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The Journal of Immunology, 2007, 179, 5274 -5280
Copyright © 2007 by The American Association of Immunologists, Inc.
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Role of Activation-Induced Deaminase Protein Kinase A Phosphorylation Sites in Ig Gene Conversion and Somatic Hypermutation1

Monalisa Chatterji*, Shyam Unniraman*, Kevin M. McBride{dagger} and David G. Schatz2,*

* Howard Hughes Medical Institute, Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510; and {dagger} Laboratory of Molecular Immunology, Rockefeller University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation-induced deaminase (AID) is thought to initiate somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR) by the transcription-coupled deamination of cytosine residues in Ig genes. Phosphorylation of AID by protein kinase A (PKA) and subsequent interaction of AID with replication protein A (RPA) have been proposed to play important roles in allowing AID to deaminate DNA during transcription. Serine 38 (S38) of mouse AID is phosphorylated in vivo and lies in a consensus target site for PKA, and mutation of this residue interferes with CSR and SHM. In this study, we demonstrate that S38 in mouse and chicken AID is phosphorylated in chicken DT40 cells and is required for efficient GCV and SHM in these cells. Paradoxically, zebra fish AID, which lacks a serine at the position corresponding to S38, has previously been shown to be active for CSR and we demonstrate that it is active for GCV/SHM. Aspartate 44 (D44) of zebra fish AID has been proposed to compensate for the absence of the S38 phosphorylation site but we demonstrate that mutation of D44 has no effect on GCV/SHM. Some features of zebra fish AID other than D44 might compensate for the absence of S38. Alternatively, the zebra fish protein might function in a manner that is independent of PKA and RPA in DT40 cells, raising the possibility that, under some circumstances, AID mediates efficient Ig gene diversification without the assistance of RPA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
After their assembly by V(D)J recombination, Ig genes can be altered by three distinct reactions: somatic hypermutation (SHM),3 gene conversion (GCV), and class switch recombination (CSR). SHM introduces nontemplated point mutations into the Ig V region exon while GCV replaces portions of this exon with stretches of pseudo-V donor sequences. CSR deletes portions of the Ig H chain (IgH) locus to change the H chain C region and hence Ab effector function (reviewed in Refs. 1 and 2). The contribution of SHM and GCV to postassembly V region diversification differs between species. For example, chickens rely heavily on GCV to generate the preimmune repertoire and use both GCV and SHM for further diversification during the immune response while in contrast, mice and humans use only SHM (3).

Despite their distinctive outcomes, SHM, GCV, and CSR have several mechanistic features in common including a strict requirement for the activation-induced deaminase (AID), a tight link to transcription, and involvement of the base excision repair factor uracil DNA glycosylase (UNG; reviewed in Refs. 2 , 4, 5, 6). AID is a ssDNA deaminase and is thought to initiate SHM, GCV, and CSR by deaminating cytosine to uracil in Ig genes (reviewed in Refs. 2 and 7).

AID is a mutagen thought to contribute to genome instability and cellular transformation by several different mechanisms (8, 9, 10, 11, 12, 13). Its activity is regulated at multiple levels including tissue-specific expression, protein subcellular localization, gene-specific targeting, and posttranslational modifications. Expression of AID is limited almost exclusively to activated B cells (14) and the protein shuttles in and out of the nucleus with the majority located in the cytoplasm (reviewed in Refs. 2 and 7). Ig loci are mutated more heavily than other expressed genes in activated B cells and it is plausible that this is a result of specific targeting of AID to Ig loci, although how such targeting might be achieved is unknown (reviewed in Ref. 5).

Several lines of evidence support the model that transcription plays a key role in generating the ssDNA substrate for AID (reviewed in Refs. 2 and 7), although it remains unclear exactly how AID activity is coupled to transcription. In a search for interacting factors that would facilitate the ability of AID to deaminate a model dsDNA SHM substrate in a transcription-coupled assay, Chaudhuri et al. (15) identified replication protein A (RPA), a ssDNA-binding factor involved in replication, recombination, and repair. The AID-RPA interaction strongly stimulated transcription-coupled deamination of dsDNA substrates but not ssDNA deamination activity and required phosphorylation of AID (15). Subsequently, AID purified from B cells was demonstrated to be phosphorylated on serine 38 and tyrosine 184 (16). S38 resides within a consensus protein kinase A (PKA) phosphorylation site (RRDS38) and PKA interacts with AID and is able to phosphorylate S38 as well as threonine 27 (16, 17), which resides within a consensus or variant PKA site (RRET27 or RHET27) in most species (see Fig. 1A).


