Fig. 4.

Effect of 14-3-3ζ on the activity of phosphorylated AANAT at physiological concentrations of substrates (4, 28). A sample containing 15 ng of phosphorylated AANAT proteins oAANAT pT31 pS205, oAANAT S205A pT31, and oAANAT T31A pS205 was incubated with an ≈10-fold molar excess of 14-3-3ζ (125 ng). The addition of 14-3-3ζ increased ¹⁴C-labeled N-acetylserotonin (N-Ac-[¹⁴C]5HT) formation by AANAT pT31 pS205 from 22.6 to 109.0 pmol (average of two assays in which the results were essentially identical); there was no increase with the AANAT T31A pS205 or pT31A S205. For additional details, see Materials and Methods.