Figure Legends for Supplementary data:
Supplementary Figure 1.
(A) Protein sequence alignment of residues surrounding the PKA sites at T27 and S38 from different species. The 'd' is the aspartate residue conserved in fish (B) Peptides corresponding to the T27 site or the S38 site, each 11 amino acid long and containing the threonine or serine residue (T27wt or S38wt) or with the site mutated to alanine (T27A or S38A), were phosphorylated by PKA in vitro in presence of [g-32P]ATP. The phosphorylation of these peptides was assayed by filter binding over a nitrocellulose membrane followed by liquid scintillation counting. For (B), values represent the mean from three independent experiments and error bars represent the standard deviation from the mean.
Supplementary Figure 2.
(A) Titration of ssDNA deamination activity of AIDWT, AIDS38A and AIDY184A at different protein concentrations. (B) RPA-dependent dsDNA deaminase activity of PKA, AID293 and PKA-phosphorylated AID293 treated with Calf Intestinal Alkaline Phosphatase (CIP). (C) Purified AIDWT and AIDS38A proteins were phosphorylated by PKA in the presence of [g-32P]ATP and run on a 15%SDS-PAGE, transferred to a membrane and assayed by autoradiography (upper panel) and the AID protein levels determined by western blotting (lower panel). (D) The purified AIDWT and AIDS38A proteins were assayed for RPA-dependent dsDNA deaminase activity and (E) the same proteins were assayed for ssDNA deaminase activity. For (B), (D) and (E), values represent the mean from three independent experiments and error bars represent the standard deviation from the mean.
Supplementary Figure 3.
(A) Splenic B cells were stimulated for CSR by treatment with IL4 and anti-CD40 antibodies and an equal number of cells were harvested after days 0, 2, 3 and 4. Nuclear and cytoplasmic extracts (30mg) from these cells were run on SDS-PAGE followed by western blotting using anti-PKA catalytic subunit antibodies (upper panel) or anti-AID antibodies (lower panel). (B) These extracts were assayed for PKA activity. 250 ng of each extract was used to phosphorylate a PKA consensus site containing peptide. The mobility of the phosphorylated peptide (+) or unphosphorylated peptide (-) indicates PKA activity in these extracts. PKA activity after days 0, 2, 3 and 4 present in the nuclear extract (top panel) and cytoplasmic extracts (bottom panel) are shown. PKA activity in the nucleus is sensitive to H89 treatment (middle panel).
Supplementary Figure 4.
(A) CH12F3 cells were stimulated to induce CSR with or without treatment with the PKA-inhibitor H89 (10mm/ml) for 48 hrs. CSR was detected by FACS for surface expression of IgA in the live cell gate. (B) CH12F3 cells were cultured under the depicted conditions and total RNA was harvested at the onset of culture and after 24 hours. RT-PCR was performed to quantify alpha germline transcripts (a-GLTs) using the housekeeping gene HPRT as a loading control. In addition, total protein was isolated and resolved by SDS-PAGE (C), transferred to a membrane and blotted with an anti-AID antibodies (top panel), using Lamin B as a loading control (lower panel).
SUPPLEMENTARY METHODS
Mass spectrometric analysis of phosphorylated AIDBcell
40 pmol of AID purified from B cells was run in a 15% SDS-PAGE, coomassie stained and analyzed by MS/MS at the Harvard Medical School Mass spectrometric analysis facility as described below1.
Protein bands were excised from gels and cut into approximately 1 mm pieces. Gel pieces were then subjected to modified in-gel trypsin digestion procedures. Gel pieces were washed and dehydrated with acetonitrile for 10 min. followed by removal of acetonitrile. Pieces were then completely dried in a speed-vac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5 ng/ml modified sequencing-grade trypsin (Promega, Madison, WI) at 4°C for 45 minutes. The excess trypsin solution was removed, and replaced with 50mM ammonium bicarbonate solution, and samples were then incubated at 37°C room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by two washes, for 20 min each, with a solution containing 50% acetonitrile and 5% formic acid. The extracts were dried and stored at 4°C until analysis.
