FIGURE 1.

PdkA and PdkB are the activation loop kinases of PkbA and PkbR1. A, PdkA and PdkB are schematically represented and share 67 and 58% similarities in a kinase (thick black bars) and PH domain (circles), respectively. B, wild-type (WT), pdkA⁻, pdkB⁻, and pdkA⁻pdkB⁻ cells were stimulated with 1 μm cAMP, and cell extracts were prepared at the indicated times. The phosphorylation levels of the AL, Thr³⁰⁹ for PkbR1 and Thr²⁷⁸ for PkbA, the HM, Thr⁴⁷⁰ for PkbR1, and the PKB substrates were detected by immunoblotting with α-phospho-protein kinase C (pan) antibody, α-phospho-PDK docking motif antibody, and α-phospho-PKB substrate antibody, respectively. pp350, pp200, pp180 (including GefN), pp140, pp110 (including GefS and PI5K), pp90, and pp65/67 (including GacQ) were already assigned as PKB substrates in α-phospho-PKB substrate antibody staining experiments. The protein-transferred membrane was stained with Coomassie Brilliant Blue (CBB) as a loading control. C, AL phosphorylations of PkbR1 and PkbA (upper panel) and PKB substrates phosphorylations (lower panel) were compared in pdkA⁻pdkB⁻ cell extracts expressing vector (V), PdkA-GFP, or PdkB-GFP prepared as in B. D, wild-type cells expressing GFP or PdkA-GFP were treated as in B, and cell extracts taken at the indicated times were probed with an α-GFP antibody.