FIG. 7.

Ceramide induces phosphorylation of PKB on Thr³⁴ in intact cells and a PKB T34A mutant is ceramide resistant. (A) L6 myotubes were incubated in the absence or presence of C₂-ceramide (Cer, 100 μM) for 2 h prior to cell lysis. Cell lysates were resolved by SDS-PAGE prior to immunoblotting with an antibody to PKB, a phospho-specific antibody to PKB-Thr³⁴, or anti-PKB-Thr³⁴ that had been preadsorbed the antigenic phospho-peptide (100 μg/ml). (B) Myotubes were treated as in panel A but, in addition, were also exposed to insulin (Ins, 100 nM for 10 min) or pretreated with Ro 31.8220 (Ro, 5 μM) prior to incubation with Cer. Cells were lysed and immunoblotted with antibodies to PKB or PKBThr³⁴. (C) HA-tagged PKB (wild type) and HA-tagged PKB T34A were transiently transfected into L6 cells as described. Cells were exposed to C₂-ceramide (Cer, 100 μM, 2 h) and or insulin (Ins, 100 nM, 10 min) prior to cell lysis and immunoprecipitation with an anti-HA antibody. Precipitated kinases were resolved by SDS-PAGE and immunoblotted with antibodies to PKB-Ser⁴⁷³ or anti-HA. Phospho-PKB-Ser⁴⁷³- and HA-immunoreactive bands were quantified and are expressed as a ratio (lower panel).