For analysis, samples were reconstituted in 5ml of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse phase HPLC capillary column was created by packing 5um C18 spherical silica beads into a fused silica capillary (75 um inner diameter, 12 cm length) with a flame drawn tip. After equilibrating the column each sample was pressure-loaded off-line into the column. The column was then reattached to the HPLC system. A gradient was formed and peptides were eluted with increasing concentration of solvent B (97.5% acetonitrile, 0.1% formic acid). As each peptide was eluted they were subjected to electrospray ionization and then they entered into an LTQ linear ion-trap mass spectrometer (Thermofinnigan, San Jose, CA). Eluting peptides were detected isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein or translated nucleotide databases with the acquired fragmentation pattern by the software program, Sequest (Thermofinnigan, San Jose, CA). Out of a possible 17 tryptic peptides from the AID protein sequence, 16 peptides were recovered and analyzed by this approach. As mentioned above, all the amino acids in the peptides recovered were analyzed for a phosphorylated residue. Based on these analyses, the two phosphorylated peptides with their most likely phosphorylated residues were (RDSATSCSLDFGHLR) and (ILLPLYEVDDLR).
AID immunoprecipitation assay
Nuclear extracts from AID cDNA-tranfected 293 cells or B cells were prepared as described earlier 2 . The extract was incubated with 10 units of purified PKA (sigma) and [g-32P] ATP for 1h at 30°C and then treated with anti-AID antibodies(20mg) and protein A-sepharose for 6h at 4°C in buffer A (20mM Tris, pH 7.5, 1mM DTT, 10 mM ZnCl2, 0.5mM EDTA, 500 mM NaCl and 10% glycerol ) and immunoprecipitated with anti-AID antibodies. The immunoprecipitate was washed with buffer A and then analyzed by SDS-gel followed by autoradiography to detect phosphorylated AID. To determine RPA interaction, the immunoprecipitate was incubated (in Buffer A+150 mM NaCl) with whole cell extracts derived from 293 cells, the immunoprecipitate was washed in the same buffer and then analyzed by western blotting using anti-RPA32 antibodies
PKA assay
The phosphorylation of florescent kempeptide containing PKA-phosphorylation sites works on the basis that addition of phosphate to the peptide alters its mobility on a neutral pH agarose gel (0.8%) and the phosphorylated peptide migrates towards the anode 3. The source of the reagents for the assay (florescent peptide and buffer) is Promega (PepTag non-Radioactive cAMP dependent Protein kinase Assay system). In Fig. 2B, approximately 500mg extracts from cells were immunoprecipaitated with anti-AID antibodies and assayed for phosphorylation of kempetide where as in Supp.Fig 3, 250ng of nuclear or cytoplasmic extracts of activated B cells were assayed for phosphorylation of kempeptide.
In vitro phosphorylation of AID peptides
The phosphorylation of AID peptides was carried out in reaction mixtures (20 ml each) containing 20mM Tris-HCl pH 7.5, 100mM KCl, 5mM DTT, 10mM MgCl2, 2mM sodium phosphate buffer pH 7.5, 1mM [g-32P] ATP, 1mg of peptide and 5 units of purified PKA (Sigma) for 30 min at 37°C. The reaction was terminated by addition of 5 ml of 20mg of BSA and 5 ml of 5%Tricholoacetic acid (TCA) solution containing 1mM sodium pyrophosphate at 4°C. After 20 min., the mixtures were passed through 5%TCA pre-soaked nitrocellulose filters. The filters were washed and dried and assayed for retained radioactivity in a liquid scintillation spectrometer. The sequences of the PKA peptides used are as follows. (a) YVVKRRDSATS (b) YVVKRRDAATS (c) AKGRHETYLCY (d) AKGRHEAYLCY.
CSR assay in CH12F3 cells treated with H89
2.5-5.0 x 10e4 CH12F3 cells were treated with or without the PKA inhibitor H-89 (Calbiochem) at 10 mm/ml for 30 minutes, and then stimulated for CSR as previously described 4, with cultured cells harvested immediately, 24 hours, and 48 hours after stimulation. Total RNA was prepared using Tripure reagent (Roche) and cDNA was generated (Superscript, Invitrogen) using previously described oligonucletide sequences 5. Cells were stained with anti-B220-CyC (Pharmingen) and anti-IgA (Southern Biotech) and analyzed on a FACScalibur using FlowJo (TreeStar) software. Total protein extract (30mg) of each culture was run on a SDS-PAGE and analyzed for expression of AID and Lamin B (loading control).
REFERENCES
1. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-858 (1996).
2. Chaudhuri, J. et al. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726-730. (2003).
3. Zhong, H., SuYang, H., Erdjument-Bromage, H., Tempst, P. & Ghosh, S. The transcriptional activity of NF-kappaB is regulated by the IkappaB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89, 413-424 (1997).
4. Nakamura, M. et al. High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. Int Immunol 8, 193-201 (1996).
5. Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553-563. (2000).