Figure 6.

PDK1 Regulates S6K1 and Its Activity Is Not Affected by Osmotic Stress.

(A) Alignment of the activation loop sequences present in the catalytic domain of different AGC kinases (Rn RSK1, rat p90 ribosomal S6 kinase1; Hs PKB, human protein kinase B; Hs PKA, human protein kinase A). The consensus sequence motif is indicated in bold, and the phosphorylation site on the motif is indicated by a gray box. Dots represent a gap of ∼150 amino acids.

(B) GFP-PDK1 was expressed in tobacco leaves (N. benthamiana) by agroinfiltration and immunoprecipitated with GFP antibody. The immunoprecipitates were then subjected to in vitro kinase assay using GST-S6K1-CD as substrate. Top panel: protein gel blot showing the equal loading of PDK1 in the kinase reactions. Bottom panel: autoradiograph showing the kinase activity over time. Time indicates the kinase reaction time at 25°C.

(C) Tobacco leaves expressing GFP-PDK1 as in (B) were treated with different stresses or inhibitors, as indicated in the figure, before subjecting the protein samples to immunoprecipitation with GFP antibody. The immunoprecipitates were then assayed for the PDK1 activity as in (B).