Fig. 6. PICK1 inhibits PKCα phosphorylation of mGluR7a.A, Inhibition of PKCα phosphorylation by PICK1. GST-ct-mGluR7a (15 pmol) was preincubated with 15 pmol MAL-PICK1 or MAL (control) in 10 μl buffer B (20 mm HEPES, pH 7.4, 120 mm NaCl, 1 mm CaCl2) for 3 hr at 4°C. The phosphorylation reaction was started by adding varying concentrations of PKCα in buffer A (supplemented with 200 μm [γ-32P]ATP, 50 mCi/mmol) and allowed to proceed for a total of 3 min. When compared with MAL alone, MAL-PICK1 inhibited PKCα-evoked phosphorylation of GST-ct-mGluR7a throughout the range of PKCα concentrations examined. B, Inhibition of PKCα phosphorylation requires PICK1/mGluR7a interaction. GST-ct-mGluR7a (15 pmol) or GST-ct-mGluR7a-mutant (H851–L892, a construct that contains the PKCα phosphorylation sites but not the interaction site for PICK1) was preincubated with 15 pmol MAL-PICK1 and 1.35 μg/ml PKCα in 10 μl buffer A (supplemented with 1 mmCaCl2) for 3 hr at 4°C. The phosphorylation reaction was started by addition of 10 μl buffer C (40 mmTris-HCl, pH 7.5, 200 μm [γ-32P]ATP, 50 mCi/mmol) and allowed to proceed for times indicated. The rate of PKC-evoked phosphorylation of GST-ct-mGluR7a was reduced as compared with the GST-ct-mGluR7a-mutant. The mean percentage inhibitions of PKCα phosphorylation of GST-ct-mGluR7a as compared with GST-ct-mGluR7a-mutant were 14.4, 16.4, 15.5, 17.9, and 19.2% at the time points 1, 2, 3, 5, and 10 min, respectively.