Figure 1
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  		FIGURE 1. AID proteins and expression analysis. A, Alignment of N-terminal sequences of AID proteins from different organisms. Conserved amino acids in the two PKA phosphorylation sites as well as the aspartate residue present at position 44 in fish AID are in bold. The residues mutated in this study are underlined. Numbers above the sequences indicate the amino acid positions for the first six AID sequences including chicken (Gallus gallus) and mouse (Mus musculus), while the fish sequences including zebra fish (Danio rerio) contain three or four additional residues at the N terminus. The other species shown are human (Homo sapiens), chimpanzee (Pan troglodytes), dog (Canis familiaris), cow (Bos taurus), frog (Xenopus laevis), cat fish (Ictalurus punctatus), and Japanese puffer fish (Takifugu rubripes). B, Representative Western blot analyses for AID and HMGB1. The clones analyzed are as indicated above the lanes. Clones WO1, WO2, and WO5 express WT chicken AID; DO3, DO5, and DO6 express S38A chicken AID; EO1, EO2, and EO7 express T27A chicken AID; and MO1 and MO4 express T27A/S38A double mutant of chicken AID (see Table I). PIE mS38A 1, 4, and 7 express S38A mouse AID and PIE cDM 10 express double mutant chicken AID (see Table II) In some cases, two different amounts of extract (differing by 2-fold) were analyzed from individual clones (indicated by triangles above the lanes). Controls were the {psi}V parental clone (parental) and AID–/– DT40 cells. The parental clone expresses AID from a retroviral expression vector, not from the endogenous AID locus (21 ). The open arrowhead indicates a background band detected by the AID Ab. Note that clone WO2 was analyzed on each blot and was used as the basis for normalization of expression levels in other clones.

 
Although mutation of Y184 to alanine had no discernable effect on AID activity, S38A and T27A mutants of mouse AID were not phosphorylated by PKA, were unable to interact with RPA, and were defective for transcription-coupled dsDNA deamination (16). Furthermore, single (16) or double (17) mutants of these two residues were compromised in their ability to mediate CSR in cultured B cells, and increased/decreased levels of PKA activity resulted in increased/decreased CSR (16, 17). Importantly, the T27A mutation (but not S38A or Y184A) resulted in a significant decrease in ssDNA deaminase activity (16), suggesting that mutation of T27 might cause structural alterations of AID that compromise the deaminase active site. These findings support a model in which PKA-mediated phosphorylation of AID at S38 allows formation of an AID-RPA complex which binds ssDNA created during transcription, leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins (16, 17). Therefore, in addition to influencing AID activity, PKA and RPA might also affect the targeting of SHM by focusing AID to transcribed sequences.

Further support for this model was obtained using Abs specific for S38-phosphorylated AID and cellular fractionation. These experiments demonstrated that S38-phosphorylated AID (which constitutes only 5–15% of the total AID protein in activated B cells) is enriched in the chromatin-bound fraction as opposed to cytosolic or soluble nuclear fractions. Furthermore, S38A and S38D mutant forms of mouse AID have diminished SHM activity on an artificial substrate in 3T3 fibroblasts as well as at the region 5' of the IgH µ switch region in activated B cells (18). These results suggest that phosphorylation of S38 plays a role in targeting AID to DNA and that the phosphorylated form contributes disproportionately to SHM.

Curiously, AID proteins from bony fish are natural S38 mutants (see Fig. 1A; position 42 of zebra fish AID corresponds to S38 in mouse AID) and yet are active for CSR in activated B cells (19, 20) and for SHM of an artificial substrate in 3T3 fibroblasts (20). It has been proposed that aspartate 44, found in AID of fish but not other species (see Fig. 1A), allows for interaction with RPA by serving as a mimic of phosphorylated S38 (16).

