residues phosphorylated following metabolic stimulation. ACF phosphorylation and whether these sites are the same as the ‘basal’ phosphorylation sites or are additional sites of phosphorylation remains to be determined. Although parallels were observed when comparing ethanol with insulin editing induction (i.e. ACF accumulation in the nucleus and ACF hyperphosphorylation), we cannot assume that the same sites of ACF phosphorylation are involved or that the phosphorylation state of additional editing factors is not affected. In fact, previous studies involving alanine and aspartic acid site-specific mutagenesis of predicted serine phosphorylation sites suggested that APOBEC-1 may have two sites of phosphorylation which had opposing effects of editing activity (35
). APOBEC-1 has never been validated as a phosphoprotein through metabolic labeling studies similar to those reported here because the expression of endogenous APOBEC-1 is prohibitively low. We evaluated phosphorylation of APOBEC-1 overexpressed in a stable McArdle cell line under basal and ethanol stimulated conditions and found no evidence for radiolabeling during a 4 h incubation period (data not shown). If APOBEC-1 is a phosphoprotein, the sites of phosphorylation may not be subjected to acute regulation and in fact Ser47 and Ser72, which were proposed to be phosphorylated in human APOBEC-1, are not conserved in rat APOBEC-1. The expression level of, and availability of high titre antibodies against, two APOBEC-1 homologs namely activation induced deaminase (AID) and APOBEC-3G have made equivalent studies possible and both proteins were identified as phosphoproteins (54
). Consequently, phosphorylation of APOBEC-1 and its role in regulating editing activity remains a formal possibility.
In conclusion, regulation of hepatic apoB mRNA editing by ethanol and insulin promotes serine phosphorylation of ACF and its localization to active nuclear 27S editosomes. The data support a role for the metabolic regulation of ACF phosphorylation that promotes its interaction with APOBEC-1, in editosome assembly, as well as ACF nuclear retention/import. Thus, phosphorylation of ACF adds a new level of understanding of the control mechanisms cells use to modulate apoB mRNA editing in the context of current models of editosome composition and assembly.