Mouse 3-Phosphoinositide-dependent Protein Kinase-1 Undergoes Dimerization and trans-Phosphorylation in the Activation Loop

Activation of mouse 3-phosphoinositide-dependent protein kinase-1 (mPDK1) requires phosphorylation at a conserved serine residue, Ser²⁴⁴, in the activation loop. However, the mechanism by which mPDK1 is phosphorylated at this site remains unclear. We have found that kinase-defective mPDK1 (mPDK1 ^KD), but not a kinase-defective mPDK1 in which Ser²⁴⁴ was replaced with alanine (mPDK1^KD/S244A), is significantly phosphorylated in intact cells and is a direct substrate of wild-type mPDK1 fused to the yellow fluorescence protein. Phosphoamino acid analysis and phosphopeptide mapping studies revealed that mPDK1 trans-autophosphorylation occurred mainly on Ser²⁴⁴. On the other hand, Ser³⁹⁹ and Thr⁵¹⁶, two recently identified autophosphorylation sites of mPDK1, are phosphorylated primarily through a cis mechanism. In vivo labeling studies revealed that insulin stimulated both mPDK1^KD and mPDK1 ^KD/S244A phosphorylation in Chinese hamster ovary cells overexpressing the insulin receptor. However, Western blot analysis using a phosphospecific antibody revealed no increase in insulin-stimulated phosphorylation of Ser²⁴⁴ in these cells overexpressing mPDK1. mPDK1 undergoes dimerization in cells and this self-association is enhanced by kinase inactivation. Deletion of the extreme C terminus disrupts mPDK1 dimerization and Ser²⁴⁴ trans-phosphorylation, suggesting that dimerization is important for mPDK1 trans-phosphorylation. Taken together, our results show that mPDK1 autophosphorylation occurs at multiple sites through both cis and trans mechanisms and suggest that dimerization and trans-phosphorylation may serve as mechanisms to regulate PDK1 activity in cells.