The results described above strongly suggest that AID is phosphorylated by PKA in vitro as well as in vivo, but the contribution of this modification to Ig diversification processes remains uncertain. In particular, the contribution of PKA phosphorylation sites in AID to Ig GCV has not been examined, nor has the significance of these sites been assessed for SHM of an endogenous Ig V region, the natural target of the reaction. In addition, the possible function of the conserved aspartate residue in bony fish AID has not been tested in any system. To address these issues, we have measured the ability of mutant AID proteins from several species to perform GCV and SHM of the endogenous Ig L chain V region in the chicken B lymphoma cell line DT40. The results demonstrate a critical role for the PKA phosphorylation sites in mouse and chicken AID but also demonstrate that aspartate 44 of zebra fish AID is not required for robust GCV/SHM activity. It is therefore unclear whether interaction with RPA is required for AID function in chicken B cells, or if it is, how this interaction is mediated by fish AID.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Plasmids, cells, and mutagenesis

pMSCVpuro, pMX-PIE, and AID–/– DT40 cells were obtained from M. Nussenzweig (Rockefeller University, New York) and CL18 and {psi}V parental DT40 cells (21) were obtained from J.-M. Buerstedde (Institute of Molecular Radiobiology, Munich, Germany). AID mutants were generated by Quik-Change mutagenesis using Pfu polymerase (Stratagene) and were sequenced to ensure the absence of additional changes. Sequences of oligonucleotides used for mutagenesis are available upon request.

Cell culture, retroviral transduction, and flow cytometry

DT40 cells were grown as described previously (22). Retroviral production in 293T cells was performed as described previously (23) by cotransfecting the retroviral plasmid of interest and packaging plasmid pKAT (24) into 293T cells in medium supplemented with 25 µM chloroquine. Retroviral supernatants were added to recipient AID–/– DT40 cells seeded out 10–14 h earlier at 1–2 x 105 cells/ml. After addition of the viral supernatant, recipient cells were centrifuged at 648 x g for 45 min at room temperature. Cells were sorted or seeded out in puromycin (0.5 µg/ml) or without drug selection after 2 days. After 7 days of growth, single clones were transferred from 96-well dishes to 24-well dishes and maintained for an additional 4 wk. DT40 cells were analyzed for surface expression of IgM as described previously (22).

Western blotting

After 34 days of culture, protein extracts were prepared by boiling cells in SDS sample buffer, resolved by SDS-PAGE (1% SDS, 15% polyacrylamide), and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with PBS containing 5% milk overnight and then incubated with either an affinity purified rabbit polyclonal Ab raised against the peptide CEDRKAEPEGLRRLHR derived from mouse/chicken AID (aa 116–132) or an affinity purified rabbit polyclonal Ab raised against the peptide KPAAKKGVVKAEKSK from human HMGB1 (spanning aa 167–182). Blots were developed with HRP-conjugated goat anti-rabbit IgG (The Jackson Laboratory) and ECL reagent (Amersham). For quantitation, two or three amounts of each extract were analyzed to determine the linear range of the assay. Band intensities were obtained by scanning the x-ray film using a Storm 820 (Molecular Dynamics) and quantitated using GeneTools software. The AID signal for each extract was normalized to its high mobility group protein B1 signal and these values were then normalized to that of a standard extract (from clone W02; Table I) that was loaded on every gel.


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  		Table I. Mutagenic events in AID–/– DT40 cells infected with pMSCV-puro AID expression vectors

 
Assay for S38 phosphorylation

Phosphorylation of S38 was assessed using specific Abs to AID phosphoserine 38 (anti-p38) as described previously (18). This Ab was raised to a region of AID (residues 30–45) in which chicken and mouse AID are identical (see Fig. 1A). Briefly, wild-type or AID–/– B cells were purified from mouse spleens by depletion with anti-CD43 beads (Miltenyi Biotec) and cultured in RPMI 1640 medium with 5 ng/ml IL-4 plus 25 µg/ml LPS (Sigma-Aldrich) for 72 h. Cells were extracted with RIPA buffer (50 mM Tris (pH 8), 200 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 1% Nonidet P-40, 25 mM NaF, 1 mM DTT). For immunoprecipitation, 2 mg of extracts were incubated with anti-AID Ab (18) and protein A-Sepharose (Amersham Pharmacia) for 2 h. For Flag immunoprecipitation, anti-Flag M2 agarose beads (Sigma-Aldrich) were incubated with extracts for 2 h. Western blots were performed on the immunoprecipitated protein with anti-AID Ab or anti-p38. Chicken AID with three copies of the Flag epitope tag at its C terminus was expressed in AIDRUng–/– cells (25) from which the AIDR expression cassette (21) had been deleted by induction of Cre recombinase by 8-hydroxy tamoxifen treatment.

