FIGURE 4.

Characteristics of the binding between the C-terminal fragment of PRK2 and PDK1. A and B, HEK293 cells were transfected with DNA constructs expressing Myc-PDK1 together with constructs expressing GST or GST-PRK2 fusion proteins, and the interaction was analyzed as described in the legend to Fig. 2. Duplicates of each condition are shown. The extent of PDK1 binding was quantified using the program MultiGauge V3.0 (Fujifilm) and normalized over the amount of immobilized PRK2. A value of 1 was assigned to wild-type PRK2. A, the GST-fused C-terminal region of PRK2 (amino acids 918–984; GST-CT-PRK2) was analyzed for binding to Myc-PDK1. Binding of GST-CT-PRK2 (wt) to PDK1 was not affected by mutation of the Z/TM phosphorylation site to Ala (T958A; 0.9-fold binding compared with wt) or Glu (T958E; 0.7-fold binding). In contrast, substitution of Ile-965 and Leu-966 to Ala (I965A/L966A) abolished this interaction (0.0-fold binding). B, full-length GST-PRK2 was mutated in its P-Z/TM binding site. Binding of PRK2 to PDK1 was increased by substitution of Lys-670 for Ser (K670S; 1.8-fold binding) or Lys-689 for Glu (K689E; 10.2-fold), similar to the mutation of the Z/TM phosphorylation site to Ala (T958A; 6.2-fold). The double mutation (K670S/K689E) did not further increase this interaction (6.5-fold binding). C, PRK2 C terminus-derived polypeptides (PIFtide, REPRILSEEEQEMFRDFDYIADWC) and PIFtide IL/AA were used to analyze the ability of the PRK2 C-terminal region to activate PDK1 in vitro. The activity of 0.2 μm PDK1 was measured in triplicates using the peptide T308tide as a substrate. Both, PIFtide wt and PIFtide mutated in Ile-965 and Leu-966 (IL/AA) activated PDK1 to a similar level. However, the AC₅₀ of PIFtide IL/AA was 0.82 ± 0.25 units/mg, considerably higher than AC₅₀ PIFtide wt (0.20 ± 0.04 units/mg). D, ability of biotinylated PIFtide or PIFtide IL/AA to bind to PDK1 was analyzed using surface plasmon resonance technology using SA Chips on a BiaCore system. The binding of PDK1 to biotin-peptide was performed at the indicated concentrations of PDK1. The inset shows the data at equilibrium and the estimated dissociation constant of PDK1 for each polypeptide. PDK1 is injected to the system during the “association” part of the curve. When PDK1 is not longer injected through the system, PDK1 dissociates from the chip (dissociation part of the curve). The higher slope of the dissociation from PIFtide IL/AA indicates a higher dissociation rate.