RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry
Figure 9
TARP is a substrate for Abl family kinases.
(A) The putative TARP consensus tyrosine phosphorylation sites are compared to the Abl kinase consensus target sequence. C. trachomatis TARP contains six copies of a ∼50 amino acid repeat. Each repeat region encodes either motif I or motif II with 1 or 2 potential tyrosine phosphorylation sites (indicated with an asterisk). Highlighted residues indicate the high homology of the TARP motifs to consensus Abl kinase target substrates at positions −3, −1, +1, and +4. (B) HeLa, NIH 3T3, and Abl/Arg−/− cells were transfected with EGFP-fused to TARP and incubated for 24 hours (green; panels B, E, H). Cells were then fixed and tyrosine phosphorylated TARP was stained with 4G10 (red; panels A, D, G) and actin was stained with phalloidin (blue; panels C, F, I). The exposure time for each filter of all images was identical. Tyrosine phosphorylation of TARP is significantly decreased in Abl/Arg−/− cells. (C) Immunoblots of protein extracts from NIH 3T3 and Abl/Arg−/− cells transfected with EGFP-TARP were probed with antibodies to 4G10, GFP, or actin (loading control). The fraction of phosphorylated TARP compared to total TARP was quantified by densitometry analysis for both cell types. Immunoblots shown are representative of three independent experiments. There was a significant reduction in the total TARP phosphorylated in the Abl/Arg−/− cells compared to the 3T3 cells, **p<0.01 (ANOVA).