Sequence analysis

IgL and IgH V regions were amplified and sequenced as described previously (22) except that primer VLA-SEQ1 (5'-CTCTGTCCCACTGCTGCGCG-3') was used to sequence the IgL V region. Sequences were aligned using ClustalW and mutations were classified as GCV, templated point mutations (TPM), or nontemplated point mutation (NPM) as described previously (22). GCV events consisted of two or more nearby mutations that exactly matched the same pseudo-V ({psi}V) gene segment and were flanked by at least 5-bp identity on the 5' side and 3-bp identity on the 3' side with the relevant {psi}V. Single nucleotide changes were scored as TPM when the mutation as well as the 5 bp upstream and downstream of it were identical with a {psi}V gene segment. Less stringent criteria were used for GCV because they involve two or more closely linked mutations that derive from the same donor and are less likely to be the result of a PCR or sequencing error than TPM. NPM were those single nucleotide point mutations not satisfying this criterion. "Other" events included ambiguous sequences, deletions, and insertions. One-tailed unpaired t tests were performed using GraphPad Prism, version 4.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Chicken AID mutants

To address the role of PKA phosphorylation sites in AID in GCV and SHM in DT40 cells, we mutated the two putative PKA phosphorylation sites in chicken AID-RRDS38 and RRET27 (Fig. 1A). Wild-type (WT) and S38A, T27A, and double mutant AID proteins were stably expressed in AID-deficient DT40 cells using the retroviral vector pMSCVpuro, in which AID was expressed from the 5' long terminal repeat and the puromycin resistant gene was expressed from an internal promoter. Puromycin-resistant single-cell colonies were expanded and analyzed for AID expression by Western blot and for mutations in the Ig L chain (IgL) V region by sequencing (after 5 wk of culture).

Clones expressing WT, S38A, T27A, and T27A/S38A double mutant chicken AID were selected for sequence analysis based on roughly equivalent levels of AID expression (Fig. 1B, Table I). The three clones expressing WT chicken AID accumulated numerous mutations in their IgL V region (Table I), which were scored as GCV events, TPM, or NPM (defined as in Materials and Methods). In total, 49 events were observed in 86 sequences for an average mutation frequency of 16.8 events/104 bp. In sharp contrast, not a single mutation was observed in 86 sequences from three clones expressing S38A AID (<0.4 events/104 bp). Nine mutation events were observed in 87 sequences from three clones expressing T27A AID (3.1 events/104 bp) while the double mutant yielded two point mutations in 59 sequences from two clones (1.0 event/104 bp) (Table I, Fig. 2).


Figure 2
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  		FIGURE 2. Scatter plot of GCV/SHM data. The total frequency of GCV and SHM events is plotted for each cell clone for WT (open symbols) and mutant (closed symbols) forms of AID as detailed at the bottom of the graph. The mean for each form of AID is indicated with a horizontal line. AID was expressed from the pMX-PIE vector except where indicated. DM, Double T27A/S38A mutant AID.

 
We also measured the gain of surface IgM expression in the clones over time in culture to assess correction of a frame-shift mutation in the IgL V region by GCV. The IgM reversion data were consistent with the sequencing results, showing activity for WT AID but little or no activity for the mutants. However, the cells expressing WT AID underwent IgM reversion at a much reduced frequency compared with parental CL18 DT40 cells (data not shown), making it difficult to assess GCV activity reliably using this assay. Subsequent analyses relied exclusively on sequencing to assess GCV/SHM frequencies.

