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Fig. 4. PKC phosphorylates -adducin at its Ser726 both in vivo and in vitro. (A) GFP, GFP-PKC or its kd mutant was transiently expressed in COS cells. 48 hours later, whole cell lysates were analyzed by immunoblotting with anti-phospho-adducin (Ser726), anti-adducin or anti-GFP. Ser726 phosphorylation of -adducin was measured and expressed as fold increase relative to the level in the cells expressing GFP. Values (means ± s.d.) are from three independent experiments. (B) Whole cell lysates from MDCK cells stably expressing GFP, GFP-PKC or its kd mutant were analyzed by immunoblotting with anti-phospho-adducin (Ser726) or anti-adducin. The Ser726 phosphorylation of adducin was measured and expressed as fold increase relative to the level in the control GFP cells. Values (means ± s.d.) are from three independent experiments. (C) MDCK cells stably expressing GFP-PKC were serum starved for 16 hours and treated with (+) or without (-) 100 nM PMA and 10 µM rottlerin for 15 minutes. The Ser726 phosphorylation of -adducin was analyzed. (D) MDCK cells stably expressing GFP-PKC were plated on collagen-coated glass coverslips for 24 hours and then stained for adducin and its Ser726 phosphorylated form. The fluorescent images were scanned by a confocal microscope on the z-axis 2 µm above the substratum. Bars, 2 µm. (E) GFP--adducin (GFP-add) or its mutants (S716A, S726A and S716A/S726A) were transiently overexpressed in HEK293 cells and immunoprecipitated by anti-GFP. The bound GFP--adducin proteins were eluted from the beads with citric acid and neutralized in Tris buffer. The purity and yield of GFP--adducin proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Blue stain. (F) GFP-PKC was transiently expressed in HEK293 cells and immobilized on protein A beads with anti-GFP. The washed immunocomplexes were analyzed by immunoblotting with anti-GFP or subjected to an in vitro kinase assay in the presence of [-³²P]ATP using purified GFP--adducin proteins as the substrates. The ³²P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The phosphorylation of GFP--adducin proteins was measured and expressed as a percentage relative to the phosphorylation of GFP--adducin (wt). Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay.

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