Autophosphorylated Pak2 was present in immunoprecipitates using eIF4G antibody, confirming the interaction between Pak2 and eIF4G (data not shown). to structural changes related to phosphorylation. The effects of Pak2 on translation were not due to changes in transcription, as shown with GFP reporter mRNA. Pamapimod (R-1503) Total RNA was extracted from the transfected cells and RTCPCR was carried out using GFP-specific primers. There Pamapimod (R-1503) was no change in GFP mRNA levels (Figure 1C). A similar pattern of Pak2 inhibition of translation was observed using luc reporter mRNA transcribed and transfected into cells (data not shown). The inhibitory effect of Pak2 on translation was evaluated further using a modified reticulocyte lysate system. To enhance regulation, excess amounts of eIFs, especially eIF4F, were reduced with m7GTP-Sepharose, as developed by Svitkin (1996). Low levels of mRNA were used to ensure that translation was susceptible to 5cap-3poly(A) synergy. Purified WT Pak2, K/R and the constitutively active mutant T402E were added to reticulocyte lysate. Increasing levels of WT Pak2 inhibited translation by up to 60%, while T/E was a potent regulator of translation with up to 82% inhibition; little effect was observed with K/R (Figure 2). Thus, the level of inhibition was consistent with the activity of Pak2, indicating that the inhibitory effect was exerted through the protein kinase activity. Open in a Pamapimod (R-1503) separate window Figure 2 Pak2 inhibits translation in reticulocyte lysate. Increasing amounts of purified WT GST-Pak2, K/R and T/E were added to the reticulocyte lysate as indicated. Protein synthesis was monitored Pamapimod (R-1503) using luc reporter mRNA (2.5 g/ml). The data are the average of three experiments; standard deviations are shown by error bars. Pak2 interacts with eIF4G The interaction of Pak2 with eIF4F was examined in 293T cells transfected with Pak2, K/R or the dominant-negative mutant T402A (T/A). Using antibody to eIF4G, WT Pak2 and K/R coimmunoprecipitated with eIF4G, whereas there was no signal with T/A or the preimmune IgG control, as shown by Western blotting (Figure 3A). When calculated on a molar basis, approximately 80% of the WT Pak2 was bound to eIF4G, as compared to 30% of K/R. Since the dominant-negative T/A was not associated with eIF4G, and the binding of K/R was significantly reduced, the data suggest that WT Pak2 preferentially bound to eIF4G, as compared to the kinase-inactive mutants. Open in a separate window Figure 3 Interaction between Pak2 and eIFs in 293T cells. (A) The interaction between Pak2 and endogenous eIF4G was analyzed by transfection of HA-Pak2 (WT, K/R, and T/A) into 293T cells; eIF4G was immunoprecipitated from cell extracts with eIF4G antibody and analyzed by SDSCPAGE. Upper panel, Western blot of Pak2 with HA-tag antibody. Lower panel, reprobe of the same membrane with eIF4G antibody. (B) Following transfection with HA-WT Pak2, immunoprecipitation was with antibodies specific for the individual eIFs, as indicated. The lanes were probed with antibody to the corresponding factor. Arrows indicate each eIF. (C) Coimmunoprecipitation of Pak2 was shown by Western blotting of (B) with anti-HA-Pak2. (D) The interaction between Rabbit Polyclonal to UGDH endogenous eIF4G and Pak2 in 3T3-L1 cells was analyzed following immunoprecipitation with N-19 antibody. Lower panel, Western blot with N-19; upper panel, the membrane was stripped and reprobed with antibody to eIF4G. Similar Pamapimod (R-1503) experiments were carried out by immunoprecipitation with antibody specific for eIF4E, eIF4A, PABP, eIF4B and eIF3. Each of these antibodies efficiently immunoprecipitated the cognate eIFs (Figure 3B). eIF4A and eIF4B were found to interact with WT Pak2, but to a lesser extent than eIF4G (Figure 3C). No interaction between WT Pak2 and eIF4E, eIF3 or PABP was observed. Approximately 5% of the total eIF4G was detected in the immunoprecipitate with anti-eIF4A, suggesting that the association could be partially through eIF4G. With antibody to eIF4B, no eIF4G was coimmunoprecipitated, suggesting a direct interaction between eIF4B and Pak2. To.
Autophosphorylated Pak2 was present in immunoprecipitates using eIF4G antibody, confirming the interaction between Pak2 and eIF4G (data not shown)