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. 2015 Jul 13;6:7721. doi: 10.1038/ncomms8721

Figure 2. C2 domains were phosphorylated on specific Thr residues.

Figure 2

(a) The PTEN and TNS3 C2 domains were phosphorylated. The C2 domains were expressed in HEK293 cells and examined for Ser, Thr or Tyr phosphorylation using specific antibodies following EGF treatment for 30 min. GFP was included as a control. (b) The Mek1/2 kinase is involved in phosphorylating the PTEN-C2 domain. HEK293 cells expressing the GFP-PTEN-C2 domain, or the T319A or T321A mutant, were co-transfected with a construct for HA-CA-Mek1/2 or treated with EGF or U0126, a Mek1/2 inhibitor. Protein phosphorylation was determined using an anti-pThr antibody. Whole-cell lysates (WCL) were IB for total Erk1/2, phosphorylated Erk1/2 (pErk1/2) or HA. (c) The PTEN-C2 domain double mutant, T319/321A was no longer Thr phosphorylated following EGF stimulation or CA-Mek1/2 overexpression. (d) The TNS3-C2 domain, but not the T323A mutant, was phosphorylated following Mek1/2 overexpression or EGF stimulation. Data shown are representative of three independent experiments.