In order to test if Gadd45γ is able to interact with p27Xic1
in similar manner to Gadd45α interacts with p21Cip1; we performed a protein interaction study. Co- immunoprecipitation is technique that uses antibodies to first purify a specific antigen, and also any proteins bound to that antigen, from a protein extract, and then a second antibody is used to detect the presence of binding partners by Western blot analysis. The gold standard of co-immunoprecipitation experiments is the use of antibodies that detect endogenous protein as opposed to over-expressed or tagged proteins. Here, we were unable to perform co-immunoprecipitations on endogenous proteins as we did not possess antibodies for Gadd45γ or p27Xic1. Instead we obtained tagged proteins (C-terminus Myc tagged (MT) Gadd45γ and N-terminus Haemagglutinin tagged (HA) p27Xic1) and over-expressed these constructs in oocytes. Oogenesis in X. laevis is separated into six stages based on size and yolk content (Dumont, 1972). Stage VI oocytes are the most mature and will only enter meiosis after incubation in progesterone, and can be utilised as translation factories. Injected oocytes were left to translate the exogenous message overnight before being homogenised and processed for protein extraction.
To test if our injected messages were being translated we performed Western blot analysis on the protein extracts to identify translated HA- or Myc-tagged proteins. Figure 4.9A shows an anti-HA Western blot performed on these protein extracts. Lane 1 was our negative control; Western blot analysis using an antibody specific for the HA epitope did not identify any proteins in un-injected oocytes, thus there is no non-specific binding of the HA antibody used in the Western blot analysis. Lane 2 shows Western blot analysis on protein extracted from oocytes injected with
Figure 4.9 Gadd45γinteracts with p27Xic1with a low affinity.Oocytes were injected with 100 ng of various mRNA messages (as indicated), left to translate overnight, then harvested for protein extraction 24 hours after injection. Protein extracts were then exposed to Western blotting to observe translation of injected messages. (A) Haemagglutinin (HA) epitopes were detected using an anti-HA antibody. Ap27Xic1ORF cDNA clone was tagged with the HA epitope
(YPYDVPDYA) by PCR cloning. mRNA synthesisedin vitrofrom this clone translatedin vivo, as observed by a band at approximately 27 kDa in lane 2. HA- p27Xic1was also detected in lanes 5 and 6 where it was co-injected with Myc tagged (MT)Gadd45γmRNA andCyclin A2mRNA. (B)Gadd45γ-MTwas a kind gift from Jose-Luis Gomez-Skarmeta, CABD Seville, andMT-Cyclin A2was a kind gift from Anna Philpott, University of Cambridge. Oocytes injected within vitrosynthesised Gadd45γ-MTmRNA successfully translated this message to produce Gadd45γ, as indicated by a distinct band in lane 3.MT-Cyclin A2mRNA also translated
successfully (lane 4). After co-injection withHA-p27Xic1mRNA, bothGadd45γ-MT (lane 5) andMT-Cyclin A2(lane 6) again successfully translated. Taken together A and B showed translation ofHA-p27Xic1mRNA,Gadd45γ-MTmRNA andMT-Cyclin A2mRNA was successful and therefore co-immunoprecipitations could be
performed. (C) Immunoprecipitation of each protein extract was performed using an anti-Myc antibody. This procedure purified the protein extract by isolating only proteins with the Myc epitope (EQKLISEEDL), or those bound to protein containing the Myc epitope. A western using an anti-HA primary antibody was then performed to observe if HA-p27Xic1co-immunoprecipitated with, and therefore bound (directly or indirectly) to, Gadd45γ-MT and MT-Cyclin A2. All six lanes of the Western blot shown in C contain bands highlighting non-specific binding. These bands (as
indicated by the red arrow) have a molecular weight of approximately 20-25 kDa and are most probably the small subunit of the Myc antibody that for unknown reasons is bound to by the HA antibody. Nevertheless, we believe that there are bands present in this Co-IP that represent real protein interactions (black arrow). In lane 6, our positive control shows HA-p27Xic1 is present in anti-Myc purified protein extracts, showing, as has already been well characterised, that p27Xic1directly interacts with Cyclin A2. Lanes 1-4 do not contain bands for HA-p27Xic1, as expected. But there is a weak band of appropriate size, present in lane 5; suggesting HA-p27Xic1interacts with Gadd45γ-MT, albeit with much lower affinity than p27Xic1
and Cyclin A2 interactions.
