Regulation of 3-Phosphoinositide–Dependent Protein Kinase 1 Activity by Homodimerization in Live Cells

Abstract

3-Phosphoinositide-dependent kinase 1 (PDK1) plays a central role in regulating the activity of protein kinases that are essential for signaling; however, how PDK1 itself is regulated is largely unknown. We found that homodimerization of PDK1 is a spatially and temporally regulated mechanism for controlling PDK1 activity. We used Förster resonance energy transfer monitored by fluorescence lifetime imaging microscopy to observe PDK1 homodimerization in live cells. A pleckstrin homology (PH) domain-dependent, basal dimeric association of PDK1 was increased upon cell stimulation with growth factors; this association was prevented by a phosphatidylinositol 3-kinase inhibitor and by a mutation in, or a complete deletion of, the PH domain of PDK1. The distinct spatial distribution of PDK1 homodimers relative to that of heterodimers of PDK1 and protein kinase B (PKB), and the ability of monomeric mutants of PDK1 to phosphorylate PKB, suggested that the monomer was the active conformation. Mutation of the autophosphorylation residue threonine-513 to glutamate, which was predicted to destabilize the homodimer interface, enhanced the interaction between PDK1 and PKB and the activity of PKB. Through in vitro, time-resolved fluorescence intensity and anisotropy measurements, combined with existing crystal structures and computational molecular modeling, we determined the geometrical arrangement of the PDK1 homodimer. With this approach, we calculated the size of the population of PDK1 dimers in cells. This description of a previously uncharacterized regulatory mechanism for the activation of PDK1 offers possibilities for controlling PDK1 activity therapeutically.

Regulation of Protein Kinase C-related Protein Kinase 2 (PRK2) by an Intermolecular PRK2-PRK2 Interaction Mediated by Its N-terminal Domain

“…Homodimers of AGC kinases have also been described in the literature for PKB/Akt, mediated by the PH domain (48,49), and for PKC␣, mediated by the C1 and C2 domains (50). Recent work using fluorescence lifetime imaging microscopy deduced that PDK1 forms homodimers in cells (51). Altogether, the oligomerization mechanisms described for tyrosine kinases and suggested for AGC kinases PKB/Akt, PDK1, and PKC␣ seem to be different from the mechanism we propose for PRK2.…”

PI3K/Akt signaling requires spatial compartmentalization in plasma membrane microdomains

“…Despite advances (23), the activation mechanisms of PDK1 are still not well understood. Here, in contrast to the previously held belief that PDK1 is constitutively active and cannot be further activated by growth factor stimulation (10), we show that PDK1 can be activated by various growth factors, and this induced activation occurs in membrane rafts.…”

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

“…The AGC kinase PDK1 is known to heterodimerize with several AGC kinases including PKCs, AKT, PKN, and S6K through interactions involving the PIF binding pocket on the PDK1 catalytic domain and the hydrophobic motif found on the C-tail of the other kinase (34). This interaction has been shown to dramatically enhance PDK1 catalytic activity and cellular function (34 between the catalytic and PH domains has been speculated to down-regulate PDK1 activity in cells (60). In the protein kinase C-related protein 2 (PRK2) an inhibitory homodimerization occurs between the N-terminal regulatory domains (including a C2 domain) and the catalytic domains (26).…”