Figure 2. PKC phosphorylates α_1c Ser⁵²⁸ and Ser⁵³³.

A, The schematic demonstrates the PKC phosphorylation sites in the α_1c I-II loop. B, upper panel, Shown is autoradiogram of PKCα in vitro kinase reaction performed with [γ-³²P] ATP and GST-fused WT, S528A, S533A and S528/S533A I-II loop. PKC phosphorylated Ser⁵²⁸ and Ser⁵³³. Lower panel, Coomassie-staining of autoradiogram demonstrating amount of fusion protein used. C-D, upper panels, WT GST fusion protein was phosphorylated with PKCα, size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose and immunoblotted using anti-phospho-Ser⁵²⁸ and Ser⁵³³ antibodies (pS528 and pS533 respectively). PKC phosphorylates Ser⁵²⁸ and Ser⁵³³ in the GST fusion protein I-II loop. Lower panels, Ponceau staining indicates equivalent loading of GST fusion proteins. E-F, Shown are pS528 and pS533 immunoblots of in vitro kinase reactions of eight PKC isoforms. Lower panels, Ponceau staining indicates equivalent loading of GST fusion proteins. G, Extracts from HEK cells transfected with WT or S528A were prepared, followed by pre-immune or α_1c immunoprecipitation and PKCα kinase reaction as indicated. Samples were size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with anti-phospho-Ser⁵²⁸ antibody (upper panel) or α_1c antibody (lower panel). H, Extracts from HEK cells transfected with WT α_1c, in the presence or absence of β_2a subunit, were prepared, followed by α_1c immunoprecipitation and PKCα kinase reaction as indicated. Samples were size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with anti-phospho-Ser⁵³³ antibody (upper panel) or α_1c antibody (lower panel). All blots are representative of 3 or more similar experiments.