FIGURE 3.

SSeCKS inhibits PKC activity through its scaffolding domains. A, confluence-induced suppression of PKC activity correlates with increased SSeCKS levels. Upper panel, IB of lysates of WT- or KO-MEF for SSeCKS, PKCα, or GAPDH; lower panel, relative PKC specific activity (total activity normalized for PKCα protein levels) in subconfluent versus confluent conditions. Error bars, S.E. from triplicate experiments. B, increased relative PKCα activity in KO-MEF based on a ³²P in vitro kinase assay using immunoprecipitated PKCα and MBP substrate. Relative protein or phosphorylation levels (numbers below the lower panels) reflect a typical result of three independent experiments. C, SSeCKS/PKCα association increases with culture confluence and correlates with decreased kinase activity. Lysates from subconfluent (subconfl.) and confluent cultures of NIH3T3 cells were directly blotted for PKCα (inset panel, “input”) or immunoprecipitated with anti-SSeCKS Ab and then probed by IB with anti-PKCα (lower inset panel) or subjected to PKC kinase assays (graph) as in A. Error bars, S.E. from triplicate experiments. D, PKC can be activated by PMA in the absence of SSeCKS. Lysates of serum-starved subconfluent WT- or KO-MEF treated with PMA (50 nm) or DMSO vehicle (“Ctrl”) for 1 or 5 min (′) were assayed for PKC activity. Error bars, S.E. from triplicate experiments. E, the PKCα-inhibitory domain on SSeCKS maps to aa 553–900. HA IPs of HEK293T lysates co-transfected with HA-PKCα, pEGFP, and either FL, Δ553–900, or Δ2–553 SSeCKS expression vectors were added to a ³²P in vitro kinase assay as in B. Error bars, S.E. from triplicate experiments. F, expression of FL or Δ553–900 SSeCKS products in WT- (“+/+”) or KO (“−/−”)-MEF probed with anti-SSeCKS Ab. G, FL but not Δ553–900 SSeCKS suppresses PKC activity in cells. Lysates of WT- or KO-MEF transfected with pCMV vector (“V”) or expression vectors encoding FL or Δ553–900 SSeCKS were assayed for total PKC specific activity as in A. Error bars, S.E. from triplicate experiments. H, the homologous motifs aa 596–605 and 745–753, respectively, are required for SSeCKS aa 553–720 and 721–900 domains to inhibit PKCα activity (act.). Molar excesses (25×) of GST or GST-SSeCKS proteins (shown in Fig. 2F) were added to ³²P in vitro kinase (IVK) assays with commercial (Biomol) PKCα and MBP substrate (lower panel, total versus ³²P levels of MBP). Error bars, S.E. from triplicate experiments. *, p < 0.01. I, phosphorylation levels of the GST or GST-SSeCKS competitors used in H.