HA-p27Xic1 mRNA. A clear band at approximately 27 kDa is visible; and is most likely HA-p27Xic1. Lane 3 and lane 4 are Western blot analyses on protein extracts taken from Gadd45γ-MT mRNA and MT-Cyclin A2 mRNA injected oocytes. Both lanes are negative for protein detectable with the HA antibody. To observe interactions between Gadd45γ and p27Xic1 we co-injected Gadd45γ-MT mRNA and HA-p27Xic1 mRNA and performed a co-immunoprecipitation (Fig 4.9C, lane 5). Figure 4.9A, lane 5 shows the presence of HA-p27Xic1 in the protein extract. As a positive control co-immunoprecipitation to show the interaction between p27Xic1 and Cyclin A2, which are known to interact and cause cell cycle exit (Philpott and Yew, 2008), was performed. Lane 6 shows the protein extracted from oocytes injected with HA-p27Xic1 mRNA and MT-Cyclin A2 mRNA, with HA-p27Xic1 clearly present. In conclusion anti-HA Western blot analysis on each protein extract shows HA-p27Xic1 is present and there is no non-specific binding of this antibody during Western blot analysis.
Figure 4.9B shows Western blot analysis on the same protein extracts, but using an antibody that detects the Myc epitope. This Western blot aimed to detect successful translation of the over-expressed Myc-tagged messages. The negative control, un-injected oocyte protein extract (lane 1) and the HA-p27Xic1 mRNA injected oocyte protein extract (lane 2) contained no Myc tagged proteins. Gadd45γ- MT was detected in oocytes injected with Gadd45γ-MTmRNA (lanes 3 and 5). The protein product of Gadd45γhas an actual size of 18 kDa (Hoffman and Liebermann, 2009). However, the protein detected by the Myc antibody used here has an apparent molecular mass that is much larger, approximately 25-30 kDa. We believe this protein is Gadd45γ-MT, as there is no non-specific binding in the negative control
(lane 1). We are unable to explain why this protein runs at an anomalous position relative to non-tagged Gadd45γ, although it is likely to be a consequence of this protein being Myc-tagged. Lanes 4 and 6 identify the presence of appropriately sized MT-Cyclin A2 (untagged Cyclin A2 is estimated to be 46.5 kDa) in the oocytes injected with MT-Cyclin A2 mRNA. In conclusion Figure 4.8B shows all Myc- tagged mRNA constructs translated appropriately.
We next performed a co-immunoprecipitation on the oocyte protein extracts described above, using the Myc antibody to purify the extracts, and the HA antibody for Western blot analysis (Fig 4.9C). No proteins were present in the un-injected control sample or the single injection controls (lanes 1-4). Our positive control with HA-p27Xic1mRNA co-injected withMT-Cyclin A2mRNA (lane 6), showed p27Xic1is able to bind Cyclin A2 as a band for HA-p27Xic1 at the appropriate size (27 kDa) is present (black arrow). This result shows HA-p27Xic1is bound to MT-Cyclin A2 when MT-Cyclin A2 is immunoprecipitated with the Myc antibody. Co- immunoprecipitation of Gadd45γ-MT with HA-p27Xic1 was less successful. A weak band of appropriate size can be observed (lane 5, white arrow) suggesting p27Xic1 is able to bind Gadd45γ, but with a much lower affinity than Cyclin A2, although this has not been confirmed biochemically.