Quantitation of Western blots suggested that S38A AID was expressed at slightly lower levels than WT AID in the clones analyzed (Table I). To address whether this might be responsible for the mutant’s lack of activity, we cloned WT and mutant forms of chicken AID into a second retroviral expression vector (pMX-PIE (26)) in which expression of AID and enhanced GFP (EGFP; separated by an internal ribosome entry site) was driven by the 5' long terminal repeat. AID–/– DT40 cells were infected with AID-expressing retroviruses and single EGFP-positive cells were isolated by flow cytometry, expanded, and AID expression levels were monitored both by measurements of EGFP fluorescence and by Western blotting (Table II). Sequencing of the IgL V region yielded results comparable to those obtained with the pMSCVpuro expression vector: WT AID supported efficient GCV/SHM (29 events in 86 sequences; 10.0 events/104 bp) while the S38A (1.5 events/104 bp) and T27A/S38A (0.3 events/104 bp) mutants yielded mutation frequencies similar to control clones infected with the empty retrovirus (0.6 events/104 bp) and the T27A mutant yielded intermediate mutation levels (4.3 events/104 bp) (Table II, Fig. 2). Both EGFP mean fluorescence intensity and Western blot signals were stronger for the S38A mutant than for WT AID (Table II), ruling out the possibility that lower levels of expression could explain the poor activity of the mutant protein. Overall, these results demonstrate that serine 38 of chicken AID plays a critical role in GCV/SHM in DT40 and that threonine 27 is also important for full activity of the protein.


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  		Table II. Mutagenic events in AID–/– DT40 cells infected with pMX-PIE AID expression vectors

 
Mouse AID mutants

As described in the Introduction, WT and PKA phosphorylation-site mutant forms of murine AID have been analyzed in vitro and in various CSR and SHM assays in mammalian cells. It was therefore of interest to assess the activity of these murine AID proteins in DT40 cells. WT, S38A, and T27A forms of murine AID were expressed in AID–/– DT40 cells using the pMX-PIE expression vector. As above, EGFP-positive single-cell clones were expanded and analyzed for EGFP and AID expression and for mutations by sequencing. We observed that WT mouse AID was able to support GCV and SHM in DT40 cells (five separate clones) and surprisingly, did so {approx}3-fold more efficiently than did WT chicken AID (30.3 events/104 bp for mouse vs 10.0 events/104 bp for chicken; p < 0.002) (Table II, Fig. 2). Consistent with the results with chicken AID (Tables I and II, Fig. 2), mutation of S38 or T27 in mouse AID reduced GCV/SHM activity levels to only 15 or 17%, respectively, of that seen with WT mouse AID (Table II, Fig. 2). We conclude that S38 and T27 play important roles for the function of both chicken and mouse AID in DT40 cells.

Zebra fish AID

As described in the Introduction, zebra fish AID is active for CSR and SHM but is a natural serine to glycine mutant at position 42 corresponding to S38 of mouse and chicken AID (19, 20). Like AID from Japanese puffer fish and cat fish, it contains conserved proline and aspartate44 (D44) residues immediately downstream of this position (Fig. 1A), raising the possibility that the negatively charged aspartate in fish AID serves as a PKA-independent functional mimic of phosphorylated S38 (16).

To test this idea and determine whether zebra fish AID can support GCV and SHM of an endogenous Ig V region, WT and D44A mutant forms of zebra fish AID were expressed in AID–/– DT40 cells using pMX-PIE. Sequence analyses after 5 wk of culture revealed that WT zebra fish AID (13.3 events/104 bp) is as active as chicken AID but less active than mouse AID, and that zebra fish AID can mediate both GCV and SHM events (Table II, Fig. 2). In cells expressing zebra fish AID, EGFP fluorescence levels were comparable to those in cells expressing chicken or mouse AID, and Western blot signals were relatively low for zebra fish AID relative to the chicken or mouse protein (Table II). We infer that zebra fish AID is not functionally overexpressed (relative to chicken or mouse AID) in our experiments.

Strikingly, the D44A zebra fish AID mutant (25.4 events/104 bp) maintained activity and was at least as active as WT zebra fish AID (Table II, Fig. 2). The mutant was not more highly expressed than the WT protein as assessed by Western blot, although EGFP levels were high in the two most active clones expressing the D44A mutant. Nonetheless, even if these two clones are ignored, it is clear that the D44A mutant is as active as WT zebra fish AID. We conclude that D44 is not required for full GCV/SHM activity by zebra fish AID and hence that this amino acid does not compensate for the absence of the S38 PKA phosphorylation site in DT40 cells.

