Figure 3. Two equivalent residues in TNS3 and PTEN dictate the binding specificity.

(a) Thr319 in PTEN, and the equivalent Tyr321 in TNS3–ABD, controlled their dynamic interactions with DLC1 in response to EGF treatment. HEK293 cells co-expressing DLC1 and wt PTEN or a mutant (T319A, T319E or T319Y), or wt TNS3–ABD or the Y321T mutant were subjected to IP/IB to examine their interactions in the absence (−) or presence (+) of EGF stimulation. DLC1 was tagged with FLAG; PTEN, TNS3 or the mutants were fused to GFP. (b) The same panel of proteins as in a were examined for binding to endogenous PI3K (via p85). (c,d) Thr321 in PTEN and Thr323 in TNS3–ABD play an important role in dictating binding to DLC1 (c) or PI3K–p85 (d). (e,f) Recombinant GST–PTEN, GST–TNS3–ABD or an indicated mutant was used to pull down FLAG-DLC1 (e) or FLAG-p85 (f) from HEK293 cells. The bound proteins were detected by anti-FLAG IBs. (g) A summary of binding specificities displayed by the panel of mutants used in a–f and in Supplementary Fig 6 and 8. Data shown are representative of three independent experiments.