Activation of mouse 3-phosphoinositide-dependent protein kinase-1 (mPDK1) requires phosphorylation at a conserved serine residue, Ser 244 , 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 kinasedefective mPDK1 in which Ser 244 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 Ser 244 . On the other hand, Ser 399 and Thr 516 , 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 Ser 244 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 Ser 244 trans-phosphorylation, suggesting that dimerization is important for mPDK1 transphosphorylation. 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.3-Phosphoinositide-dependent protein kinase-1 (PDK1) 1 is a serine/threonine kinase that acts downstream of PI 3-kinase in insulin and IGF-1 signaling pathways. PDK1 phosphorylates many important cellular kinases such as protein kinase B (Akt/PKB), PKC isoforms, and p70 S6 kinase in the activation loop and contributes to their activation in a PI 3-kinase-dependent manner. These results suggest that PDK1 may play a key role in the regulation of various cellular events such as cell proliferation, differentiation, and apoptosis (1, 2).While PDK1 has been shown to activate PKB/Akt and its other substrates by catalyzing the phosphorylation of a conserved residue in the activation loop of these kinases, how PDK1 itself is regulated remains largely unknown. Recent studies suggest that phosphorylation may play a role in the regulation of PDK1 function in cells (3). Multiple phosphorylation sites have been identified on PDK1 and several heterologous kinases that mediate some of these phosphorylation events have been identified. Src, Fyn, and Abl tyrosine kinases can phosphorylate PDK1 in vitro and overexpression of v-Src results in tyrosine phosphorylation of PDK1 in cells (4 -6). Specific serine/threonine kinases that phosphorylate PDK1 have not been identified. However, PDK1 has been shown to possess the intrinsic ability to phosphorylate itself (7,8).The mechanism ...