Analysis of single point mutations (both TPM and NPM) generated by WT chicken and mouse AID, as well as WT and D44A zebra fish AID, revealed a strong bias in favor of mutation of GC bp, particularly in the NPM mutations, as well as a bias toward transversion mutations (Table III). These results are in good agreement with earlier mutation analyses in DT40 cells (21, 27) and argue that similar repair pathways operate downstream of the different forms of AID examined here.


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  		Table III. Spectrum of point mutationsa

 
S38 is phosphorylated in DT40 cells

Given the robust activity associated D44A zebra fish AID, it was important to determine whether S38 of mouse and chicken AID is phosphorylated in DT40 cells. To accomplish this, we used a recently described assay involving immunoprecipitation of AID followed by Western blotting with Abs specific for AID phosphoserine-38 (anti-p38) (18). WT mouse AID immunoprecipitated from cell clone PIE mW 17 was detected by the anti-p38 Ab (Fig. 3, lane 4) but the S38A mutant form of AID from clone PIE mS38A 7 was not (lane 5). As controls, the assay was also performed with LPS plus IL4-stimulated B cells from AID–/– and WT mice (lanes 2 and 3). Flag-epitope tagged chicken AID was also detected by the anti-p38 Ab in two independent DT40 cell lines (lanes 7 and 8), but no corresponding band was visible in AID–/– DT40 cells (lane 6). We conclude that S38 of mouse and chicken AID is phosphorylated in DT40 cells and that the level of phosphorylation is comparable to that of mouse AID in splenic B cells.


Figure 3
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  		FIGURE 3. AID serine 38 is phosphorylated in DT40 cells. Anti-p38 and anti-AID immunoblots of rAID from E. coli (rAID) (lane 1) or AID purified by anti-AID immunoprecipitation from AID–/– (lane 2) or WT mouse (lane 3) splenocytes cultured for 72 h with LPS and IL-4, DT40 (AID–/–) cells expressing WT mouse AID (clone PIE mW 17, lane 4) or S38A mouse AID (clone PIE mS38A 7, lane 5) or purified by anti-Flag immunoprecipitation from DT40 (AID–/–, UNG–/–) cells alone (lane 6) or expressing Flag-tagged chicken AID, clone 11A (lane 7) and clone 20E (lane 8).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Both biochemical and cell culture experiments have provided support for the model that PKA phosphorylation of mouse AID at S38 is important for its function (28). This residue is phosphorylated in activated B cells and its mutation reduces AID activity in a variety of assays of CSR and SHM. Furthermore, modulation of PKA activity in cultured B cells alters CSR activity in a parallel fashion (see Introduction). Consistent with this, our results demonstrate that S38 is phosphorylated in DT40 cells and that efficient GCV/SHM of an endogenous Ig V region by chicken or mouse AID requires the integrity of this residue. Our data reveal that the S38A mutation reduces GCV/SHM activity to 15% or less of that seen with WT AID, while mutation of T27 has a somewhat smaller effect and the T27A/S38A double mutant is essentially inactive for GCV/SHM. Hence, in species where it is present, serine 38 is required for full AID activity in all three AID-dependent reactions: CSR, SHM, and GCV.

The above experiments also revealed a higher rate of GCV and SHM in DT40 cells expressing mouse AID compared with those expressing chicken AID. This difference was not due to variations in experimental conditions as experiments involving mouse and chicken AID were performed in parallel. Nor could it easily be explained by AID levels because chicken AID was expressed as well as the mouse protein in most clones, as assessed both by EGFP fluorescence and Western blotting (Table II). One possible explanation is that the mouse AID protein escapes some form of negative regulation (29) that acts on the chicken protein in chicken cells.

We also attempted to address the role of AID phosphorylation by PKA in DT40 cells using two PKA inhibitors, H89 and Rp-cAMP. We were, however, unable to identify concentrations of these inhibitors that strongly inhibited PKA activity (as assessed by biochemical assays of PKA activity in cellular extracts) while avoiding significant cell toxicity. Parental CL18 DT40 cells cultured for 4 wk under conditions that reduced PKA activity to 50% of normal (200 µM Rp-cAMP) had no discernable deficit in GCV/SHM (data not shown).

