If our mutations really affect the tyrosine phosphorylation of CTD by c- Abl, in response to DNA damage, then the level of phosphorylation on some mutants should be reduced compared to those containing a wt last repeat. Cells expressing the previously untested CKII and 'last repeat' Pol II LS* mutants were exposed to IR before harvesting one hour later, a time point where the most tyrosine phosphorylation is to be seen (Baskaran et al., 1993; Baskaran et al., 1999). In order to detect the phospho-tyrosine content of the expressed mutant, immunoprecipitation (IP) is first required to remove the background signal from other cellular proteins.
Figure 20. Tyrosine phosphorylation of Pol II LS* CTD mutants following IR. Cells expressing Pol II LS* CTD mutants were irradiated with 12 Gy IR, then harvested 1h later. Immunoprecipitation (IP) was performed using the anti-HA antibody, 3F10. Samples were separated by SDS-PAGE and Western blots thereof were stained with either (A) anti-HA (3F10) mAb, to control IP yield, or (B) the anti-pY antibody, pY100.
Western analysis of the immunoprecipitates reveals successful IP of the mutants (Fig. 20A), as visualised using antibody against the HA-tag. The IIb form is also immunoprecipitated, since the HA tag is N-terminal. IIb is only produced in cells expressing mutants LS*49+50ATM, LS*49+NS52. A band migrating slightly faster than IIb can be seen for some mutants, but is a degradation product other than IIb, which may result from incomplete inhibition of proteases during IP.
Immunoprecipitated material from irradiated cells was probed with anti- phospho-tyrosine antibody to determine the tyrosine phosphorylation induced. The levels of tyrosine phosphorylation detected were overall very weak for all mutants tested, leading to a low signal/ background ratio. The blocking procedures for the anti-HA antibody produced an unacceptable level of background when used with the anti-phospho-tyrosine antibody, and vice-versa, thereby preventing stripping and re-probing of the same blots. Following testing of several commercially available antibodies, the best results were obtained using the P-Tyr-100 antibody. This antibody was raised against a synthetic peptide, and has been shown to recognise a synthetically phosphorylated CTD peptide (manufacturer’s claim). However, it is not clear how the recognition by this antibody, and other anti-phospho-amino acid antibodies, is affected by other changes in the surrounding sequence (for example, phosphorylation of other residues). Despite the weak signals obtained, it could be shown that only mutants containing an intact CTD last repeat become tyrosine-phosphorylated following exposure to IR (Fig. 20B). Signals could be seen for CKII mutants LS*49+52 -S9A, -S13A, and -S9/13A, indicating that these mutations do not interfere with c-Abl interaction.
2.7
CKII phosphorylation of the CTD
Through point mutation of the CKII consensus sites in the Pol II LS final repeat we hoped to destroy this interaction in vivo. CKII has been shown only to phosphorylate fragments containing the C-terminal, largely non-consensus region of CTD, and not N-terminal consensus fragments (Kuenzel et al., 1987). It is thus still not clear whether the consensus CKII sites are actually real targets. Work on the CTD last repeat by the Bensaude group resulted in the production of rabbit polyclonal antibodies to address this question. Two synthetic peptides of the last repeat were produced for immunisation: one
phosphorylated on every serine, the other possessing no phosphorylated amino acids. The resulting serum was shown in their own tests to specifically recognise phosphorylated, and non-phosphorylated forms of Pol II LS, however the CKII consensus sites used could not be determined. To address this question, these antibodies were tested for their ability to recognise our last repeat mutants. Rosi cells were induced to express the Pol II LS mutants for 24h before addition of α-amanatin for a further 24h, to remove the background of the endogenous Pol II LS. Western blots using either polyclonal antibody against the phosphorylated (DEEP1), or dephosphorylated (DEEN4) peptide are shown in figure 21A, compared to the loading control using anti-HA antibody. The mutants lacking the last repeat (LS*49+50, LS*49+50ATM and LS*49+NS52) or both CKII sites (LS*49+52 S9/13A), show no response to either antibody as expected. However, those containing the final repeat show a mixed reaction: LS*49M+52 and LS*49+52 react to both antibodies; LS*wtMCS to just DEEN4. This result is interesting, since the Bensaude lab failed to identify an in
vivo situation where the DEEN4 antibody recognised the endogenous Pol II LS.
This may be due to a structural artefact of our mutants, or that as a result of over expression, not all Pol II LS* has been able to become phosphorylated. The absence of reactivity to the DEEP1 antibody seen with LS*wtMCS may be explainable after considering the CKII point mutants: Pol II LS*49+52 S9A only reacts with DEEP1, while Pol II LS*49+52 S13A only reacts with DEEN4. This suggests that only serine 13 of the last repeat is a CKII target. The extra seven amino acids in the LS*wtMCS last repeat may interfere with its structure, or prevent the interaction with CKII. Figure 21B shows a summary of the reactivity of the antibodies which each of the mutants and the epitopes present. However, it is important to note that these antibodies are polyclonal mixtures raised against a wt last repeat peptide, and that there reactivity to synthetic point mutants has not been characterised. For the purposes of this experiment we assume structural similarity of the point-mutated sequence to that of wt.
Figure 21. Phosphorylation specific antibodies determine the level of phosphorylation at the final repeat CKII sites in vivo. (A) Expression of Pol II LS* CTD mutants in stably transfected Rosi cell lines. Cells were
harvested 48h after induction of expression, and incubation with α-
Amanatin for the last 24h. Expression levels were determined by staining Western blots with the high-affinity anti-HA antibody 3F10. Polyclonal antibodies raised against a repeat 52 peptide, phosphorylated at positions 5, 9 and 13 (DEEP), were used to determine the level of phosphorylation resulting from CKII activity. Polyclonal antibodies raised against an equivalent, non-phosphorylated peptide (DEEN), were used as a control. (B) The table reflects the potential CKII target epitopes present in each CTD mutant, and their recognition by the phosphorylation-specific antibodies.