Fig. 2. Sgk1 phosphorylates Nedd4-2, but not Nedd4-1. (A) Oocytes expressing either wild-type Nedd4-2 (N4-2) or the phosphorylation mutants Nedd4-2-S338A-S444A [N4-2(S-A)₂], Nedd4-2-S338A, Nedd4-2-S444A and myc-Sgk1 [wild-type, catalytically inactive (KA, K130A) or PY motif-mutated (ΔPY, Y301A)] were incubated with [³²P]ortho–phosphate and treated as follows: top, immunoprecipitation from lysates with anti-Nedd4-2 antibodies and autoradiography; middle, western blot on lysates with anti-myc antibody (recognizing Sgk1); bottom, western blot on lysates with anti-Nedd4-2 antibodies. (B) Mouse Nedd4-1 (mNedd4-1/T7) or Nedd4-2 (mNedd4-2/T7), both epitope-tagged with a T7 epitope (Novagen), were expressed with or without Sgk1 (as indicated) and phosphorylation was followed as described in (A), except that the Nedd4 proteins were immunoprecipitated with anti-T7 antibody (top). Expression of mNedd4-1, mNedd4-2 and Sgk1 was followed by western blot analysis on lysates, using both anti-T7 (Nedd4-1 or Nedd4-2) and anti-myc (Sgk1) antibodies. (C) Phosphorylation of a synthetic peptide substrate (Sgktide) by Sgk1. Wild-type and mutant myc-Sgk1 (lacking a functional PY motif) were expressed in Xenopus oocytes and immunoprecipitated with anti-myc antibodies. The immunoprecipitated kinases were assayed in a kinase assay with Sgktide or a mutant peptide lacking the phosphorylation site as described in Materials and methods. Phosphorylated peptides were then analyzed by separation on a tricine acrylamide gel followed by autoradiography. Both wild-type and mutant Sgk1 were able to phosphorylate Sgktide, but not its mutant (top). Bottom, Coomassie Blue staining.