Zebra fish AID presents a challenge to the hypothesis that phosphorylation of S38 is required for robust AID activity because it lacks a serine at position 42, corresponding to S38 of mouse or chicken AID, and yet is active for CSR and SHM (19, 20). Our data extend this by demonstrating that zebra fish AID performs GCV/SHM of the DT40 IgL V region, and therefore that a PKA phosphorylation site corresponding to S38 is not required for any of the known functions of AID. If S38 exerts its function by virtue of being phosphorylated by PKA, then some other feature of fish AID would be predicted to compensate for the lack of this phosphorylation site. An appealing candidate is D44 (16), a negatively charged residue just 2 aa away and found only in fish AID. Our results, however, show that the activity of zebra fish AID in GCV/SHM in DT40 cells is unaltered by mutation of D44.

Because S38 mutants are hypomorphs that retain full ssDNA deaminase activity in vitro, high-level overexpression of this protein could obscure significant functional defects (2, 28), perhaps explaining the recent finding that retrovirally expressed S38A mutant human AID exhibited 80% of WT CSR activity in ex vivo-stimulated B cells (30). Clearly, the expression levels obtained in our experiments did not conceal the GCV/SHM defect caused by mutation of S38 in mouse or chicken AID, and hence overexpression was unlikely to be a major issue for these proteins. Zebra fish AID was not more active than the chicken and mouse proteins and it is unlikely that overexpression can explain the robust activity associated with the zebra fish D44A mutant protein.

The anti-AID Ab used for Western blot quantitation of AID expression (Fig. 1, Tables I and II) was raised to a peptide derived from mouse/chicken AID, and zebra fish AID has multiple amino acid changes in this region (31). Therefore, the Western blot analysis might underestimate expression of the zebra fish protein. The relatively modest levels of activity observed for zebra fish AID (similar to chicken AID, lower than mouse AID) might stem from the thermolability of fish AID at 37°C (19, 32) and the fact that our experiments were performed in cells cultured at 41°C.

We consider two explanations for the finding that zebra fish D44A mutant AID retains full activity. One possibility is that some other feature of fish AID supports the interaction of AID with RPA in DT40 cells. Conceivably, the conserved threonine corresponding to mouse and chicken T27 could perform this function, although it does not reside within a PKA phosphorylation motif in all fish species (e.g., catfish AID; Fig. 1A). A second possibility is that zebra fish AID functions in a PKA- and RPA-independent manner in DT40 cells. Fish AID differs from its mouse and chicken counterparts at numerous residues, most strikingly with an 8-aa insertion in the cytidine deaminase motif (31). One or more of these changes might allow fish AID to gain access to transcription-induced ssDNA in V regions without interaction with RPA. It was recently observed that phosphorylation of recombinant human AID by PKA and addition of RPA did not increase deamination activity in a transcription-coupled dsDNA deamination assay using Escherichia coli RNA polymerase (33). By extension, it is possible that S38 has PKA- and RPA-independent functions in DT40 cells and hence that the decrease in activity observed when chicken or mouse AID is mutated at S38 is due to a loss of these other functions. It will therefore be of considerable importance to test directly the role of RPA in AID-dependent Ig diversification reactions in vivo.


   Acknowledgments
 
We thank M. Nussenzweig for pMX-PIE and AID–/– DT40 cells; J.-M. Buerstedde for CL18, {psi}V parental, and AIDRUNG–/– DT40 cells; S. Yang for AID–/– EGFP-positive clones and many helpful suggestions; M. Strout for DT40 cells expressing Flag-tagged chicken AID; D. Chen for help with initial experiments; and members of Schatz laboratory for help and encouragement.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. David G. Schatz, Howard Hughes Medical Institute, Department of Immunobiology, Yale Medical School, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011. E-mail address: david.schatz{at}yale.edu Back

3 Abbreviations used in this paper: SHM, somatic hypermutation; CSR, class switch recombination; GCV, gene conversion; AID, activation-induced deaminase; UNG, uracil DNA glycosylase; PKA, protein kinase A; RPA, replication protein A; TPM, templated point mutation; NPM, nontemplated point mutation; WT, wild type; EGFP, enhanced GFP. Back

Received for publication March 30, 2007. Accepted for publication August 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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