FIGURE 2.

SSeCKS/PKC binding: domain mapping and effects of culture confluence. A, domains of SSeCKS that associate with PKCα. Lysates of HEK293T cells transiently transfected with a PKCα-encoding expression vector were incubated with beads containing GST alone or GST fusions of various SSeCKS domains (lower panel, Coomassie-stained GST proteins). The beads were then subjected to IB analysis and probed with anti-PKCα Ab (upper panel). B, SSeCKS aa 553–900 are required or PKCα binding. Left panel, ³⁵S-labeled FL SSeCKS and SSeCKS[Δaa553–900] were produced in TnT reactions; right panel, IP of ³⁵S-labeled proteins with beads containing GST or GST-PKCα. C, effect of PKC activity and PS (100 μg/ml) on SSeCKS domain binding. Left panel, slot blots containing GST or GST-SSeCKS fusion proteins blotted with anti-GST Ab; right panel, overlay assays incubated with various PKC preparations and probed by IB with PKC Abs. D, upper panel, homologous sequences within the aa 553–720 and 721–900 domains (*, identical; #, homologous). Ser-613, putative PKC phosphorylation site (underlined); bold, residues represented in the peptides in the lower panel. Lower panel, peptide competitors used in E (homologous residues of the 599 and 748 peptides in bold). E, aa 596–605 and 745–753 required for SSeCKS to bind PKCα. GST- or GST-SSeCKS beads were used to bind cell lysates of HEK293T cells transiently overexpressing PKCα followed by IB for PKCα. Δ596–605 and Δ745–753 are internal deletions to GST-SSeCKS[553–720] and SSeCKS[721–900], respectively. Right side, addition of molar excess (100×) of peptides 599, 748, and 835 described in D. The relative levels of bound PKC are shown in the lower box; *, S.E. from two independent experiments. F, Coomassie-stained SDS-PAGE of GST and GST-SSeCKS fusion proteins used in E. *, purified GST proteins.