FIG. 11.
Effects of ceramide on kinase activity of PKC subspecies, in vitro and in vivo. (A) Effects of C2-ceramide on kinase activity of δPKC-GFP assessed by in vitro kinase assay. Kinase activities of the immunoprecipitated δPKC-GFP were measured in the presence of various concentrations of C2-ceramide (C2-Cer) or activators of δPKC such as PS and DO. Data are expressed as percentages of the control level. Statistical significance: ∗, P < 0.05 versus kinase activity of PS and DO. (B) Changes in kinase activity of δPKC-GFP in HeLa cells after C2-ceramide treatment assessed by in vivo kinase assay. δPKC-GFP was immunoprecipitated from HeLa cells overexpressing δPKC-GFP at various time points after ceramide treatment. The kinase activity of δPKC-GFP was assayed with H1 histone as the substrate without any activators such as PS or DO. Data are expressed as percentages of the control level (the kinase activity before stimulation). (C) Effects of C2-ceramide on kinase activity of endogenous PKC subtypes assessed by in vivo kinase assay. Endogenous αPKC, δPKC, and ζPKC were immunoprecipitated from HeLa cells before and after C2-ceramide (C2-Cer) treatment. The kinase activity of αPKC, δPKC, and ζPKC was assayed with H1 histone (αPKC and δPKC) or MBP (ζPKC) as the substrate without any activators such as PS or DO. Data are expressed as percentages of the control level (the kinase activity of each PKC subtype immunoprecipitated from untreated cells). Statistical significance: ∗, P < 0.01 versus kinase activity of control. All results represent the means and standard errors of more than three determinations.